Methods and compositions for expansion of cell population

FIELD OF THE INVENTION

The present invention relates to methods and compositions for expanding a cell population, and particularly stem cell populations such as a hematopoietic stem cell population.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing text file “1065334.000151-seq.txt”, file size of 4,097 bytes, created on Nov. 25, 2024. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

Hematopoietic stem cells (HSCs) are clonogenic cells, which possess the properties of both self-renewal (expansion) and multilineage potential giving rise to all types of mature blood cells. HSCs are responsible for hematopoiesis and undergo proliferation and differentiation to produce mature blood cells of various lineages while still maintaining their capacity for self-renewal. The ability to self-renew maintains the HSC population for the lifespan of an animal and also allows HSCs to repopulate the bone marrow of lethally irradiated congenic hosts.

Early HSC development displays a hierarchical arrangement, starting from long-term (LT-) HSCs, which have extensive self-renewal capability, followed by the expansion state, which corresponds to short-term (ST-) HSCs (having limited self-renewal ability) and proliferative multipotent progenitors (MPPs) (having multipotent potential but no self-renewal capability). MPP is also a stage of priming or preparation for differentiation. An MPP differentiates and commits to become either a common lymphoid progenitor (CLP), which gives rise to all the lymphoid lineages, or a common myeloid progenitor (CMP), which produces all the myeloid lineages. During this process, the more primitive population gives rise to a less primitive population of cells, which is unable to give rise to a more primitive population of cells. The intrinsic genetic programs that control these processes including the multipotential, self-renewal, and activation (or transient amplification) of HSCs, and lineage commitment from MPP to CLP or CMP, remain largely unknown.

To sustain constant generation of blood cells for the lifetime of an individual, HSCs located in bone marrow niches (Zhang, J. et al. Nature 425, 836-841, 2003; Calvi, L. M. et al. Nature 425, 841-846, 2003; Kiel, M. J., et al. Cell 121, 1109-1121, 2005; Arai, F. et al. Cell 118, 149-161, 2004) must achieve a balance between quiescence and activation so that immediate demands for hematopoiesis are fulfilled, while long-term stem cell maintenance is also assured. In adults, homeostasis between the quiescent and activated states of stem cells is important to protect HSCs from losing their potential for self-renewal and, at the same time, support ongoing tissue regeneration (Li, L. and Xie, T. Annu. Rev. Cell. Dev. Biol. 21, 605-631, 2005). Over-activation and expansion of stem cells risks both eventual depletion of the stem cell population and a predisposition to tumorigenesis. Although some factors important for stem cell activation have been identified (Heissig, B. et al. Cell 109, 625-637, 2002), the molecular events governing the transition between quiescence and activation are poorly understood.

HSCs are responsible for life-long hematopoiesis under homeostatic and stress conditions, which relies on an exquisite balance between stem cell self-renewal and differentiation (Li et al. Science, 327: 542-545, 2010; Weissman et al. Cell, 100: 157-168, 2000). Thus, HSC transplantation is a life-saving therapy for a broad spectrum of disorders, including hematologic, immune, and genetic diseases, as well as cancers (Walasek et al. Annals of the New York Academy of Sciences 1266: 138-150, 2012). However, HSC-based treatment can be limited primarily by the lack of HLA-matched donor bone marrow (BM). Allogeneic transplantation offers an alternative approach, but graft vs host disease (GvHD) remains a life-time challenge, since taking immune suppression medicine has numerous side effects, such as delayed immunological recovery, thrombotic microangiopathy (Sung et al. Stem Cells Translational Medicine, 2: 25-32 (2013); Shlomchik et al. Nature Reviews. Immunology, 7: 340-352, 2007). Transplantation of HSCs from hUCB reduces the risk of GvHD; however, the lower number of HSCs in hUCB than in BM or mobilized peripheral blood limits its application (Walasek et al. Annals of the New York Academy of Sciences, 1266: 138-150, 2012). Targeting single molecules or pathways has been studied for hUCB HSC expansion (Huang et al. Leukemia, 30: 144-153, 2016; Boitano et al. Science, 329: 1345-1348, 2010; Fares et al. Science, 345; 1509-1512, 2014; Ansellem et al. Nature Medicine, 9: 1423-1427, 2003; Antonchuk et al. Cell, 109: 39-45, 2002; Rentas et al. Nature, 532: 508-511, 2016; Himburg et al. Nature Medicine, 16: 475-482, 2010; North et al. Nature, 447: 1007-1011, 2007; Guo et al. Nature Medicine, 2018); Varnum-Finney et al. Nature Medicine, 6: 1278-1281, 2000; and Chou et al. Experimental Hematology, 41: 479-490 e474, 2013). However, other approaches are sought in order to relatively favor stem cell self-renewal versus differentiation (Zhao et al. Molecular Cell Biology, 18: 31-42, 2017).

m⁶A is a prevalent internal modification in mRNAs that regulates the outcome of gene expression by modulating RNA processing, localization, translation, and eventual decay, which is modulated by “writers,” “erasers” and “readers” of the mark (Roundtree et al. Cell, 169: 1187-1200, 2017; Li et al. Annual Review of Genomics and Human Genetics, 15: 127-150, 2014). Recent studies have elucidated the roles of m⁶A modification in stem cell fate determination and endothelial-to-hematopoietic transition during embryogenesis (Batista et al. Cell Stem Cell, 15: 707-719, 2014; Geula et al. Science, 347: 1002-1006, 2015; Yoon et al. Cell, 2017; Zhang et al. Nature, 549: 273-276, 2017; Zhao et al. Nature, 542: 475-478, 2017) as well as in leukemia development (Li et al. Cancer Cell, 31: 127-141, 2017; Vu et al. Nature Medicine, 2017; Barbieri et al. Nature, 2017; Weng et al. Cell Stem Cell, 22: 191-205 e199, 2018). Interestingly, deficiency in m⁶A writer complex, Mettl3 and Mettl14, leads to distinct outcomes in different types of stem cells. For example, Mettl3 or Mettl14 KO promoted differentiation in HSCs (Vu et al. Nature Medicine, 2017; Weng et al. Cell Stem Cell, 22: 191-205 e199, 2018; Barbieri et al. Nature, 2017) while resulting in enhanced stem cell self-renewal and maintenance in mouse embryonic stem cells (mESCs) and embryonic neuronal stem cells (NSCs) (Batista et al. Cell Stem Cell, 15: 707-719, 2014; Yoon et al. Cell, 2017). Besides, the physiological function of m⁶A in stem cells and leukemia are mediated through different mechanisms. In stem cells, m⁶A modifications regulate stem cell fate determination by m⁶A-mediated decay of mRNAs encoding stem cell fate determinant (Batista et al. Cell Stem Cell, 15: 707-719, 2014; Yoon et al. Cell, 2017) while in acute myeloid leukemia (AML), Mettl3 and Mettl14 promote leukemogenesis as m⁶A modifications stabilize the mRNAs of oncogenes and/or increase their translation (Vu et al. Nature Medicine, 2017; Barbieri et al. Nature, 2017; Weng et al. Cell Stem Cell, 22: 191-205 e199, 2018). Furthermore, previous studies have reported that the leukemogenic functions of FTO and Mettl14 are independent of YTHDF reader proteins (Li et al. Cancer Cell, 31: 127-141, 2017; Weng et al. Cell Stem Cell, 22: 191-205 e199, 2018).

As the m⁶A RNA modification is modulated by “writers,” “erasers” and “readers” of the mark (Wang et al. Nature, 505(7481): 117-120, 2014), processes that install, recognize and remove this and other marks may have various implications for cellular, developmental, and disease processes. For example, studies have shown that the m⁶A mark may act as a key post-transcriptional modification to promote initiation of microRNA (miRNA) biogenesis (Alarcon et al. Nature, 519(7544): 482-485, 2015). Evidence also points to m⁶A RNA modifications possibly being involved in the differentiation of stem cells to specific lineages (Batista, Cell stem Cell, 15(6): 707-719, 2014; Zhang et al. Nature, 549(7671): 273-276, 2017), and in regulating gene expression (Dominissini et al., Nature 485(7397):201-206, 2012; Haussmann et al, Nature 540(7632): 301-304, 2016). A m⁶A transferase METTL3 has been identified as a regulator for terminating murine naïve pluripotency (Geula et al. Science, 347(6225): 1002-1006, 2015). The m⁶A “writer” protein METTL3 has also been demonstrated in mouse T cells to disrupt T cell homeostasis and differentiation (Li et al. Nature, 548(7667): 338-342, 2017), and m⁶A RNA methylation has been found to promote XIST-mediated transcriptional repression (Patil et al. Nature, 537(7620): 369-373, 2016). M⁶A RNA modifications have also been shown to regulate the ultraviolet-induced DNA damage response (Xiang et al. Nature, 543(7646): 573-576, 2017). Study of the maternal-to-zygotic transition (MZT) as in zebrafish also indicated a role for m⁶A mRNA methylation in transcriptome switching and animal development (Zhao et al. Nature, 542(7642): 475-478, 2017). Accordingly, while accumulative evidence has brought insights into the biological functions of m⁶A (Lence et al. Nature, 540 (7632); 242-247, 2016), the function of m⁶A in adult stems cells are largely unknown.

Hematopoietic stem cells (HSCs) in bone marrow (BM) maintain homeostasis hematopoiesis throughout life and also support regeneration after myeloablation (Weissman, 2000). Quiescent HSCs perform superiorly to proliferative HSCs in lethally irradiated mice, which largely attributes to the quiescent state that protects HSCs from DNA damage (Arai et al., 2004; Fleming et al., 1993; Wilson et al., 2008) (Walter et al., 2015). However, a recent study showed that DNA damage accumulation in HSCs was associated with broad attenuation of DNA repair and response pathways that were dependent upon HSC quiescence (Beerman et al., 2014). In fact, the majority of HSCs, despite their quiescence, are sensitive to DNA damage from chemotherapeutic drugs, such as 5-Fluorouracil (5FU) (Lerner and Harrison, 1990). The unresolved issue is how the hematopoietic system overcomes the consequence of myeloablation. In respect to the remarkable heterogeneity of HSCs during development and in adult (Benveniste et al., 2010; Benz et al., 2012; Fleming et al., 1993; Morita et al., 2010; Zhou et al., 2016), the existence of a reserve HSC (rHSC) subpopulation was proposed, with the features of drug-resistance and capacity to regenerate the bulk of HSCs to overcome stress-caused myeloablation (Haug et al., 2008; Li and Clevers, 2010; Wilson et al., 2008). Thus far, however, no functional evidence has been provided in supporting existence of rHSCs in the blood system.

HSCs are preserved in complex BM niches for their maintenance and regeneration (Li and Clevers, 2010; Mendelson and Frenette, 2014; Morrison and Scadden, 2014; Scadden, 2014; Schofield, 1978). In the past decades, multiple studies have uncovered the complexity of HSC bone marrow niche components, including: endosteal (inner bone surface) cells (Calvi et al., 2003; Zhang et al., 2003), sinusoidal endothelial cells (Hooper et al., 2009; Kiel et al., 2005), Cxcl12 abundant reticular (CAR) cells (Sugiyama et al., 2006), Nestin⁺ and NG2⁺ perivascular cells (Kunisaki et al., 2013; Mendez-Ferrer et al., 2010), LepR⁺ and Prx-1⁺ mesenchymal stem and progenitor cells (Ding and Morrison, 2013; Ding et al., 2012; Greenbaum et al., 2013), non-myelinating Schwann cells (Yamazaki et al., 2011), and megakaryocytes (Bruns et al., 2014; Zhao et al., 2014). However, whether and how the BM niche complexity contributes to HSC heterogeneity regulation remain largely unclear (Itkin et al., 2016). Furthermore, the first HSC niche was initially identified as the spindle shaped N-Cadherin⁺ (N-cad⁺) pre-osteoblastic cells in the endosteum of the trabecular bone region (Calvi et al., 2003; Xie et al., 2009; Zhang et al., 2003), but the nature and function of N-cad⁺ niche cells in BM remain unclear.

Accordingly, there remains a need for elucidation and understanding of the role of m⁶A and m⁶A mRNA pathways to provide insight into molecular regulation of stem cell proliferation and differentiation. There remains a further need for methods of expanding populations of stem cells, both in vivo and ex vivo, and methods of providing treatment with such expanded stem cell populations, such as via transplant into a suitable subject.

SUMMARY

In one embodiment of the present disclosure, a method for expanding a population of stem cells is provided, the population of stem cells being obtained from a tissue selected from the group consisting of peripheral blood, cord blood and bone marrow. The method includes modulating a N-Methyladenosine (m⁶A) mRNA modification pathway in the population of stem cells, to expand the number of stem cells.

In yet another embodiment, a method for ex vivo expansion of a substantially undifferentiated stem cell population is provided, comprising modulating a N⁶-Methyladenosine (m⁶A) mRNA modification pathway in the undifferentiated stem cell population to expand the number of undifferentiated stem cells without significant differentiation of the stem cell population.

According to yet another embodiment, a method for ex vivo expansion of an hematopoietic stem cell (HSC) population is provided, the HSC population being obtained from a tissue selected from the group consisting of peripheral blood, cord blood, and bone marrow, the method comprising modulating a N⁶-Methyladenosine (m⁶A) mRNA modification pathway in the HSC population to expand the HSC population to a sufficient quantity while maintaining a multilineage differentiation potential in the HSC population, which is sufficient for subsequent transplantation into a subject in need thereof.

According to yet another embodiment, a method for ex vivo expansion of hematopoietic stem cells (HSCs) by at least 2-fold is provided, the expanded HSCs being competent to reconstitute an HSC lineage upon transplantation into a mammal in need thereof, the method comprising introducing a mutation into the stem cells that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader and culturing the population of HSCs in a suitable culture medium.

According to a further embodiment, a kit for expanding an hematopoietic stem cell population (HSC) population for subsequent transplantation into a subject in need thereof is provided, the kit comprising a system for introducing a mutation into the HSC population that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader, and instructions for use thereof.

According to yet another embodiment, a kit for expanding an hematopoietic stem cell population (HSC) population for subsequent transplantation into a subject in need thereof is provided, the kit comprising an inhibitor of a m⁶A mRNA modification reader, and instructions for use thereof.

In yet a further embodiment, a method for administering an hematopoietic stem cell (HSC) to a subject in need thereof is provided, the method comprising: (a) introducing, into a sample containing an HSC population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader; (b) culturing the sample in a suitable culture media for a period of time sufficient to expand the number of HSCs in the sample to a number sufficient to transplant into the subject; and (c) administering the HSCs to the subject.

In yet a further embodiment, a method for administering an hematopoietic stem cell (HSC) to a subject in need thereof is provided, the method comprising: (a) culturing, in a suitable culture media, a sample containing an HSC population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of HSCs in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the HSCs to the subject.

In another embodiment, a method for reconstituting bone marrow in a subject in need thereof is provided, comprising: (a) introducing, into a sample containing an HSC population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader; (b) culturing the sample in a suitable culture media for a period of time sufficient to expand the number of HSCs in the sample to a number sufficient to transplant into the subject; and (c) administering the HSCs to the subject.

In another embodiment, a method for reconstituting bone marrow in a subject in need thereof is provided, comprising: (a) culturing, in a suitable culture media, a sample containing an HSC population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of HSCs in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the HSCs to the subject.

In one embodiment, a method for expanding a population of hematopoietic cells (HSCs) comprising culturing the population of HSCs under conditions sufficient to result in an expansion of the HSC population by at least 2-fold is provided, wherein the expanded population of HSCs is suitable for transplantation into a mammal in need thereof.

In a further embodiment, a method for expanding a population of hematopoietic stem cells (HSCs) is provided, comprising: (a) obtaining from a mammal a tissue sample comprising an HSC population; (b) expanding, in vitro, the HSC population from the sample, wherein: (i) the HSC population expands by at least 2-fold; and (ii) the expanded HSC population has at least a 5-fold increase in total colony-forming units.

In a further embodiment, a method for reconstituting a hematopoietic stem cell lineage in a subject in need thereof is provided, the method comprising: (a) obtaining from a mammal a tissue sample comprising an HSC population; (b) expanding, in vitro, the HSC population from the sample, wherein: (i) the HSC population expands by at least 2-fold; and (ii) the expanded HSC population has at least a 5-fold increase in total colony-forming units; and (c) transplanting the expanded HSC population into a subject in need thereof.

In yet another embodiment, a method for expanding a hematopoietic stem cell population in a mammal in need of such expansion is provided, comprising administering to the mammal a therapeutically effective amount of a modulator of a N⁶-Methyladenosine (m⁶A) mRNA modification pathway for a period of time sufficient to expand the HSC population by at least 2-fold with HSCs that possess the ability to reconstitute a hematopoietic lineage in the mammal.

According to yet another embodiment, a method for ex vivo expansion of a mesenchymal stem cell (MSC) population is provided, the MSC population being obtained from a tissue selected from the group consisting of peripheral blood, cord blood, and bone marrow, the method comprising modulating a N⁶-Methyladenosine (m⁶A) mRNA modification pathway in the MSC population to expand the MSC population to a sufficient quantity while maintaining a multilineage differentiation potential in the MSC population, which is sufficient for subsequent transplantation into a subject in need thereof.

According to yet another embodiment, a method for ex vivo expansion of mesenchymal stem cells (MSCs) by at least 2-fold is provided, the expanded MSCs being competent to reconstitute a MSC lineage upon transplantation into a mammal in need thereof, the method comprising introducing a mutation into the stem cells that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader and culturing the population of MSCs in a suitable culture medium.

According to a further embodiment, a kit for expanding a mesenchymal stem cell population (MSC) population for subsequent transplantation into a subject in need thereof is provided, the kit comprising a system for introducing a mutation into the MSC population that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader, and instructions for use thereof.

According to yet another embodiment, a kit for expanding an mesenchymal stem cell population (MSC) population for subsequent transplantation into a subject in need thereof is provided, the kit comprising an inhibitor of a m⁶A mRNA modification reader, and instructions for use thereof.

In yet a further embodiment, a method for administering a mesenchymal stem cell (MSC) to a subject in need thereof is provided, the method comprising: (a) introducing, into a sample containing a MSC population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader; (b) culturing the sample in a suitable culture media for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient to transplant into the subject; and (c) administering the MSCs to the subject.

In yet a further embodiment, a method for administering a mesenchymal stem cell (MSC) to a subject in need thereof is provided, the method comprising: (a) culturing, in a suitable culture media, a sample containing a MSC population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the MSCs to the subject.

In another embodiment, a method for reconstituting bone marrow in a subject in need thereof is provided, comprising: (a) introducing, into a sample containing a MSC population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader; (b) culturing the sample in a suitable culture media for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient to transplant into the subject; and (c) administering the MSCs to the subject.

In another embodiment, a method for reconstituting bone marrow in a subject in need thereof is provided, comprising: (a) culturing, in a suitable culture media, a sample containing a MSC population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the MSCs to the subject.

In one embodiment, a method for expanding a population of mesenchymal cells (MSCs) comprising culturing the population of MSCs under conditions sufficient to result in an expansion of the MSC population by at least 2-fold is provided, wherein the expanded population of MSCs is suitable for transplantation into a mammal in need thereof.

In a further embodiment, a method for expanding a population of mesenchymal stem cells (MSCs) is provided, comprising: (a) obtaining from a mammal a tissue sample comprising a MSC population; (b) expanding, in vitro, the MSC population from the sample, wherein: (i) the MSC population expands by at least 2-fold; and (ii) the expanded MSC population has at least a 5-fold increase in total colony-forming units.

In a further embodiment, a method for reconstituting a mesenchymal stem cell lineage in a subject in need thereof is provided, the method comprising: (a) obtaining from a mammal a tissue sample comprising a MSC population; (b) expanding, in vitro, the MSC population from the sample, wherein: (i) the MSC population expands by at least 2-fold; and (ii) the expanded MSC population has at least a 5-fold increase in total colony-forming units; and (c) transplanting the expanded MSC population into a subject in need thereof.

In yet another embodiment, a method for expanding a mesenchymal stem cell population in a mammal in need of such expansion is provided, comprising administering to the mammal a therapeutically effective amount of a modulator of a N⁶-Methyladenosine (m⁶A) mRNA modification pathway for a period of time sufficient to expand the MSC population by at least 2-fold with HSCs that possess the ability to reconstitute a mesenchymal lineage in the mammal.

According to year another embodiment, a method for ex vivo expansion of a mesenchymal stem cell (MSC) population is provided, the MSC population being obtained from a tissue selected from the group consisting of peripheral blood, cord blood, and bone marrow, the method comprising modulating a N⁶-Methyladenosine (m⁶A) mRNA modification pathway in the MSC population to expand the MSC population to a sufficient quantity while maintaining a multilineage differentiation potential in the MSC population, which is sufficient for subsequent transplantation into a subject in need thereof.

In another embodiment, a method for reconstituting bone marrow in a subject in need thereof is provided, comprising: (a) culturing, in a suitable culture media, a sample containing a MSC population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the MSCs to the subject.

In one embodiment, a method for expanding a population of mesenchymal cells (MSCs) comprising culturing the population of MSCs under conditions sufficient to result in an expansion of the HSC population by at least 2-fold is provided, wherein the expanded population of MSCs is suitable for transplantation into a mammal in need thereof.

In a further embodiment, a method for reconstituting a mesenchymal stem cell lineage in a subject in need thereof is provided, the method comprising: (a) obtaining from a mammal a tissue sample comprising a MSC population; (b) expanding, in vitro, the MSC population from the sample, wherein: (i) the MSC population expands by at least 2-fold; and (c) transplanting the expanded MSC population into a subject in need thereof.

In yet another embodiment, a method for expanding a mesenchymal stem cell population in a mammal in need of such expansion is provided, comprising administering to the mammal a therapeutically effective amount of a modulator of a N⁶-Methyladenosine (m⁶A) mRNA modification pathway for a period of time sufficient to expand the MSC population by at least 2-fold with MSCs that possess the ability to reconstitute a mesenchymal lineage in the mammal.

In a further embodiment, a method of isolating mesenchymal stem cells (MSCs) from a biological sample is provided, the method comprising contacting the biological sample having a population of MSCs with one or more N-cadherin antibodies.

In a further embodiment, an isolated population of mesenchymal stem cells is provided, as made by any of the processes described herein. According to yet another embodiment, an expanded, isolated population of mesenchymal stem cells is provided, as made by any of the processes described herein.

According to yet another embodiment, a kit for isolating a mesenchymal stem cell (MSC) population for subsequent transplantation into a subject in need thereof is provided. The kit comprises a system for contacting a biological sample comprising MSCs with one or more N-cadherin antibodies, and instructions for use thereof.

According to yet another embodiment, a method for administering a mesenchymal stem cell (MSC) to a subject in need thereof is provided. The method comprises: (a) isolating MSCs from a biological sample comprising a population of MSCs, by contacting the biological sample with one or more N-cadherin antibodies, and (b) administering the isolated MSCs to the subject.

According to yet another embodiment, a method for reconstituting bone marrow in a subject in need thereof is provided. The method comprises: (a) isolating mesenchymal stem cells (MSCs) from a biological sample comprising a population of MSCs, by contacting the biological sample with one or more N-cadherin antibodies, and (b) administering the isolated MSCs to the subject.

According to yet another embodiment, a method for treating a subject in need of a transplant, selected from the group consisting of a bone marrow transplant, a peripheral blood transplant and an umbilical cord blood transplant, is provided. The method comprises administering to the subject a population of isolated MSCs obtained by any of the methods described herein.

In yet another embodiment, a method for expanding a population of chimeric antigen receptor (CAR) T-cells prepared by modifying T-cells obtained from a tissue selected from the group consisting of peripheral blood, cord blood and bone marrow, is provided. The method comprises modulating a N⁶-Methyladenosine (m⁶A) mRNA modification pathway in the population of CAR T-cells, to expand the number of CAR-T cells.

In a further embodiment a method for ex vivo expansion of a chimeric antigen receptor (CAR) T-cell population is provided. The method comprises modulating a N⁶-Methyladenosine (m⁶A) mRNA modification pathway in the CAR T-cell population to expand the number of CAR T-cells.

According to yet another embodiment, a method for ex vivo expansion of a chimeric antigen receptor (CAR) T-cell population prepared by modifying T-cells obtained from a tissue selected from the group consisting of peripheral blood, cord blood, and bone marrow, is provided. The method comprises modulating a N⁶-Methyladenosine (m⁶A) mRNA modification pathway in the CAR T-cell population to expand the CAR T-cell population to a sufficient quantity which is sufficient for subsequent transplantation into a subject in need thereof.

In yet another embodiment, a method for ex vivo expansion of chimeric antigen receptor (CAR) T-cells, the expanded CAR T-cells being competent to treat a cancer and/or blood disorder upon transplantation into a mammal in need thereof, is provided. The method comprises introducing a mutation into the CAR T-cells that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader and culturing the population of CAR T-cells in a suitable culture medium.

In a further embodiment, a kit for expanding a chimeric antigen receptor (CAR) T-cell (HSC) population for subsequent transplantation into a subject in need thereof, is provided. The kit comprises a system for introducing a mutation into the CAR T-cell population that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader, and instructions for use thereof.

In yet a further embodiment, a method for administering chimeric antigen receptor (CAR) T-cell to a subject in need thereof is provided. The method comprises: (a) introducing, into a sample containing CAR T-cell population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader; (b) culturing the sample in a suitable culture media for a period of time sufficient to expand the number of CAR T-cells in the sample to a number sufficient to transplant into the subject; and (c) administering the CAR T-cells to the subject.

In another embodiment, a method for administering a CAR T-cell to a subject in need thereof, is provided. The method comprises: (a) culturing, in a suitable culture media, a sample containing a CAR T-cell population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of CAR T-cells in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the CAR T-cells to the subject.

In a further embodiment, a method for treating cancer and/or a blood disorder in a subject in need thereof is provided. The method comprises: (a) introducing, into a sample containing a CAR T-cell population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader; (b) culturing the sample in a suitable culture media for a period of time sufficient to expand the number of CAR T-cells in the sample to a number sufficient to transplant into the subject; and (c) administering the CAR T-cells to the subject.

In yet a further embodiment, a method for treating cancer and/or a blood disorder in a subject in need thereof, is provided. The method comprises: (a) culturing, in a suitable culture media, a sample containing a CAR T-cell population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of CAR T-cells in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the CAR T-cells to the subject.

In one embodiment, a method for expanding a population of chimeric antigen receptor (CAR) T-cells is provided. The method comprises culturing the population of CAR T-cells under conditions sufficient to result in an expansion of the CAR T-cell population by at least 2-fold, wherein the expanded population of CAR T-cells is suitable for transplantation into a mammal in need thereof.

In yet another embodiment, a method for expanding a population of chimeric antigen receptor (CAR) T-cells is provided. The method comprises: (a) obtaining from a mammal a tissue sample comprising a T-cell population; (b) modifying the T-cell population with chimeric antigen receptors to provide CAR T-cell population; and (c) expanding, in vitro, the CAR T-cell population from the sample, wherein: (i) the CAR T-cell population expands by at least 2-fold.

In a further embodiment, a method for treating a subject suffering from cancer and/or a blood disorder is provided. The method comprises: (a) obtaining from a mammal a tissue sample comprising a T-cell population; (b) modifying the T-cell population with a chimeric antigen receptor (CAR) to form a CAR T-cell population; (c) expanding, in vitro, the CAR T-cell population from the sample, wherein: (i) the CAR T-cell population expands by at least 2-fold; and (d) transplanting the expanded CAR T-cell population into the subject.

In yet a further embodiment, a method for expanding a chimeric antigen receptor (CAR) T-cell population in a mammal in need of such expansion is provided. The method comprises administering to the mammal a therapeutically effective amount of a modulator of a N⁶-Methyladenosine (m⁶A) mRNA modification pathway for a period of time sufficient to expand the CAR T-cell population by at least 2-fold with CAR T-cells that possess the ability to treat cancer and/or a blood disorder in the mammal.

In yet another embodiment, a method of treating a subject suffering from a blood disorder is provided. The method comprises (a) obtaining a population of cells selected from the group consisting of stem cells and T-cells, from a tissue selected from the group consisting of peripheral blood, cord blood and bone marrow; (b) optionally, in a case where the population of cells comprises T-cells, modifying the T-cells with a chimeric antigen receptor (CAR) to provide CAR T-cells; (c) expanding the population of cells by modulating a N-Methyladenosine (m⁶A) mRNA modification pathway in the cells, to expand the number of cells; and (d) transplanting the expanded cells to the subject to treat the blood disorder.

These and other aspects of the invention are further disclosed in the detailed description and examples which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A-1H: Ythdf2 KO leads to increase in phenotypic HSCs in mice. FIG. 1A is a schematic showing a deletion of Ythdf2 in the HSPCs of Mx1-cre;Ythdf2^f/fconditional KO (cKO) mice; FIG. 1B shows a Western blot (left) and histogram (right) showing intracellular flow validation of knockout Ythdf2 in mouse HSPCs; FIG. 1C are representative flow cytometric analysis plots of HSPCs in BM from wt and Ythdf2 KO mice (n=5 for each group); FIGS. 1D and 1E are bar graphs showing the frequency in total nucleated cells (TNC) (1D) and absolute cell number (1E) of HSPCs in BM from wt and Ythdf2 KO mice (n=5 for each group); FIG. 1F is a bar graph showing the absolute number of BM TNC from wt and Ythdf2 KO mice (n=5 for each group); and FIGS. 1G and 1H are bar graphs showing the absolute number of committed progenitors (1G) and lineage cells (1H) in BM of wt and Ythdf2 KO mice (n=5 for each group). Data shown as mean±s.e.m. Unpaired t-test. n.s., nonsignificant

FIGS. 2A-2J: Ythdf2 KO results in expansion of functional HSCs in mice. FIG. 2A depicts an experimental scheme for limiting dilution transplantation assay (LDA) to determine the frequency of functional HSCs; FIG. 2B is a graph showing Primary LDA to determine the CRU frequency by ELDA (Extreme Limiting Dilution Analysis) (n=10 per group) at 16 weeks post transplantation; FIG. 2C is a graph showing a competitive reconstitution assay by transplanting 200K whole bone marrow (WBM) cells with 200K rescue cells into irradiated recipients. (n=10 for each group); FIGS. 2D and 2E are bar graphs showing the frequency in TNC (2D) and absolute cell number (2E) of donor derived HSPCs in BM from transplantation recipient mice as (2C) (n=10 for each group); FIGS. 2F and 2G are bar graphs showing absolute cell number of donor derived (CD45.2⁺) committed progenitors (2F) and lineage cells (2G) in BM from primary 200K BM transplantation recipient mice (n=9-10 for each group); and FIG. 2H shows a plot with secondary LDA to determine the long-term CRU frequency by ELDA at 16 weeks after secondary transplantation (n=10); FIGS. 2I and 2J are, respectively, a bar graph showing quantification of functional mouse hematopoietic stem cells (HSCs) by transplantation assay with peripheral blood analysis for total engrafted donor cells at 4 weeks after transplantation (2I), and a bar graph showing quantification of functional mouse HSCs by transplantation assay with the percentage of B, T and myeloid lineage cells at 4 weeks after transplantation (2J). Data shown as mean±s.e.m. Unpaired t-test. n.s., nonsignificant.

FIGS. 3A-3I: Ythdf2 functions as an m⁶A reader and regulates HSC gene expression by mediating mRNA decay. FIG. 3A is a schematic of irCLIP-seq workflow; FIG. 3B is a schematic showing Ythdf2 binding motif (SEQ ID NO: 1) identified by MEME with all irCLIP peaks found in all three replicates; FIG. 3C is a pie chart depicting the fraction of Ythdf2 binding peaks in each of five transcript segments; FIG. 3D is a chart showing a GO enrichment analysis of Ythdf2 targets from intersect genes of three Ythdf2 irCLIP-seq replicates; FIG. 3E shows representative tracks of Tal1 harboring m⁶A peaks and Ythdf2 irCLIP peaks, with coverage of m⁶A immunoprecipitation and input fragments indicated in red and grey, respectively, and Ythdf2 irCLIP reads highlighted in yellow; FIG. 3F is a chart showing qPCR analysis of total mRNA of sorted LSK cells from wt and Ythdf2 KO mice. All Ct values were first normalized to Actb control (not m⁶A-tagged). Then the ratio (Ythdf2 KO over wt) was calculated. (n=3); FIG. 3G shows representative images (left) and quantification via bar graph (right) of staining intensity of wt (n=65) and Ythdf2 KO (n=54) HSPCs for TAL1 (green); FIG. 3H shows images depicting fluorescence in situ hybridization of Tal1 mRNA (red) and fluorescence immunostaining of Dcp1a (P-body marker) (magenta), Ythdf2 (green) in wt and Ythdf2 KO HSPCs, where arrows indicate co-localized staining. Scale bars, 5 μm; FIG. 3I shows Quantification of Tal1 mRNA and DCP1a co-localization in sorted LSK cells from wt and Ythdf2 KO mice. Percentage indicates the average frequency of the Tal1 mRNA that co-localized with DCP1a over total Tal1 mRNA level in each LSK cells (n=9-17). Data shown as mean±s.e.m. Unpaired t-test.

FIGS. 4A-4I: Role of YTHDF2 in human cord blood HSCs by m⁶A-seq and RNA-seq analysis. FIG. 4A shows metagene profiles depicting sequence coverage in windows surrounding the TSS (left) and stop codon (right), where coverage of m⁶A IP and control (input) fragments indicated in red and grey, respectively; FIG. 4B depicts pie charts presenting the fraction of m⁶A peaks in each of five non-overlapping transcript segments; FIG. 4C depicts a Venn diagram showing shared and unique m⁶A-tagged genes in mouse and hUCB HSPCs; FIG. 4D depicts a chart with a GO enrichment analysis of m⁶A-tagged transcripts shared in both mouse and hUCB HSPCs; FIG. 4E shows representative tracks of HOXB4 harboring m⁶A peaks, where color codes are the same as in FIG. 4A; FIG. 4F depicts a schematic of lentivirus mediated YTHDF2 KD in hUCB CD34⁺ HSPCs for RNA-seq; FIG. 4G is a plot of a cumulative distribution of log 2 (fold change) for m⁶A-marked genes (purple line) and non-m⁶A-marked genes (black line), with control and YTHDF2 KD hUCB CD34⁺ cells; FIG. 4H shows representative coverage plots from the RNA-seq analysis, showing increased reads of m⁶A-tagged gene HOXB4 but not a non-m⁶A-tagged gene ACTB in YTHDF2 KD compared to control hUCB CD34⁺ cells; and FIG. 4I is a bar graph showing relative mRNA expression levels of non-m⁶A labeled ACTB (as control) and m⁶A-marked transcription factor related to stem cell self-renewal in control and YTHDF2 KD hUCB CD34⁺ cells. RPKM from RNA-seq analysis were normalized to controls. Adjusted P value were indicated. n.s., nonsignificant.

FIGS. 5A-5F: YTHDF2 KD facilitates expansion of human cord blood HSCs ex vivo. FIGS. 5A-5B are bar graphs depicting the fold change of frequency (5A) and absolute number (5B) of indicated cells in YTHDF2 KD over control cells after 7 days culture; FIG. 5C shows a bar graph depicting CFU output from transduced CD34⁺CD38⁻ hUCB cells and images of CFU-granulocyte erythrocyte monocyte megakaryocyte (GEMMs) (scale bar, 200 μm); FIG. 5D includes images of burst forming unit-erythroid (BFU-E) (left) and colony-forming unit-granulocyte/macrophage (CFU-GM) (right) from 7-days cultured control or YTHDF2 KD hUCB cells (scale bar, 200 μm), where independent cord blood samples were used and repeated twice for the panels; FIG. 5E is a bar graph showing apoptosis analysis of CD34⁺CD38⁻ cells in 7-day cultures of transduced CD34⁺ hUCB cells by Annexin V staining (n=3 independent CB samples); FIG. 5F is a bar graph showing the fold change of Tthdf2 knockdown (KD) to control in indicated human cord blood HSCs (n=3 individual human samples), where lentivirus was used to deliver control shRNA or human Ythdf2 shRNA into sorted CD34⁺CD38⁻ blood cord HSCs. Dashed lines indicate 95% confidence intervals. Data shown as mean±s.e.m. Unpaired t-test. n.s., nonsignificant.

FIGS. 6A-6G: YTHDF2 KD facilitates expansion of human cord blood functional long-term HSCs. FIG. 6A is an image showing an experimental scheme for measuring frequency of HSCs after in vivo expansion; FIG. 6B includes representative flow plots of hCD45⁺ GFP+ reconstitution from primary recipient mice receiving the highest two cell doses. hCD45=human CD45; FIG. 6C is a plot showing hCD45⁺ GFP⁺ engraftment in BM from the primary recipient mice that received the highest two doses (n=8); FIG. 6D is a plot showing HSC frequency determined by primary LDA. Dashed lines indicate 95% confidence intervals; FIG. 6E includes representative flow plots of hCD45⁺ GFP⁺ reconstitution from secondary recipient mice receiving the highest two cell doses; FIG. 6F is a plot showing hCD45⁺ GFP⁺ engraftment in BM from the secondary recipient mice that received the highest two doses (n=6); FIG. 6D is a graph showing HSC frequency determined by secondary LDA. Dashed lines indicate 95% confidence intervals. Data shown as mean±s.e.m. Unpaired t-test. n.s., nonsignificant.

FIGS. 7A-7I: Ythdf2 KO HSCs show no signs of lineage bias or differences in quiescence and homing ability but exhibit lower apoptotic rate. FIG. 7A is bar graph showing absolute cell number of HSPCs in BM from Mx1-cre⁻; Ythdf2^f/fand Mx1-cre⁺; Ythdf2^f/fmice without pl:pC injection (n=3 per group); FIG. 7B is a bar graph showing cell cycle analysis of HSPCs in wt (n=3) and Ythdf2 KO (n=4) mice; FIG. 7C is a bar graph showing apoptosis analysis of BM HSPCs in wt and Ythdf2 KO mice (n=5 for each group); FIG. 7D shows images of spleens from wt and Ythdf2 KO mice; FIGS. 7E-7H are bar graphs showing absolute number of TNC (7E), LSK CD48⁻ CD150⁺ HSCs (7F), committed progenitors (7G) and lineage cells (7H) in the spleen of wt (n=3) and Ythdf2 KO (n=4) mice; FIG. 7I is a bar graph showing Homing ability of wt and Ythdf2 KO cells was determined by transplanting 1×10⁶CFDA SE-labelled BM cells into lethally irradiated mice. 18 hours later, BM was analyzed for homed events (n=6 mice per group). Data shown as mean±s.e.m. Unpaired t-test. n.s., nonsignificant.

FIGS. 8A-8F: Transplantation recipient mice of Ythdf2 KO BM display no lineage changes or defects 16 weeks post transplantation. FIGS. 8A to 8C are bar graphs depicting absolute cell number of donor derived (CD45.2⁺) TNC (8A), committed progenitors (8B) and lineage cells (8C) in the BM from secondary 200K transplantation recipient mice at 16 weeks after secondary transplantation (n=7-10 for each group); FIGS. 8D to 8F are bar graphs depicting absolute cell number of donor derived (CD45.2⁺) TNC (8D), committed progenitors (8E), and lineage cells (8F) in the spleen from secondary 200K transplantation recipient mice at 16 weeks after secondary transplantation (n=7-10 for each group). Data shown as mean±s.e.m. Unpaired t-test. n.s., nonsignificant.

FIGS. 9A-9K: Ythdf2 KO has long-term effect on mouse HSC expansion in vivo without inducing lineage bias. FIG. 9A is a schematic showing BM and spleen collected from wt and Ythdf2 KO mice were analyzed by flow cytometry at 5-7 months post pl:pC inductions; FIGS. 9B to 9E are bar graphs showing: absolute cell number of TNC (FIG. 9B), HSPCs (FIG. 9C), committed progenitors (FIG. 9D) and lineage cells (FIG. 9E) in the BM of wt and Ythdf2 KO mice at 5-7 months post induction. (n=4-7 mice per group); FIG. 9F is a bar graph showing the weight of spleens from wt and Ythdf2 KO mice at 5-7 months post induction. (n=4-7 mice per group); FIGS. 9G to 9J are bar graphs showing: absolute cell number of TNC (FIG. 9G), LSK CD48⁻ CD150⁺ HSCs (FIG. 9H), committed progenitors (FIG. 9I) and lineage cells (FIG. 9J) in the spleen of wt and Ythdf2 KO mice at 5-7 months post induction. (n=4-7 mice per group); FIG. 9K is a schematic and graph showing 5 months post pl:pC injection, 75 k WBM from wt and Ythdf2 KO mice were transplanted with 200K rescue cells into lethally irradiated recipients. Peripheral blood from transplantation recipients were analyzed every 4 weeks post transplantation to determine the donor derived engraftment (n=10 for each group). Data shown as mean±s.e.m. Unpaired t-test. n.s., nonsignificant.

FIGS. 10A-10D: Molecular characterization of m⁶A modification in mouse HSPCs. FIG. 10A shows plots of metagene profiles depicting sequence coverage in windows surrounding the TSS (up) and stop codon (down), where coverage of m⁶A IP and control (input) fragments indicated in red and grey, respectively; FIG. 10B shows pie charts presenting the fraction of m⁶A peaks in each of five transcript segments; FIG. 10C shows plots of the fraction of genes in mouse HPSCs with m⁶A peaks in each of the segments as a function of expression level; FIG. 10D shows graphs of m⁶A-tagged and non-m⁶A-tagged mRNA degradation rates as determined by analysis of the expression level at 0 hour and 4 hours post actinomycin D treatment in HSPCs.

FIGS. 11A-11E: Define Ythdf2 functionality in mouse HSPCs by irCLIP-seq. FIG. 11A is an image showing immunoprecipitation of Ythdf2 in control or Flag-Ythdf2 overexpressed HPC7 cells; FIG. 11B is an irCLIP membrane image showing IR800 labeled RNA-Ythdf2 complex, where red box indicate the RNA-Ythdf2 complex collected for library construction, and with samples without UV crosslinking serve as controls; FIG. 11C is a Venn diagram showing intersection genes identified in three independent Ythdf2 irCLIP-seq experiments; FIG. 11D is a Venn diagram showing overlap of Ythdf2 binding targets and m⁶A labeled mRNAs;

FIG. 11E shows representative tracks of Gata2 harboring m⁶A peaks and Ythdf2 irCLIP peaks, where coverage of m⁶A immunoprecipitation and input fragments are indicated in red and grey, respectively, and Ythdf2 irCLIP reads are highlighted in yellow.

FIGS. 12A-12F: Ythdf2 KO increased m⁶A-tagged mRNA expression, contributing to HSC expansion. FIG. 12A is a bar graph showing total RNA was extracted from 15,000 sorted BM LSK Flk2⁻ cells; FIG. 12B shows quantification of m⁶A RNA methylation in wt and Ythdf2 KO Lin⁻ cells (n=6); FIG. 12C shows quantification (right) and histogram (left) showing intracellular flow validation of increased expression of TAL1, GATA2, RUNX1 and STAT5 in Ythdf2 KO LT-HSCs comparing to wt LT-HSCs (n=3 mice per group); FIG. 12D shows fluorescence in situ hybridization of Gata2 mRNA (red) and fluorescence immunostaining of Dcp1a (P-body marker) (magenta), Ythdf2 (green) in wt and Ythdf2 KO HSPCs. Arrows indicate co-localized staining. Scale bars, 5 μm; FIG. 12E shows quantification of Gata2 mRNA and DCP1a co-localization in sorted LSK cells from wt and Ythdf2 KO mice. Percentage indicates the average frequency of the Gata2 mRNA that co-localized with DCP1a over total Gata2 mRNA level in each LSK cells (n=12-20); FIG. 12F shows percentage of GFP⁺ cells in the CD45⁺ population at 4 weeks post transplantation (n=10). Data shown as mean±s.e.m. Unpaired t-test. n.s., nonsignificant.

FIGS. 13A-13G: YTHDF2 regulates expression of transcription factors related to stem cell self-renewal in human cord blood stem cells. FIG. 13A is a Venn diagram showing intersection genes identified in three independent m⁶A-seq experiments, using three independent cord blood samples; FIG. 13B shows pie charts with the percentage of mRNAs and non-coding RNAs containing m⁶A peaks; FIG. 13C is a bar graph showing GO enrichment analysis of the transcription factors harboring m⁶A modifications in hUCB CD34⁺ cells; FIG. 13D shows images of Western blotting of YTHDF2 (up) and β-Actin (down) in sorted GFP⁺ control and YTHDF2 KD hUCB cells, showing knockdown efficiency of YTHDF2; FIG. 13E shows a bar graph with expression level (left) and representative track plots (right) of YTHDF2 from RNA-seq analysis of control and YTHDF2 KD hUCB CD34⁺ cells, showing knockdown efficiency of YTHDF2. FIGS. 13F through 13G are representative track plots of indicated transcription factors harboring m⁶A peaks (up) and their representative coverage plots from the RNA-seq analysis (bottom). Adjusted P values are indicated.

FIGS. 14A-14C: YTHDF2 KD in hUCB cells resulted in HSC expansion without changing lineage output. FIG. 14A depicts representative flow plots of GFP⁺ CD34⁺CD38⁻ CD45RA⁻ EPCR⁺ HSCs in control and YTHDF2 KD hUCB cells post 7 days culture; FIG. 14B depicts confirmation of YTHDF2 protein knockdown and overexpression in transduced Hela cells; FIG. 14C depicts CFU production by YTHDF2 OE and control transduced CD34⁺CD38⁻ CB from day 10 cultures (n=3 independent human samples). Data shown as mean±s.e.m. Unpaired t-test. n.s., nonsignificant.

FIGS. 15A-15D: YTHDF2 KD in hUCB cells resulted in HSC expansion without changing lineage output. FIG. 15A includes representative flow plots of hCD45⁺ GFP⁺ monocyte, megakaryocyte (MK cell), B cell and erythrocyte in primary NSG recipient BM.; FIGS. 15B and 15C are bar graphs depicting the percentage of lineage cells in hCD45⁺ GFP⁺ (FIG. 15B) and in total CD45⁺ (FIG. 15C) BM cells from primary NSG recipients at 10 weeks post transplantation (n=13-15); FIG. 15D is a bar graph depicting a summary of human donor derived lineage chimerisms in total CD45⁺ BM cells from secondary NSG recipients at 12 weeks post transplantation (n=6). Data shown as mean±s.e.m. Unpaired t-test. n.s., nonsignificant.

FIG. 16: expansion of mesenchymal stem cells in vivo. FIG. 16 is a bar graph showing the frequency of N-Cad+ CD105+ in TNC with both wt Ythdf2 and a Ythdf2 knockout (KO).

FIGS. 17A-17J: functional identification of rHSC population. FIG. 17A is a schematic representation of FACS sorting for rHSCs, pHSCs, ST-HSCs and MPPs; FIG. 17B shows a quantification of rHSCs and pHSCs function by transplantation assay. PB analysis for total engrafted donor cells at the indicated number of weeks post transplantation and the percentage of donor-derived B, T and myeloid lineage cells at 20 weeks post transplantation (n=10 mice per group); FIG. 17C shows donor derived cells from rHSCs or pHSCs transplanted mice at 40 weeks post transplantation; FIG. 17D H2B-GFP label retaining cells in rHSCs and pHSCs at 130 days post chasing (n=4 mice per group); FIG. 17E shows cell cycle gene expression in rHSCs and pHSCs (n=3 replicates from 20 mice); FIG. 17F shows rHSCs and pHSCs transplanted recipients received 5FU injection at 4 weeks post transplantation as indicated. PB analysis for donor engraft cells at indicated weeks post transplantation. The percentage of donor-derived B, T and myeloid lineage cells were shown at 20 weeks post transplantation (n=10 mice per group); FIG. 17G shows rHSCs and pHSCs at day 3 post 5FU treatment (pool from 15 mice); FIG. 17H shows DNA damage gene expression in rHSCs and pHSCs (n=3 replicated from 20 mice); FIG. 17I shows DNA damage genes in rHSCs from control mice and mice at day 3 post 5FU (n=20 in control mouse group, n=40 in D3 5FU mouse group). * P<0.05, ** P<0.01, ***P<0.001. Error bars, s.e.m; FIG. 17J shows heat map of stress response genes in rHSCs, pHSCs and 5FU rHSCs.

FIGS. 18A-18I: rHSCs located by endosteal region in BM niche. FIG. 18A shows representative whole-mount images of mouse sternal bone marrow (BM), with bone (white, generated by second harmonic generation, SHG), MKs (yellow, distinguished by size, morphology and CD41 expression). Green arrowheads denoted phenotypic Lin⁻ CD48⁻CD41⁻CD150⁺CD49b⁻rHSCs, White arrowheads denoted phenotypic Lin⁻CD48⁻ CD41⁻CD150⁺CD49b+ pHSCs; FIG. 18B shows representative image of rHSCs, pHSCs and 5FU rHSCs. White arrows denoted phenotypic Lin⁻CD48⁻CD41⁻CD150⁺CD49b⁻rHSCs, phenotypic Lin⁻CD48⁻CD41⁻CD150⁺CD49b⁺ pHSCs and phenotypic Lin⁻CD48⁻CD41⁻ CD150⁺CD49b⁻ 5FU rHSCs; FIGS. 18C-E show relative distance between rHSCs, pHSCs and 5FU rHSCs to vessels, MKs or bones (n=144 rHSCs, n=685 pHSCs, n=707 5FU rHSCs); FIG. 18F shows absolute number of healthy, apoptotic and dead VE-Cad⁺ CD31⁺ vessel cells in control mice and mice at 1 day post 5FU treatment; FIGS. 18F-G show absolute number of AnnexinV⁺ SytoxG⁻ apoptotic Ve-Cad⁺ CD31⁺ endothelial cells in central marrow (CM) or N-cad− tomato+ cells in both CM and bone from control mice and mice 1 day post 5FU treatment. N=3 in each group. * P<0.05, ** P<0.01, ***P<0.001. Error bars, s.e.m; FIGS. 18H-I show representative image of N-cad− tomato+ and Ve-Cad⁺ CD31⁺ vessels in control mice and in mice at 3 days post 5FU treatment.

FIGS. 19A-19J: N-cad⁺ cells maintain functional HSCs in BM niche. FIG. 19A shows a scheme for DT administration to N-cad-CreER^T;iDTR mice used for the experiments shown in B-G. D indicated day (e.g., DO indicates day 0); FIG. 19B shows N-cad⁺ cell ablation efficiency as indicated by Tomato⁺ cells in N-cad-CreER^T;iDTR mice; FIGS. 19C-D show flow cytometric analyses to determine the absolute numbers of total nucleated cells (TNC) and HSPCs in the bone marrow (BM) from N-cad-CreER^T;iDTR mice post TMX and DT injections (n=5 mice per group); FIGS. 19E-H show quantification of functional HSCs by transplantation assay in primary 1° and secondary 2° transplantation. Total BM cells from PB analysis for total engrafted donor cells at the indicated number of weeks post transplantation and the percentage of donor-derived B, T and myeloid lineage cells at 16 weeks post transplantation (n=10 mice per group); FIGS. 19I-J show flow cytometric analyses to determine the absolute numbers of HSPCs in the bone marrow (BM) from N-cad-CreER^T;SCFf/f (N-cad-CreER^T_;SCFf/f, n=4 mice, N-cad-CreER^T+;SCFf/f, n=6 mice) and N-cad-CreER^T+;Cxcl12f/f mice (N-cad-CreER^T_;Cxcl12f/f, n=3 mice, N-cad-CreER^T+;Cxcl12f/f, n=6 mice) post TMX and DT injections. (* P<0.05, ** P<0.01, ***P<0.001. Error bars, s.e.m.).

FIGS. 20A-20K: transcriptome analysis for hematopoietic cells and niche cells. FIGS. 20A-B shows pearson distance tree and PCA analysis for hematopoietic stem and progenitor cells (HSPCs); FIGS. 20C-D show pearson distance tree and PCA analysis for BM niche cells; FIG. 20E shows HSC signature gene expression in hematopoietic stem and progenitor cells; FIG. 20F shows BM niche signature gene expression in niche cells; FIG. 20G shows term analysis for BM niche cells; FIG. 20H shows stromal cell development gene expression in niche cells from endosteal and perivascular zones; FIG. 20I shows lineage tracing for N-cad-CreER^T; R26-tdT; Nestin-GFP mice after 3 TMX injections, and N-cad-CreER^T; R26-ZsG; Cxcl12-DsR mice after 3 TMX injections; FIG. 20J shows enzymatically digested bone marrow cells from N-cad-CreERT; R26-tdT mice post TMX injections. LepR and Pdfgrα stained by antibodies shown as red peak. Isotype control shown as gray peak; FIG. 20K shows CFU-F activity in niche cells from endosteal and perivascular zones.

FIG. 21A-21G: in vitro differentiation potential and localization of N-cad⁺ derived cells. FIG. 21A shows experimental design; FIG. 21B shows live CFU-F colonies cultured from enzymatically digested bone marrow, stained with Cell Trace™ and Tomato⁺ cells in one colony in high magnification; FIGS. 21C-E show in vitro differentiation of stromal cells derived from N-cad-CreER^T; R26-tdT mice: live tdTomato⁺ cells and Alkaline Phosphatase staining of culture 21 days after osteo-differentiation (21C); live Tomato⁺ cells and Oil Red O lipid staining 21 days after adipo-differentiation (21D); aggrecan antibody stained and toluidine blue stained chondrocytes at 21 days after chondro-differentiation of Tomato⁺ cells (21E); FIGS. 21F-G show(F-G) experimental design; FIG. 21H shows localization of cells derived from N-cad-CreERT; R26-tdT mice post TMX injection at 6 hours, 14 hours, 24 hours, 1 week and 4 weeks; FIG. 21 I shows percentage of Tomato⁺ cells post TMX injection at 1 week, 2 weeks, 4 weeks and 6 weeks in trabecular, cortical bone and central marrow (n=2 mice in 1 and 2 weeks group, n=3 mice in 4 and 6 weeks group. * P<0.05, ** P<0.01, ***P<0.001. Error bars, s.e.m.).

FIGS. 22A-22K: N-cad⁺ stromal cells give rise to osteoblasts and adipocytes in adult mice. FIG. 22A shows representative femur sections from N-cad-CreER^T; R26-tdT; Col2.3-GFP mice of different time points post TMX injection showing the anatomical distribution and increasing generation of Tomato⁺Col2.3-GFP⁺ osteoblasts. Scale bar, 100 μm; FIGS. 22B-C show higher-power images of N-cad-CreER^T; R26-tdT; Col2.3-GFP mice at early time points post TMX in region i, ii, iii, iv, v and vi shown in 6 hr (22B) and 14 hr (22C). Hollow arrowheads show Tomato⁺Col2.3GFP⁺ osteoblasts (yellow cells) at 6 hours and 14 hours post TMX, whereas solid arrowheads indicate the N-cad recombined both in the trabecular (ii, iv, v) and cortical (iii, vi) region (green). These cells are potentially undifferentiated as shown by the absence of Col2.3-GFP. Scale bar, 50 μm; FIGS. 22D-E show image quantification showing percentage of Tomato⁺Col2.3-GFP⁺ and Tomato⁺Col2.3-GFP⁻ cells in trabecular (22D) and compact bone (22E) at 6 hours, 14 hours, 24 hours, 2 weeks and 4 weeks post TMX injection; FIG. 22F shows image quantification comparing percentage of potential undifferentiated Tomato⁺Col2.3-GFP⁻ between trabecular and compact bone. * P<0.05, ** P<0.01, ***P<0.001. Error bars, s.e.m; FIG. 22G shows representative image of trabecular bone (TB) and cortical bone (CB) in N-cad-CreER^T; R26-tdT; Col2.3-GFP mice at 4 weeks post TMX injection. Scale bar, 20 μm; FIG. 22H shows representative low and high-power image of femur section from N-cad-CreER^T; R26-tdT mice with Perilipin staining at 4 weeks post TMX injection. At periosteal region (i), bone marrow near trabecular region (ii) and central marrow(iii), solid arrowheads showed Tomato⁺Perilipin⁺ derived from N-cad⁺ MSCs; FIG. 22I shows BODIPY staining showed the lipid droplet (green) inside the Tomato⁺Perilipin⁺ adipocyte (arrowheads). Scale bar, 20 μm; FIGS. 22J-K show image quantification of Tomato⁺Perilipin⁺ adipocytes in trabecular bone (22J) and periosteal region (22K) at 6 hours, 14 hours, 24 hours, 2 weeks and 4 weeks post TMX injection. (* P<0.05, ** P<0.01, ***P<0.001. Error bars, s.e.m.).

FIGS. 23A-23J: N-cad⁺ MSCs give rise to chondrocytes during development and post injury. FIG. 23A shows experimental design; FIG. 23B shows tomato⁺ partially colocalized with Aggrecan⁺ chondrocytes in rib at E14.5(2 days post TMX induction); FIG. 23C shows an Illustration of chondrocyte development in femur; FIG. 23D shows a representative femur section from 2-day-old N-cad-CreER^T; R26-tdT mice with TMX induction at E12.5. Note that Tomato⁺ cells gave rise to Aggrecan⁺ cartilage cells in articular surface (i) and the developing secondary ossification center (ii). Arrowheads indicated Tomato⁺ Aggrecan⁺ cells; FIG. 23E shows a representative femur section from 10-month-old N-cad-CreER^T; R26-tdT mice with TMX induction at E12.5. Tomato⁺ cells from early embryonic stage differentiated to Perilipin⁺ adipocytes; FIG. 23F shows a representative femur section from 2-month-old N-cad-CreER^T; R26-tdT mice with TMX induction at E12.5. Tomato⁺ cells from early embryonic stage differentiated to Osteopontin₊ hypertrophic chondrocytes (i and ii) and osteoblasts (iii, iv, v and vi); FIG. 23G shows an experimental design for femoral groove injury to N-cad-CreER^T; R26-tdT mice with TMX induction at E12.5; FIG. 23H shows quantification of Tomato⁺ chondrocytes in control mice and in mice 3 weeks after knee cartilage injury. (1) Representative section of distal femur from N-cad-CreER^T; R26-tdT mice without cartilage injury or 3 weeks after cartilage injury. Note that the clustered Tomato⁺ Aggrecan⁺ cells at the knee surface of control mice (i) significantly increased at the correspondent region in mice 3 weeks after knee cartilage injury (ii); FIG. 23J shows AHO staining of the sections in FIG. 6H showing the Alcian blue positive chondrocytes in control (i) and in the location of cartilage injury(ii).

FIGS. 24A-24K: BrdU assay and N-cad reporter mouse line post 5FU. FIGS. 24A-F show representative images showing BrdU⁺ cells in femur of mice at day 0, 2, 3, 4 and 6 post 5FU treatment. Note that BrdU⁺ cells reduced at day 2 post 5FU, and reactivated from day 3 to day 6 post 5FU near bone (red dotted line) or vessel/adipose structure (green dotted line); FIG. 24G shows a model of dynamic of BrdU⁺ cells; FIG. 24H shows a ratio of BrdU⁺ cells in bone marrow to bone surface from day 3 to 6 post 5FU treatment; FIG. 24I shows a percentage of BrdU⁺ cells near bone surface and near vessel/adipose structures from day 3 to 6 post 5FU treatment; FIG. 24J shows representative whole bone section showing N-cad driven tomato⁺ cells in both bone surface and central marrow; FIG. 24K shows absolute number of N-cad driven tomato⁺ cells in central marrow (CM) and in bone of N-cad-TdT mice of control group and mice 3 days post 5FU treatment. N=3 in each group. * P<0.05, ** P<0.01, ***P<0.001. Error bars, s.e.m.

FIGS. 25A-25B: generation of N-cad-CreER^Tmouse strain. FIG. 25A shows a generation of N-cad-CreER^Tmouse strain; FIG. 25B shows lineage tracing for N-cad-CreER^T, R26-tdT; Col2.3-GFP mice after three TMX injections; blood vessels stained by CD31 and Ve-cadherin antibodies.

FIGS. 26A-26E: sorting strategy and signature gene expression of niche cells. FIG. 26A shows niche cells in endosteal zone harvested from digested bone cells; FIG. 26 B shows niche cells from Perivascular and sinusoid zones harvested from digested bone marrow cells; FIG. 26C shows localization of NG2-RFP⁺ cells in endosteal and peri-arterial regions; FIGS. 26D-E show heatmaps of osteo-chondrogenic progenitor gene and adipogenic progenitor gene expression in niche cells.

FIGS. 27A-27E: lineage tracing in N-cad-CreER^T; R26-tdT mice at early time point post TMX induction. FIGS. 27A-B show representative images of trabecular bone (TB) and cortical bone(CB) in N-cad-CreER^T; R26-tdT; Col2.3-GFP mice at 24 hours and 2 weeks post TMX induction. Scale bar, 20 μm; FIGS. 27C-E show representative femur section with high power images of N-cad-CreER^T; R26-tdT mice at 6 hours, 14 hours and 24 hours post TMX induction. Adipocytes shown with perilipin antibody staining.

FIGS. 28A-28D: characterization of N-cad⁺ MSCs from early development and in injury repair. FIG. 28A shows representative images of femur section showing N-cad⁺ MSCs with Aggrecan staining in N-cad-CreER^T; R26-tdT 24 hours post TMX induction at postnatal D2; FIG. 28B shows a representative femur section from 2-month-old N-cad-CreER^T; R26-tdT mice with TMX induction at E12.5. Adipocytes shown with Perilipin antibody staining; FIG. 28C shows a representative femur section from 2-day-old N-cad-CreER^T; R26-tdT mice with TMX induction at E12.5. Developing bone cells shown with Osteopontin antibody staining; FIG. 28D shows a representative sagittal knee sections in E12.5 TMX induced N-cad-CreER^T; R26-tdT mice of control group and 2 weeks after knee cartilage injury. Scale bar, 1 mm.

FIG. 29: Image of surviving rHSCs (green, CD150⁺ Lin⁻ CD49b⁻) at day 3 post 5FU treatment were often detected as single cells adjacent to the bone surface (white, SHG), and proliferating HSCs were often associated with MKs (CD150⁺ Lin⁺) or near the vessels (red, CD31+).

FIG. 30: Plot showing production of human Thpo by 10K ncad+ hMSCs and 1000 k total hMSCs in vivo.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the invention is a method for expanding a population of stem cells obtained from a tissue selected from the group consisting of peripheral blood, cord blood, and bone marrow. This method comprises modulating a N6-methyladenosine (m⁶A) mRNA modification pathway in the population of stem cells to expand the number of stem cells.

Another embodiment of the invention is a method for expanding a population of chimeric antigen receptor (CAR) T-cells cells obtained by modifying T-cells obtained from a tissue selected from the group consisting of peripheral blood, cord blood, and bone marrow. This method comprises modulating a N6-methyladenosine (m⁶A) mRNA modification pathway in the population of CAR T-cells to expand the number of stem cells.

In the present invention the population of stem cells and/or T-cells may be obtained from any mammal, such as, e.g., a human, and from any tissue that contains stem cells and/or progenitor cells and/or T-cells. As noted above, in a preferred embodiment the tissue may be peripheral blood, cord blood or bone marrow.

As used herein, “expand”, “expanding” and like terms means to increase the number of stem cells and/or CAR-T cells in the population relative to the number of stem cells and/or CAR T-cells in the original population either in vivo or ex vivo using any of the methods disclosed herein. The expansion may be at least 40-fold compared to the original number of stem cells and/or CAR T-cells in the population. More preferably, the expansion is at least 2-fold, 4-fold, 5-fold, 8-fold, 10-fold, 15-fold, 20-fold or more compared to the original number of stem cells.

In the present invention “a population of stem cells” means a group of substantially undifferentiated cells that possess the ability to give rise to many different types of cells and which have the ability to self-renew. Representative, non-limiting examples of stem cells according to the present invention include bronchioalveolar stem cells (BASCs), bulge epithelial stem cells (bESCs), corneal epithelial stem cells (CESCs), cardiac stem cells (CSCs), epidermal neural crest stem cells (eNCSCs), embryonic stem cells (ESCs), endothelial progenitor cells (EPCs), hepatic oval cells (HOCs), hematopoetic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), keratinocyte stem cells (KSCs), mesenchymal stem cells (MSCs), neuronal stem cells (NSCs), pancreatic stem cells (PSCs), retinal stem cells (RSCs), and skin-derived precursors (SKPs).

Hematopoietic stem cells, for example, have the ability to self-renew (i.e., expand) and can give rise to all the types of progenitor cells (such as, e.g., CMP, GMP, MEP and CLP) and ultimately all the types of blood cells (such as e.g., red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, and platelets) in the hematopoietic system. Mesenchymal stem cells, as another example, are multipotent stromal cells that can differentiate into a variety of cell types (such as, e.g., osteoblasts, chondrocytes, myocytes and adipocytes).

In the present invention “a population of chimeric antigen receptor (CAR) T-cells” means a group of T-cells that have been modified with chimeric antigen receptors capable of binding specific antigens, such as antigens on the surface of cancer cells, and may possess the ability to target and kill such cancer cells. The CAR T-cells can be prepared by modifying T-cells with the chimeric antigen receptors, such as by introducing DNA coding for the chimeric antigen receptors into the T-cells, to express the chimeric antigen receptors on the surface of the T-cells (Davila et al. Sci Transl Med 6(224), 224ra25 (2014); Tasian et al. Ther Adv Hematol 6(5), 228-241 (2015).

In the present invention, “modulating”, “modulation” and like terms mean altering the signal transduction pathway, e.g., a protein in the m⁶A mRNA modification pathway, including but not limited to lowering or increasing the expression level of a protein, altering the sequence of such a protein (by mutation, pre-translational or post-translational modification or otherwise), or inhibiting or activating such a protein (whether by binding, phosphorylation, glycosylation, translocation or otherwise). Such modulation may be achieved genetically or pharmacologically.

In one aspect of the present invention, modulating the m⁶A mRNA modification pathway comprises introducing a mutation into a population of stem cells and/or CAR-T cells, which mutation results in modulation of a molecule in the m⁶A mRNA modification pathway. In another aspect present invention, modulation of the m⁶A mRNA modification pathway also includes contacting the stem cells and/or CAR T-cells with a modulator of a molecule in the m⁶A mRNA pathway. Representative, non-limiting examples of such modulators include a small molecule, a biologic, an antisense RNA, a small interfering RNA (siRNA), and combinations thereof.

In the present invention, the phrase “modulation of a molecule in the m⁶A mRNA modification pathway” means altering the function of a member of the m⁶A mRNA modification pathway, which altered function may have an effect similar to inhibiting or decreasing the function of a molecule involved in a process upstream and/or downstream of m⁶A modification of mRNA. Non-limiting examples of such “modulation” include increasing or decreasing the expression or function of proteins involved in any of: the incorporation of N⁶-methyladenosine modifications in mRNA, removal of N⁶-methyladenosine mRNA modifications to mRNA, and/or the recognizing and processing of N⁶-methyladenosine modified mRNA. For example, the modulation may include increasing or decreasing N⁶-methyladenosine modifications in mRNA, and/or affecting the type and/or distributions of such modifications in mRNA, such as by modulating the activity of one or more of a m⁶A writer (e.g. methyltransferase) and m⁶A eraser (e.g. demethylase). As another example, the modulation may increase or decreases expression or function of proteins that recognize N⁶-methyladenosine modifications to mRNA to mediate m⁶A-dependent functions, such as by modulating the activity of a m⁶A reader (e.g., an RNA binding protein that recognizes methylated adenosine). Thus, modulation of the molecule in the m⁶A mRNA modification pathway modulator may result in modulation of the activity and/or expression of a molecule upstream or downstream of an m⁶A mRNA modification process.

In one aspect, the modulation of the m⁶A mRNA modification pathway involves modulation of a molecule selected from the group consisting of m⁶A mRNA modification readers, m⁶A mRNA modification writers, m⁶A mRNA modification erasers and combinations thereof. Non-limiting examples of m⁶A modification writers include methyltransferases that are capable of post-transcriptionally installing the m⁶A modification in messenger RNA, and can include any selected from the group consisting of METTL3, METTL14, WTAP, KIAA1429 and combinations thereof. Non-limiting examples of m⁶A modification erasers include demethylases that are capable of reversing the methylation, and can include any selected from the group consisting of FTI, ALKBH5 and combinations thereof. M⁶A modification readers include proteins that are capable of selectively binding m⁶A-methylated mRNA to exert regulatory functions through selective recognition of methylated mRNA. Suitable m⁶A modification readers can include any selected from the group consisting of Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, elF3 and combinations thereof. According to one aspect, the m⁶A modification readers comprise proteins of the YTH domain family of proteins, which includes Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2 and combinations thereof. (see, e.g., Wang et al. Nature, 505(7481):117-120, 2014; Frayling et al. Science, 316: 889-894, 2007; Zheng et al. Mol. Cell., 49: 18-29, 2012; Cao et al. Open Biol., 6(4): 160003, 2016; Maity et al. The FEBS Journal, 283(9): 1607-1630, 2016).

As used herein, “introducing a mutation” means any conventional method for producing an alteration in the genetic makeup of the stem cell population and/or CAR T-cell population. Non-limiting examples for introducing a mutation into a stem cell population and/or CAR T-cell population include mutagenesis via ultra-violet light irradiation, chemical mutagenesis, targeted mutagenesis such as site directed mutagenesis of a stem cell and/or CAR T-cell, and creation of a transgenic mouse. According to one aspect, a mutation may be introduced into the stem cell and/or CAR T-cell to delete, replace or reduce expression of a gene that expresses a molecule in the m⁶A mRNA modification pathway, such as a molecule selected from the group consisting of a m⁶A mRNA modification reader, a m⁶A mRNA modification writer, a m⁶A mRNA modification eraser and combinations thereof. In one aspect, the mutation is introduced to delete, replace or reduce expression of a gene that expresses a m⁶A mRNA modification reader, such as any selected from the group consisting of Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, elF3 and combinations thereof. In a preferred aspect, a mutation is introduced to delete, replace or reduce expression of a gene that expresses Ythdf2. In yet another aspect, the mutation is introduced to delete, replace or reduce expression of a gene that expresses a m⁶A mRNA modification writer, such as any selected from the group consisting of METTL3, METTL14, WTAP, KIAA1429 and combinations thereof. In yet another aspect, the mutation is introduced to delete, replace or reduce expression of a gene that expresses a m⁶A mRNA modification eraser, such as any selected from the group consisting of FTI, ALKBH5 and combinations thereof.

In one aspect, the mutation can be introduced by exposing the stem cells and/or CAR T-cells to a Mx1-Cre targeting system (see, e.g., Kuhn et al. Science, 269(5229): 1427-1429, 1995) that inactivates or deletes at least a portion of a gene that expresses a molecule in the m⁶A mRNA modification pathway. In yet another aspect, a mutation is introduced that incorporates short hairpin RNA (shRNA) into the stem cells and/or CAR T-cells to reduce expression of a gene that expresses a molecule in the m⁶A mRNA modification pathway. For example, the shRNA may be introduced by exposing the stem cells and/or CAR T-cells to a vector to deliver shRNA, which may be a viral vector such as lentivirus (see, e.g., Chira et al. Oncotarget, 6(31): 30675-30703, 2015). The shRNA may be capable of triggering gene silencing to regulate gene expression (see, e.g., Paddison et al. Genes Dev., 16(8): 948-958, 2002).

As used herein, “a modulator of a NM-Methyladenosine mRNA modification pathway” (or “m⁶A mRNA modification pathway modulator”) is any agent that regulates the activity of any member of the m⁶A mRNA modification pathway, which results in, e.g., an increase or decrease in N⁶-methyladenosine modifications in mRNA, and/or a change in the types and/or distributions of such modifications in mRNA, such as by modulating the activity of one or more of a m⁶A writer (e.g. methyltransferase) and m⁶A eraser (e.g. demethylase). As another example, the agent may be one that increases or decreases activity of proteins that recognize N⁶-methyladenosine modifications to mRNA to mediate m⁶A-dependent functions, such as by modulating the activity of a m⁶A reader (e.g., an RNA binding protein that recognizes methylated adenosine). Thus, the m⁶A mRNA modification pathway modulator may act on, or upstream of, or downstream of, an agent that affects the m⁶A modification to mRNA.

In one embodiment, the m⁶A mRNA modification pathway may be modulated by down-regulating and/or inhibiting a member of the m⁶A mRNA modification pathway, such as down-regulating and/or inhibiting a m⁶A mRNA modification reader. As used herein, “down-regulating” means inhibiting or reducing the amount of or inhibiting or decreasing the activity of a member of the m⁶A mRNA modification pathway. Such down-regulation may be accomplished using, e.g. antisense RNA, siRNA, antibodies, or small molecules. As another example, the m⁶A mRNA modification reader may be down-regulated by contacting the stem cells and/or CAR T-cells with an inhibitor of an m⁶A mRNA reader, to inhibit binding and/or recognizing of the m⁶A modified mRNA by the m⁶A mRNA reader. In one aspect, the m⁶A mRNA modification reader that is down-regulated is selected from the group consisting of Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, elF3 and combinations thereof. In a preferred aspect, the m⁶A mRNA modification reader that is down-regulated is Ythdf2. Inhibitors of the m⁶A mRNA modification reader may be any selected from the group consisting of: (inhibitors of HNRNPC) hsa-let-7e-5p (MIRT051596), hsa-mir-455-3p (MIRT037890), hsa-mir-30c-5p (MIRT047904), hsa-mir-186-5p (MIRT045150), hsa-mir-744-5p (MIRT037494), hsa-mir-18a-3p (MIRT040851), hsa-mir-484 (MIRT042196), hsa-mir-505-5p (MIRT037959), hsa-mir-615-3p (MIRT039991), hsa-mir-342-3p (MIRT043694), hsa-miR-3607-3p, hsa-miR-30d, hsa-miR-3916, hsa-miR-3162-5p, hsa-miR-1273d, hsa-miR-3161, hsa-miR-30a, hsa-miR-629, hsa-miR-208b, hsa-miR-489, hsa-miR-3148, hsa-miR-2113, hsa-miR-877, hsa-miR-455-5p, hsa-miR-186, hsa-miR-548o, hsa-miR-3139, hsa-miR-320a, hsa-miR-4311, hsa-miR-555, hsa-miR-3605-5p, hsa-miR-515-5p, hsa-miR-144, hsa-miR-499-5p, hsa-miR-1323, hsa-miR-548x, hsa-miR-299-5p, hsa-miR-653, hsa-miR-576-5p, hsa-miR-548p, hsa-miR-586, hsa-miR-888, hsa-miR-3647-3p, hsa-miR-484, hsa-miR-320b, hsa-miR-620, hsa-miR-30b, hsa-miR-548q, hsa-miR-29b-1, hsa-miR-570, hsa-miR-183, hsa-miR-1276, hsa-miR-208a, hsa-miR-186, hsa-miR-28-5p, hsa-miR-330-3p, hsa-miR-548am, hsa-miR-320d, hsa-miR-3175, hsa-miR-3155, hsa-miR-548aa, hsa-miR-519e, hsa-miR-1270, hsa-miR-513b, hsa-miR-599, hsa-miR-518f, hsa-miR-4301, hsa-miR-30c, hsa-miR-3135, hsa-miR-4286, hsa-miR-202, hsa-miR-4263, hsa-miR-4299, hsa-miR-606, hsa-miR-3133, hsa-miR-583, hsa-miR-3125, hsa-miR-501-5p, hsa-miR-7-1, hsa-miR-514b-3p, hsa-miR-3155b, hsa-miR-548d-3p, hsa-miR-224, hsa-miR-7-2, hsa-miR-708, hsa-miR-3199, hsa-miR-514, hsa-miR-30e (see, e.g. Helwak et al. Cell, 153(3): 654-655, 2013; Whisnant et al., M Bio 4(2), 2013:e000193); (inhibitors of HNRNPA2B1) hsa-mir-92a-3p (MIRT049721), hsa-mir-30c-5p (MIRT048009), hsa-mir-191-5p (MIRT045809), hsa-let-7f-5p (MIRT051404), hsa-mir-27b-3p (MIRT046213), hsa-mir-877-3p (MIRT037116), hsa-mir-615-3p (MIRT040278), hsa-mir-1260b (MIRT052680), hsa-mir-103a-3p (MIRT027027), hsa-mir-16-5p (MIRT031508), hsa-mir-1296-5p (MIRT036075), hsa-mir-197-3p (MIRT048098), hsa-miR-548j, hsa-miR-3678-3p, hsa-miR-607, hsa-miR-188-5p, hsa-miR-15a, hsa-miR-3653, hsa-miR-371-5p, hsa-miR-550a, hsa-miR-3622b-3p, hsa-miR-548a-5p, hsa-miR-3170, hsa-miR-3148, hsa-miR-556-3p, hsa-miR-490-3p, hsa-miR-559, hsa-miR-200c, hsa-miR-130a, hsa-miR-548y, hsa-miR-548o, hsa-miR-23c, hsa-miR-491-3p, hsa-miR-335, hsa-miR-3667-3p, hsa-miR-466, hsa-miR-23b, hsa-miR-4310, hsa-miR-127-5p, hsa-miR-548b-5p, hsa-miR-616, hsa-miR-16, hsa-miR-338-3p, hsa-miR-3200-5p, hsa-miR-362-3p, hsa-miR-448, hsa-miR-1306, hsa-miR-944, hsa-miR-3684, hsa-miR-373, hsa-miR-103a, hsa-miR-380, hsa-miR-499-5p, hsa-miR-1323, hsa-miR-323-5p, hsa-miR-3674, hsa-miR-1252, hsa-miR-33b, hsa-miR-580, hsa-miR-548c-3p, hsa-miR-103a-2, hsa-miR-548w, hsa-miR-600, hsa-miR-634, hsa-miR-586, hsa-miR-497, hsa-miR-720, hsa-miR-654-3p, hsa-miR-524-5p, hsa-miR-543, hsa-miR-548q, hsa-let-7f-2, hsa-miR-330-5p, hsa-miR-500a, hsa-miR-548I, hsa-miR-570, hsa-miR-374a, hsa-miR-1184, hsa-miR-649, hsa-miR-424, hsa-miR-3658, hsa-miR-186, hsa-miR-326, hsa-miR-548d-5p, hsa-miR-23a, hsa-miR-15b, hsa-miR-190, hsa-miR-203, hsa-miR-548h, hsa-miR-3136-5p, hsa-miR-618, hsa-miR-551b, hsa-miR-211, hsa-miR-1305, hsa-miR-513b, hsa-miR-96, hsa-miR-2117, hsa-miR-548n, hsa-miR-3910, hsa-miR-217, hsa-miR-892b, hsa-miR-502-5p, hsa-miR-548i, hsa-miR-520d-5p, hsa-miR-4299, hsa-miR-1285, hsa-miR-3133, hsa-miR-483-3p (see, e.g., Hafner et al. Cell, 141(1): 129-141, 2010; Helwak et al. Cell, 153(3): 654-655, 2013); (inhibitors of Ythdf1) hsa-miR-548g, hsa-miR-204, hsa-miR-3143, hsa-miR-521, hsa-miR-195, hsa-miR-3182, hsa-miR-3941, hsa-miR-34c-3p, hsa-miR-767-3p, hsa-miR-563, hsa-miR-548c-5p, hsa-miR-1911, hsa-miR-26b, hsa-miR-190b, hsa-miR-33a, hsa-miR-329, hsa-miR-221, hsa-miR-612, hsa-miR-3185, hsa-miR-3156-5p, hsa-miR-107, hsa-miR-664, hsa-miR-3657; (inhibitors of Ythdf2) hsa-mir-615-3p (MIRT040054), hsa-mir-106b-5p (MIRT044257), hsa-mir-1 (MIRT023842), miR-145, hsa-miR-3607-3p, hsa-miR-200a, hsa-miR-301a, hsa-miR-519a, hsa-miR-141, hsa-miR-130b, hsa-miR-181b, hsa-miR-301b, hsa-miR-3117-3p, hsa-miR-1236, hsa-miR-181a, hsa-miR-519c-3p, hsa-miR-551b, hsa-miR-519e, hsa-miR-519b-3p, hsa-miR-19b, hsa-miR-1303, hsa-miR-608, hsa-miR-145, hsa-miR-130a, hsa-miR-181c, hsa-miR-323b-3p, hsa-miR-421, hsa-miR-515-5p, hsa-miR-3666, hsa-miR-181d, hsa-miR-146a, hsa-miR-4295, hsa-miR-454, hsa-miR-3919, hsa-miR-19a, hsa-miR-543, hsa-miR-4262 (see, e.g. Helwak et al. Cell, 153(3): 654-655, 2013; Selbach et al. Nature, 455(7209): 58-63, 2008; Yang et al. J Biol Chem., 292(9): 3614-3623, 2017); (inhibitors of Ythdf3) hsa-miR-582-3p, hsa-miR-579, hsa-miR-520e, hsa-miR-520f, hsa-miR-3152-3p, hsa-miR-106a, hsa-miR-30d, hsa-miR-30a, hsa-miR-93, hsa-miR-508-5p, hsa-miR-29a, hsa-miR-3148, hsa-miR-490-5p, hsa-miR-520b, hsa-miR-20a, hsa-miR-409-3p, hsa-miR-4255, hsa-let-7i, hsa-miR-373, hsa-let-7e, hsa-miR-520c-3p, hsa-miR-3920, hsa-miR-127-5p, hsa-miR-380, hsa-miR-616, hsa-miR-4277, hsa-miR-448, hsa-miR-16-2, hsa-let-7c, hsa-miR-340, hsa-miR-373, hsa-miR-520a-3p, hsa-miR-144, hsa-miR-1265, hsa-miR-548x, hsa-miR-362-5p, hsa-miR-33b, hsa-miR-26b, hsa-miR-17, hsa-miR-569, hsa-miR-3618, hsa-miR-576-5p, hsa-miR-922, hsa-miR-302a, hsa-miR-106b, hsa-miR-888, hsa-miR-484, hsa-let-7b, hsa-miR-582-5p, hsa-let-7f, hsa-miR-30b, hsa-miR-524-5p, hsa-miR-302d, hsa-let-7d, hsa-miR-513a-5p, hsa-miR-500a, hsa-miR-570, hsa-miR-548I, hsa-miR-105, hsa-miR-374c, hsa-let-7g hsa-miR-372, hsa-miR-3658, hsa-let-7a, hsa-miR-3908, hsa-miR-302b, hsa-miR-526b, hsa-miR-190, hsa-miR-181 b, hsa-miR-433, hsa-miR-98, hsa-miR-3606, hsa-miR-595, hsa-miR-548am, hsa-miR-187, hsa-miR-561, hsa-miR-181a, hsa-miR-3155, hsa-miR-655, hsa-miR-302c, hsa-miR-195, hsa-miR-26a, hsa-miR-590-3p, hsa-miR-30c, hsa-miR-502-5p, hsa-miR-495, hsa-miR-137, hsa-miR-181c, hsa-miR-520d-5p, hsa-miR-3942-5p, hsa-miR-202, hsa-miR-302e, hsa-miR-513c, hsa-miR-885-5p, hsa-miR-520a-5p, hsa-miR-583, hsa-miR-1297, hsa-miR-7-1, hsa-miR-520d-3p, hsa-miR-3155b, hsa-miR-3182, hsa-miR-519d, hsa-miR-550a, hsa-miR-7-2, hsa-miR-181d, hsa-miR-190b, hsa-miR-1912, hsa-miR-151-3p, hsa-miR-33a, hsa-miR-525-5p, hsa-miR-20b, hsa-miR-514b-5p, hsa-miR-30e, hsa-miR-4262, hsa-miR-636; (inhibitor of elF3) hsa-mir-92b-3p (MIRT040734), hsa-mir-16-5p (MIRT031705), hsa-mir-18a-3p (MIRT040974), hsa-mir-155-5p (MIRT020771), hsa-mir-484 (MIRT042324), hsa-let-7c-5p (MIRT051776), hsa-miR-3910, hsa-miR-148b, hsa-miR-136, hsa-miR-15a, hsa-miR-488, hsa-miR-500a, hsa-miR-1297, hsa-miR-3159, hsa-miR-374c, hsa-miR-424, hsa-miR-7-1, hsa-miR-186, hsa-miR-195, hsa-miR-15b, hsa-miR-26b, hsa-miR-505, hsa-miR-1206, hsa-miR-653, hsa-miR-1283, hsa-miR-7-2, hsa-miR-196a, hsa-miR-497, hsa-miR-33a, hsa-miR-655, hsa-miR-26a hsa-miR-16, hsa-mir-151a-3p (MIRT043600), hsa-mir-92a-3p (MIRT049064), hsa-mir-615-3p (MIRT039779), hsa-mir-877-3p (MIRT036964), hsa-mir-222-3p (MIRT046746), hsa-mir-423-3p (MIRT042468), hsa-mir-324-3p (MIRT042887), hsa-mir-124-3p (MIRT022932), hsa-miR-3140-3p, hsa-miR-124, hsa-miR-198, hsa-miR-525-5p, hsa-miR-506, hsa-miR-520a-5p, hsa-miR-196a* hsa-miR-3117-3p, hsa-mir-342-5p (MIRT038210), hsa-mir-378a-5p (MIRT043981), hsa-mir-615-3p (MIRT040086), hsa-let-7b-5p (MIRT052211), hsa-mir-455-3p (MIRT037879), hsa-miR-4267, hsa-miR-590-3p, hsa-mir-106b-5p (MIRT044355), hsa-mir-320a (MIRT044466), hsa-mir-16-5p (MIRT032018), hsa-mir-155-5p (MIRT021009), hsa-miR-4302, hsa-mir-191-5p (MIRT045793), hsa-mir-1303 (MIRT035890), hsa-mir-193b-3p (MIRT016316), hsa-mir-222-3p (MIRT046640), hsa-mir-532-3p (MIRT037924), hsa-mir-18a-3p (MIRT040929), hsa-mir-92a-3p (MIRT049001), hsa-miR-582-3p, hsa-miR-4265, hsa-miR-218-2, hsa-miR-1271, hsa-miR-340, hsa-miR-221, hsa-miR-20b, hsa-miR-508-3p, hsa-miR-141, hsa-miR-4325, hsa-miR-889, hsa-miR-29a, hsa-miR-129-3p, hsa-miR-129, hsa-miR-96, hsa-miR-3163, hsa-miR-187, hsa-miR-196a, hsa-miR-222, hsa-miR-1179, hsa-miR-182, hsa-miR-9* hsa-miR-32, hsa-miR-143, hsa-miR-4296 (see, e.g., Helwak et al. Cell, 153(3): 654-656, 2013; Selbach et al. Nature, 455 (7209):58-63, 2008; Baek et al, Nature, 455(7209):64-71, 2008; Leivonen et al. Mol Cell Proteomics, 10(7), 2011: M110.005322): (inhibitors of YTHDC1) hsa-mir-20a-3p (MIRT038967), hsa-mir-103a-3p (MIRT027037), hsa-mir-1 (MIRT023492), hsa-mir-19b-3p (MIRT031105), hsa-mir-100-5p (MIRT048400), hsa-mir-93-5p (MIRT027989), hsa-mir-16-5p (MIRT031379), hsa-let-7b-5p (MIRT052150), hsa-miR-520f, hsa-miR-300, hsa-miR-15a, hsa-miR-200a, hsa-miR-605, hsa-miR-30d, hsa-miR-30a, hsa-miR-3613-3p, hsa-miR-509-3-5p, hsa-miR-34c-5p, hsa-miR-324-3p, hsa-miR-1248, hsa-miR-152, hsa-miR-548t, hsa-miR-4310, hsa-miR-145, hsa-miR-516a-3p, hsa-miR-16, hsa-miR-3668, hsa-miR-4277, hsa-miR-448, hsa-miR-16-2, hsa-miR-148b, hsa-miR-509-5p, hsa-miR-103a, hsa-miR-1265, hsa-miR-2115, hsa-miR-548c-3p, hsa-miR-148a, hsa-miR-548p, hsa-miR-513a-3p, hsa-miR-497, hsa-miR-3647-3p, hsa-miR-382, hsa-miR-30b, hsa-miR-543, hsa-let-7f-2, hsa-miR-1269, hsa-miR-3164, hsa-miR-503, hsa-miR-500a, hsa-miR-449a, hsa-miR-141, hsa-miR-424, hsa-miR-3908, hsa-miR-889, hsa-miR-2116, hsa-miR-330-3p, hsa-miR-15b, hsa-miR-181b, hsa-miR-187, hsa-miR-1237, hsa-miR-449b, hsa-miR-101, hsa-miR-381, hsa-miR-618, hsa-miR-222, hsa-miR-181a, hsa-miR-432, hsa-miR-96, hsa-miR-19b, hsa-miR-195, hsa-miR-548n, hsa-miR-485-5p, hsa-miR-217, hsa-miR-30c, hsa-miR-495, hsa-miR-137, hsa-miR-1288, hsa-miR-181c, hsa-miR-3942-5p, hsa-miR-548v, hsa-miR-487a, hsa-miR-221, hsa-miR-891 b, hsa-miR-205, hsa-miR-195, hsa-miR-4271, hsa-miR-3611, hsa-miR-516b, hsa-miR-181d, hsa-miR-154, hsa-miR-646, hsa-miR-153, hsa-miR-34a, hsa-miR-19a, hsa-miR-107, hsa-miR-30e and hsa-miR-4262 (see, e.g. Helwak et al. Cell, 153(3): 654-655, 2013; Hafner et al. Cell, 141(1): 129-141, 2010; Kishore et al, Nat Methods, 8(7):559-64, 2011; Memczak et al. Nature, 495(7441):333-8, 2013; Selbach et al. Nature, 455(7209):58-63, 2008; Chi et al. Nature. 460(7254):479-86, 2009).

In the present invention, the term “small molecule” includes any chemical or other moiety, other than polypeptides and nucleic acids, that can act to affect biological processes, particularly to modulate members of the m⁶A mRNA modification pathway. Small molecules can include any number of therapeutic agents presently known and used, or that can be synthesized in a library of such molecules for the purpose of screening for biological function(s). Small molecules are distinguished from macromolecules by size. The small molecules of the present invention usually have a molecular weight less than about 5,000 daltons (Da), preferably less than about 2,500 Da, more preferably less than 1,000 Da, most preferably less than about 500 Da.

Small molecules include without limitation organic compounds, peptidomimetics and conjugates thereof. As used herein, the term “organic compound” refers to any carbon-based compound other than macromolecules such as nucleic acids and polypeptides. In addition to carbon, organic compounds may contain calcium, chlorine, fluorine, copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and other elements. An organic compound may be in an aromatic or aliphatic form. Non-limiting examples of organic compounds include acetones, alcohols, anilines, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, nucleosides, nucleotides, lipids, retinoids, steroids, proteoglycans, ketones, aldehydes, saturated, unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters, ethers, thiols, sulfides, cyclic compounds, heterocyclic compounds, imidizoles, and phenols. An organic compound as used herein also includes nitrated organic compounds and halogenated (e.g., chlorinated) organic compounds.

Preferred small molecules are relatively easier and less expensively manufactured, formulated or otherwise prepared. Preferred small molecules are stable under a variety of storage conditions. Preferred small molecules may be placed in tight association with macromolecules to form molecules that are biologically active and that have improved pharmaceutical properties. Improved pharmaceutical properties include changes in circulation time, distribution, metabolism, modification, excretion, secretion, elimination, and stability that are favorable to the desired biological activity. Improved pharmaceutical properties include changes in the toxicological and efficacy characteristics of the chemical entity.

In general, a polypeptide mimetic (“peptidomimetic”) is a molecule that mimics the biological activity of a polypeptide, but that is not peptidic in chemical nature. While, in certain embodiments, a peptidomimetic is a molecule that contains no peptide bonds (that is, amide bonds between amino acids), the term peptidomimetic may include molecules that are not completely peptidic in character, such as pseudo-peptides, semi-peptides, and peptoids.

As used herein, the term “biologic” means products derived from living sources as opposed to a chemical process. Non-limiting examples of a “biologic” include proteins, conditioned media, and partially purified products from tissues.

The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein. In the present invention, these terms mean a linked sequence of amino acids, which may be natural, synthetic, or a modification or combination of natural and synthetic. The term includes antibodies, antibody mimetics, domain antibodies, lipocalins, and targeted proteases. The term also includes vaccines containing a peptide or peptide fragment intended to raise antibodies against the peptide or peptide fragment.

“Antibody” as used herein includes an antibody of classes IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof, including Fab, F(ab′)2, Fd, and single chain antibodies, diabodies, bispecific antibodies, and bifunctional antibodies. The antibody may be a monoclonal antibody, polyclonal antibody, affinity purified antibody, or mixtures thereof, which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom. The antibody may also be a chimeric antibody. The antibody may be derivatized by the attachment of one or more chemical, peptide, or polypeptide moieties known in the art. The antibody may be conjugated with a chemical moiety. The antibody may be a human or humanized antibody. These and other antibodies are disclosed in U.S. Published Patent Application No. 20070065447.

Other antibody-like molecules are also within the scope of the present invention. Such antibody-like molecules include, e.g., receptor traps (such as entanercept), antibody mimetics (such as adnectins, fibronectin based “addressable” therapeutic binding molecules from, e.g., Compound Therapeutics, Inc.), domain antibodies (the smallest functional fragment of a naturally occurring single-domain antibody (such as, e.g., nanobodies; see, e.g., Cortez-Retamozo et al., Cancer Res. 2004 Apr. 15; 64(8):2853-7)).

Suitable antibody mimetics generally can be used as surrogates for the antibodies and antibody fragments described herein. Such antibody mimetics may be associated with advantageous properties (e.g., they may be water soluble, resistant to proteolysis, and/or be nonimmunogenic). For example, peptides comprising a synthetic beta-loop structure that mimics the second complementarity-determining region (CDR) of monoclonal antibodies have been proposed and generated. See, e.g., Saragovi et al., Science. Aug. 16, 1991; 253(5021):792-5. Peptide antibody mimetics also have been generated by use of peptide mapping to determine “active” antigen recognition residues, molecular modeling, and a molecular dynamics trajectory analysis, so as to design a peptide mimic containing antigen contact residues from multiple CDRs. See, e.g., Cassett et al., Biochem Biophys Res Commun. Jul. 18, 2003; 307(1):198-205. Additional discussion of related principles, methods, etc., that may be applicable in the context of this invention are provided in, e.g., Fassina, Immunomethods. October 1994; 5(2):121-9.

As used herein, “peptide” includes targeted proteases, which are capable of, e.g., substrate-targeted inhibition of post-translational modification such as disclosed in, e.g., U.S. Patent Application Publication No. 20060275823.

“Antisense” molecules as used herein include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen, Cancer Res. 48:2659, (1988) and van der Krol et al., BioTechniques 6:958, (1988).

Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides. These molecules function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, November 1994, BioPharm, 20-33) either by steric blocking or by activating an RNase H enzyme. Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190). In addition, binding of single stranded DNA to RNA can result in nuclease-mediated degradation of the heteroduplex (Wu-Pong, supra). Backbone modified DNA chemistry, which have thus far been shown to act as substrates for RNase H are phosphorothioates, phosphorodithioates, borontrifluoridates, and 2′-arabino and 2′-fluoro arabino-containing oligonucleotides.

Antisense molecules may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described, e.g., in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described, e.g., in WO 90/10448.

The term small interfering RNA (“siRNA”) refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. (Elbashir, S. M. et al. Nature 411:494-498 (2001); Caplen, N. J. et al. Proc. Natl. Acad. Sci. USA 98:9742-9747 (2001); Harborth, J. et al. J Cell Sci. 114:4557-4565 (2001).) These molecules can vary in length (generally 18-30 base pairs) and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5′ or 3′ end of the sense strand and/or the antisense strand. The term “siRNA” includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region. As used herein, siRNA molecules are not limited to RNA molecules but further encompass chemically modified nucleotides and non-nucleotides. siRNA gene-targeting may be carried out by transient siRNA transfer into cells (achieved by such classic methods as liposome-mediated transfection, electroporation, or microinjection).

In an additional aspect of the present invention, the number of stem cells and/or CAR T-cells is increased by a factor of at least 2-fold. Preferably, the number of stem cells and/or CAR T-cells is increased by a factor of at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4-fold, such as at least 5-fold, including at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold, or more. Surprisingly and unexpectedly such levels of stem cell and/or CAR T-cell expansion are achieved using the methods of the present invention.

As noted above, the methods of the present invention may be used to expand any population of stem cells. Preferably, the stem cells that may be expanded according to the methods of the present invention may selected from hematopoietic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), cardiac stem cells (CSCs), neuronal stem cells (NSCs), and combinations thereof. According to one aspect, the stem cells are HSCs. According to yet another aspect, the stem cells are MSCs.

The methods of the present invention may also be capable of expanding stem cells such that the expanded cells have at least a 5-fold increase in total colony-forming units (CFU), such as an 8-fold, 10-fold, and even 15-fold or more increase. Further, the methods may be capable of providing an increase in CFU-granulocyte erythrocyte monocyte megakaryocyte (GEMM) colonies of at least 3.8-fold, such as at least 4-fold, 5-fold, and even at least 8-fold or more.

Another embodiment of the invention is a method for ex vivo expansion of a substantially undifferentiated stem cell population. This method comprises modulating a N⁶-methyladenosine mRNA modification pathway in the undifferentiated stem cell population to expand the number of undifferentiated stem cells without significant differentiation of the stem cell population.

In this embodiment, a stem cell population is “substantially undifferentiated” if a sufficient number of cells in that population retain the ability to self-renew and can give rise to various differentiated cell types when transplanted into a recipient, for example, in the case of an HSC population, repopulating the HSC lineage when transplanted (or in the case of an MSC population, repopulating the MSC lineage when transplanted). As used herein, “without significant differentiation” means the expanded stem cell population has a sufficient number of cells that maintain a multi-lineage differentiation potential so that the full scope of a target stem lineage may be regenerated upon transplantation of the expanded stem cell population into a recipient. Thus, e.g., in the case of an HSC population, the expanded HSC population, when transplanted into a recipient, is capable of regenerating the entire hematopoietic cell lineage. In the case of a MSC population, the expanded MSC population, when transplanted into a recipient, is capable of regenerating the entire mesenchymal cell lineage.

Another embodiment is a method for ex vivo expansion of a chimeric antigen receptor (CAR) T-cell population. This method comprises modulating a N⁶-Methyladenosine (m⁶A) mRNA modification pathway in the CAR T-cell population to expand the number of CAR T-cells.

A further embodiment of the invention is a method for ex vivo expansion of an hematopoietic stem cell (HSC) population obtained from a tissue selected from the group consisting of peripheral blood, cord blood, and bone marrow. This method comprises modulating a N⁶-methyladenosine mRNA modification pathway in the HSC population to expand the HSC population to a sufficient quantity while maintaining a multilineage differentiation potential in the HSC population, which is sufficient for subsequent transplantation into a subject in need thereof.

A further embodiment of the invention is a method for ex vivo expansion of a mesenchymal stem cell (MSC) population obtained from a tissue selected from the group consisting of peripheral blood, cord blood, and bone marrow. This method comprises modulating a N⁶-methyladenosine mRNA modification pathway in the MSC population to expand the MSC population to a sufficient quantity while maintaining a multilineage differentiation potential in the MSC population, which is sufficient for subsequent transplantation into a subject in need thereof.

A further embodiment of the invention is a method for ex vivo expansion of a chimeric antigen receptor (CAR) T-cell population prepared by modifying T-cells obtained from a tissue selected from the group consisting of peripheral blood, cord blood, and bone marrow. The method comprising modulating a N⁶-Methyladenosine (m⁶A) mRNA modification pathway in the CAR T-cell population to expand the CAR T-cell population to a sufficient quantity which is sufficient for subsequent transplantation into a subject in need thereof.

As used herein, “obtained” from a tissue means any conventional method of harvesting or partitioning tissue from a donor. As noted previously, the tissue may be any tissue that contains a stem cell such as an HSC and/or MSC, and/or a T-cell that is capable of being modified with chimeric antigen receptors. Thus, for example, the tissue may be obtained from a blood sample, such as a peripheral or cord blood sample, or harvested from bone marrow. Methods for obtaining such samples are well known to the artisan. In the present invention, the samples may be fresh, i.e., obtained from the donor without freezing. Moreover, the samples may be further manipulated to remove extraneous or unwanted components prior to expansion. The samples may also be obtained from a preserved stock. For example, in the case of peripheral or cord blood, the samples may be withdrawn from a cryogenically or otherwise preserved bank of such blood. Such samples may be obtained from any suitable donor. Preferably, the donor is a mammal, for example, a primate, such as a human. Furthermore, the sample may be obtained from an autologous or allogeneic donor or source. Preferably, the sample is obtained from an autologous source.

In this method, “maintaining a multilineage differentiation potential” means that the expanded HSC and/or MSC population has the ability, when transplanted into a subject in need of such a transplant, to regenerate all the types of progenitor cells e.g., CMP, GMP, MEP, and CLP, and ultimately all the types of blood cells including, e.g., red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, and platelets in the hematopoietic system.

In the present invention, that quantity of expanded HSCs and/or MSCs and/or CAR T-cells, which is “sufficient for subsequent transplantation” generally corresponds to that number of HSCs and/or MSCs and/or CAR T-cells, which would result in greater than about 1% engraftment after transplantation. This is one accepted measure of a successful transplant. In the present invention, any conventional method may be used to determine the % engraftment, including the one set forth in the Examples. Such a measure may be carried out with or without competitor cells, typically and preferably, without competitor cells. (Zhang, C. C., et al., Nat Med, 12(2): 240-5, 2006. Zhang, C. C. and H. F. Lodish, Blood, 105(11): 4314-20, 2005).

In the above described ex vivo expansion methods, modulating the N⁶-methyladenosine mRNA modification pathway may be achieved as previously set forth. Modulating the N⁶-methyladenosine mRNA modification pathway may include introducing a mutation into the stem cells and/or CAR T-cells that results in modulation of a molecule in the m⁶A mRNA modification pathway, or contacting the stem cells and/or CAR T-cells with a modulator of a molecule in the m⁶A mRNA modification pathway selected from the group consisting of a small molecule, a biologic, an antisense RNA, a small interfering RNA (siRNA), and combinations thereof.

In one aspect of the ex vivo expansion methods, the modulation of the m⁶A mRNA modification pathway involves modulation of a molecule selected from the group consisting of m⁶A mRNA modification readers, m⁶A mRNA modification writers, m⁶A mRNA modification erasers, and combinations thereof. Non-limiting examples of m⁶A modification writers include methyltransferases that are capable of post-transcriptionally installing the m⁶A modification in messenger RNA, and can include any selected from the group consisting of METTL3, METTL14, WTAP, KIAA1429 and combinations thereof. Non-limiting examples of m⁶A modification erasers include demethylases that are capable of reversing the methylation, and can include any selected from the group consisting of FTI, ALKBH5 and combinations thereof. M⁶A modification readers include proteins that are capable of selectively binding m⁶A-methylated mRNA to exert regulatory functions through selective recognition of methylated mRNA. Suitable m⁶A modification readers can include any selected from the group consisting of Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, elF3 and combinations thereof. According to one aspect, the m⁶A modification readers comprise proteins of the YTH domain family of proteins, which includes Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2 and combinations thereof. (see, e.g., Wang et al. Nature, 505(7481):117-120, 2014; Frayling et al. Science, 316: 889-894, 2007; Zheng et al. Mol. Cell., 49: 18-29, 2012; Cao et al. Open Biol., 6(4): 160003, 2016; Maity et al. The FEBS Journal, 283(9): 1607-1630, 2016).

In one aspect of the ex vivo methods, modulating the m⁶A mRNA modification pathway comprises introducing a mutation into the stem cells and/or CAR-T cells to delete, replace, or reduce expression of a gene that expresses a molecule in the m⁶A mRNA modification pathway. For example, the mutation deletes, replaces or reduces expression of a gene that expresses a molecule selected from the group consisting of a m⁶A mRNA modification reader, a m⁶A mRNA modification writer, a m⁶A mRNA eraser, and combinations thereof. In one aspect, the mutation deletes, replaces or reduces expression of a gene that expresses a m⁶A mRNA modification reader, such as a m⁶A mRNA modification reader selected from the group consisting of Ythdf1, Ythdf2, Ythdf3, Ythdc1, HNRNPC, HNRNPA2B1, elF3, and combinations thereof. In a preferred aspect, the mutation deletes, replaces or reduces expression of a gene that expresses Ythdf2. In yet another aspect, the mutation deletes, replaces or reduces expression of a gene that expresses a m⁶A mRNA modification eraser, such as a m⁶A mRNA modification eraser selected from the group consisting of FTO, ALKBH5 and combinations thereof. In yet another aspect, the mutation deletes, replaces or reduces expression of a gene that expresses a m⁶A mRNA modification writer, such as a m⁶A mRNA modification writer selected from the group consisting of METTL3, METTL14, WTAP, KIAA1429 and combinations thereof.

In one aspect of the ex vivo expansion methods, the mutation can be introduced by any of the methods previously disclosed herein. For example, the mutation can be introduced by exposing the stem cells and/or CAR T-cells to a Mx1-Cre targeting system (see, e.g., Kuhn et al. Science, 269(5229): 1427-1429, 1995) that inactivates or deletes at least a portion of a gene that expresses a molecule in the m⁶A mRNA modification pathway. In yet another aspect, a mutation is introduced that incorporates short hairpin RNA (shRNA) into the stem cells and/or CAR T-cells to reduce expression of a gene that expresses a molecule in the m⁶A mRNA modification pathway. For example, the shRNA may be introduced by exposing the stem cells and/or CAR-T cells to a vector to deliver shRNA, which may be a viral vector such as lentivirus (see, e.g., Chira et al. Oncotarget, 6(31): 30675-30703, 2015). The shRNA may be capable of triggering gene silencing to regulate gene expression (see, e.g., Paddison et al. Genes Dev., 16(8): 948-958, 2002).

In these ex vivo expansion methods, according to one aspect, modulating of the m⁶A mRNA modification pathway comprises down-regulating and/or inhibiting a member of the m⁶A mRNA modification pathway, such as down-regulating and/or inhibiting a m⁶A mRNA modification reader. As used herein, “down-regulating” means inhibiting or reducing the amount of or inhibiting or decreasing the activity of a member of the m⁶A mRNA modification pathway. Such down-regulation may be accomplished using, e.g. antisense RNA, siRNA, antibodies, or small molecules. As another example, the m⁶A mRNA modification reader may be down-regulated by contacting the stem cells and/or CAR T-cells with an inhibitor of an m⁶A mRNA reader, to inhibit binding and/or recognizing of the m⁶A modified mRNA by the m⁶A mRNA reader. In one aspect, the m⁶A mRNA modification reader that is down-regulated is selected from the group consisting of Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, elF3 and combinations thereof. In a preferred aspect, the m⁶A mRNA modification reader that is down-regulated is Ythdf2. The RNA decay role of Ythdf2 has been previously elucidated (see, e.g., Wang et al. Nature 505(7481): 117-120, 2014). Inhibitors of the m⁶A mRNA modification reader may be any selected from the group consisting of: (inhibitors of HNRNPC) hsa-let-7e-5p (MIRT051596), hsa-mir-455-3p (MIRT037890), hsa-mir-30c-5p (MIRT047904), hsa-mir-186-5p (MIRT045150), hsa-mir-744-5p (MIRT037494), hsa-mir-18a-3p (MIRT040851), hsa-mir-484 (MIRT042196), hsa-mir-505-5p (MIRT037959), hsa-mir-615-3p (MIRT039991), hsa-mir-342-3p (MIRT043694), hsa-miR-3607-3p, hsa-miR-30d, hsa-miR-3916, hsa-miR-3162-5p, hsa-miR-1273d, hsa-miR-3161, hsa-miR-30a, hsa-miR-629, hsa-miR-208b, hsa-miR-489, hsa-miR-3148, hsa-miR-2113, hsa-miR-877, hsa-miR-455-5p, hsa-miR-186, hsa-miR-548o, hsa-miR-3139, hsa-miR-320a, hsa-miR-4311, hsa-miR-555, hsa-miR-3605-5p, hsa-miR-515-5p, hsa-miR-144, hsa-miR-499-5p, hsa-miR-1323, hsa-miR-548x, hsa-miR-299-5p, hsa-miR-653, hsa-miR-576-5p, hsa-miR-548p, hsa-miR-586, hsa-miR-888, hsa-miR-3647-3p, hsa-miR-484, hsa-miR-320b, hsa-miR-620, hsa-miR-30b, hsa-miR-548q, hsa-miR-29b-1, hsa-miR-570, hsa-miR-183, hsa-miR-1276, hsa-miR-208a, hsa-miR-186, hsa-miR-28-5p, hsa-miR-330-3p, hsa-miR-548am, hsa-miR-320d, hsa-miR-3175, hsa-miR-3155, hsa-miR-548aa, hsa-miR-519e, hsa-miR-1270, hsa-miR-513b, hsa-miR-599, hsa-miR-518f, hsa-miR-4301, hsa-miR-30c, hsa-miR-3135, hsa-miR-4286, hsa-miR-202, hsa-miR-4263, hsa-miR-4299, hsa-miR-606, hsa-miR-3133, hsa-miR-583, hsa-miR-3125, hsa-miR-501-5p, hsa-miR-7-1, hsa-miR-514b-3p, hsa-miR-3155b, hsa-miR-548d-3p, hsa-miR-224, hsa-miR-7-2, hsa-miR-708, hsa-miR-3199, hsa-miR-514, hsa-miR-30e (see, e.g. Helwak et al. Cell, 153(3): 654-655, 2013; Whisnant et al., M Bio 4(2), 2013:e000193); (inhibitors of HNRNPA2B1) hsa-mir-92a-3p (MIRT049721), hsa-mir-30c-5p (MIRT048009), hsa-mir-191-5p (MIRT045809), hsa-let-7f-5p (MIRT051404), hsa-mir-27b-3p (MIRT046213), hsa-mir-877-3p (MIRT037116), hsa-mir-615-3p (MIRT040278), hsa-mir-1260b (MIRT052680), hsa-mir-103a-3p (MIRT027027), hsa-mir-16-5p (MIRT031508), hsa-mir-1296-5p (MIRT036075), hsa-mir-197-3p (MIRT048098), hsa-miR-548j, hsa-miR-3678-3p, hsa-miR-607, hsa-miR-188-5p, hsa-miR-15a, hsa-miR-3653, hsa-miR-371-5p, hsa-miR-550a, hsa-miR-3622b-3p, hsa-miR-548a-5p, hsa-miR-3170, hsa-miR-3148, hsa-miR-556-3p, hsa-miR-490-3p, hsa-miR-559, hsa-miR-200c, hsa-miR-130a, hsa-miR-548y, hsa-miR-548o, hsa-miR-23c, hsa-miR-491-3p, hsa-miR-335, hsa-miR-3667-3p, hsa-miR-466, hsa-miR-23b, hsa-miR-4310, hsa-miR-127-5p, hsa-miR-548b-5p, hsa-miR-616, hsa-miR-16, hsa-miR-338-3p, hsa-miR-3200-5p, hsa-miR-362-3p, hsa-miR-448, hsa-miR-1306, hsa-miR-944, hsa-miR-3684, hsa-miR-373, hsa-miR-103a, hsa-miR-380, hsa-miR-499-5p, hsa-miR-1323, hsa-miR-323-5p, hsa-miR-3674, hsa-miR-1252, hsa-miR-33b, hsa-miR-580, hsa-miR-548c-3p, hsa-miR-103a-2, hsa-miR-548w, hsa-miR-600, hsa-miR-634, hsa-miR-586, hsa-miR-497, hsa-miR-720, hsa-miR-654-3p, hsa-miR-524-5p, hsa-miR-543, hsa-miR-548q, hsa-let-7f-2, hsa-miR-330-5p, hsa-miR-500a, hsa-miR-548I, hsa-miR-570, hsa-miR-374a, hsa-miR-1184, hsa-miR-649, hsa-miR-424, hsa-miR-3658, hsa-miR-186, hsa-miR-326, hsa-miR-548d-5p, hsa-miR-23a, hsa-miR-15b, hsa-miR-190, hsa-miR-203, hsa-miR-548h, hsa-miR-3136-5p, hsa-miR-618, hsa-miR-551b, hsa-miR-211, hsa-miR-1305, hsa-miR-513b, hsa-miR-96, hsa-miR-2117, hsa-miR-548n, hsa-miR-3910, hsa-miR-217, hsa-miR-892b, hsa-miR-502-5p, hsa-miR-548i, hsa-miR-520d-5p, hsa-miR-4299, hsa-miR-1285, hsa-miR-3133, hsa-miR-483-3p (see, e.g., Hafner et al. Cell, 141(1): 129-141, 2010; Helwak et al. Cell, 153(3): 654-655, 2013); (inhibitors of Ythdf1) hsa-miR-548g, hsa-miR-204, hsa-miR-3143, hsa-miR-521, hsa-miR-195, hsa-miR-3182, hsa-miR-3941, hsa-miR-34c-3p, hsa-miR-767-3p, hsa-miR-563, hsa-miR-548c-5p, hsa-miR-1911, hsa-miR-26b, hsa-miR-190b, hsa-miR-33a, hsa-miR-329, hsa-miR-221, hsa-miR-612, hsa-miR-3185, hsa-miR-3156-5p, hsa-miR-107, hsa-miR-664, hsa-miR-3657; (inhibitors of Ythdf2) hsa-mir-615-3p (MIRT040054), hsa-mir-106b-5p (MIRT044257), hsa-mir-1 (MIRT023842), miR-145, hsa-miR-3607-3p, hsa-miR-200a, hsa-miR-301a, hsa-miR-519a, hsa-miR-141, hsa-miR-130b, hsa-miR-181b, hsa-miR-301b, hsa-miR-3117-3p, hsa-miR-1236, hsa-miR-181a, hsa-miR-519c-3p, hsa-miR-551b, hsa-miR-519e, hsa-miR-519b-3p, hsa-miR-19b, hsa-miR-1303, hsa-miR-608, hsa-miR-145, hsa-miR-130a, hsa-miR-181c, hsa-miR-323b-3p, hsa-miR-421, hsa-miR-515-5p, hsa-miR-3666, hsa-miR-181d, hsa-miR-146a, hsa-miR-4295, hsa-miR-454, hsa-miR-3919, hsa-miR-19a, hsa-miR-543, hsa-miR-4262 (see, e.g. Helwak et al. Cell, 153(3): 654-655, 2013; Selbach et al. Nature, 455(7209): 58-63, 2008; Yang et al. J Biol Chem., 292(9): 3614-3623, 2017); (inhibitors of Ythdf3) hsa-miR-582-3p, hsa-miR-579, hsa-miR-520e, hsa-miR-520f, hsa-miR-3152-3p, hsa-miR-106a, hsa-miR-30d, hsa-miR-30a, hsa-miR-93, hsa-miR-508-5p, hsa-miR-29a, hsa-miR-3148, hsa-miR-490-5p, hsa-miR-520b, hsa-miR-20a, hsa-miR-409-3p, hsa-miR-4255, hsa-let-7i, hsa-miR-373, hsa-let-7e, hsa-miR-520c-3p, hsa-miR-3920, hsa-miR-127-5p, hsa-miR-380, hsa-miR-616, hsa-miR-4277, hsa-miR-448, hsa-miR-16-2, hsa-let-7c, hsa-miR-340, hsa-miR-373, hsa-miR-520a-3p, hsa-miR-144, hsa-miR-1265, hsa-miR-548x, hsa-miR-362-5p, hsa-miR-33b, hsa-miR-26b, hsa-miR-17, hsa-miR-569, hsa-miR-3618, hsa-miR-576-5p, hsa-miR-922, hsa-miR-302a, hsa-miR-106b, hsa-miR-888, hsa-miR-484, hsa-let-7b, hsa-miR-582-5p, hsa-let-7f, hsa-miR-30b, hsa-miR-524-5p, hsa-miR-302d, hsa-let-7d, hsa-miR-513a-5p, hsa-miR-500a, hsa-miR-570, hsa-miR-548I, hsa-miR-105, hsa-miR-374c, hsa-let-7g hsa-miR-372, hsa-miR-3658, hsa-let-7a, hsa-miR-3908, hsa-miR-302b, hsa-miR-526b, hsa-miR-190, hsa-miR-181 b, hsa-miR-433, hsa-miR-98, hsa-miR-3606, hsa-miR-595, hsa-miR-548am, hsa-miR-187, hsa-miR-561, hsa-miR-181a, hsa-miR-3155, hsa-miR-655, hsa-miR-302c, hsa-miR-195, hsa-miR-26a, hsa-miR-590-3p, hsa-miR-30c, hsa-miR-502-5p, hsa-miR-495, hsa-miR-137, hsa-miR-181c, hsa-miR-520d-5p, hsa-miR-3942-5p, hsa-miR-202, hsa-miR-302e, hsa-miR-513c, hsa-miR-885-5p, hsa-miR-520a-5p, hsa-miR-583, hsa-miR-1297, hsa-miR-7-1, hsa-miR-520d-3p, hsa-miR-3155b, hsa-miR-3182, hsa-miR-519d, hsa-miR-550a, hsa-miR-7-2, hsa-miR-181d, hsa-miR-190b, hsa-miR-1912, hsa-miR-151-3p, hsa-miR-33a, hsa-miR-525-5p, hsa-miR-20b, hsa-miR-514b-5p, hsa-miR-30e, hsa-miR-4262, hsa-miR-636; (inhibitor of elF3) hsa-mir-92b-3p (MIRT040734), hsa-mir-16-5p (MIRT031705), hsa-mir-18a-3p (MIRT040974), hsa-mir-155-5p (MIRT020771), hsa-mir-484 (MIRT042324), hsa-let-7c-5p (MIRT051776), hsa-miR-3910, hsa-miR-148b, hsa-miR-136, hsa-miR-15a, hsa-miR-488, hsa-miR-500a, hsa-miR-1297, hsa-miR-3159, hsa-miR-374c, hsa-miR-424, hsa-miR-7-1, hsa-miR-186, hsa-miR-195, hsa-miR-15b, hsa-miR-26b, hsa-miR-505, hsa-miR-1206, hsa-miR-653, hsa-miR-1283, hsa-miR-7-2, hsa-miR-196a, hsa-miR-497, hsa-miR-33a, hsa-miR-655, hsa-miR-26a hsa-miR-16, hsa-mir-151a-3p (MIRT043600), hsa-mir-92a-3p (MIRT049064), hsa-mir-615-3p (MIRT039779), hsa-mir-877-3p (MIRT036964), hsa-mir-222-3p (MIRT046746), hsa-mir-423-3p (MIRT042468), hsa-mir-324-3p (MIRT042887), hsa-mir-124-3p (MIRT022932), hsa-miR-3140-3p, hsa-miR-124, hsa-miR-198, hsa-miR-525-5p, hsa-miR-506, hsa-miR-520a-5p, hsa-miR-196a* hsa-miR-3117-3p, hsa-mir-342-5p (MIRT038210), hsa-mir-378a-5p (MIRT043981), hsa-mir-615-3p (MIRT040086), hsa-Iet-7b-5p (MIRT052211), hsa-mir-455-3p (MIRT037879), hsa-miR-4267, hsa-miR-590-3p, hsa-mir-106b-5p (MIRT044355), hsa-mir-320a (MIRT044466), hsa-mir-16-5p (MIRT032018), hsa-mir-155-5p (MIRT021009), hsa-miR-4302, hsa-mir-191-5p (MIRT045793), hsa-mir-1303 (MIRT035890), hsa-mir-193b-3p (MIRT016316), hsa-mir-222-3p (MIRT046640), hsa-mir-532-3p (MIRT037924), hsa-mir-18a-3p (MIRT040929), hsa-mir-92a-3p (MIRT049001), hsa-miR-582-3p, hsa-miR-4265, hsa-miR-218-2, hsa-miR-1271, hsa-miR-340, hsa-miR-221, hsa-miR-20b, hsa-miR-508-3p, hsa-miR-141, hsa-miR-4325, hsa-miR-889, hsa-miR-29a, hsa-miR-129-3p, hsa-miR-129, hsa-miR-96, hsa-miR-3163, hsa-miR-187, hsa-miR-196a, hsa-miR-222, hsa-miR-1179, hsa-miR-182, hsa-miR-9* hsa-miR-32, hsa-miR-143, hsa-miR-4296 (see, e.g., Helwak et al. Cell, 153(3): 654-656, 2013; Selbach et al. Nature, 455 (7209):58-63, 2008; Baek et al, Nature, 455(7209):64-71, 2008; Leivonen et al. Mol Cell Proteomics, 10(7), 2011: M110.005322): (inhibitors of YTHDC1) hsa-mir-20a-3p (MIRT038967), hsa-mir-103a-3p (MIRT027037), hsa-mir-1 (MIRT023492), hsa-mir-19b-3p (MIRT031105), hsa-mir-100-5p (MIRT048400), hsa-mir-93-5p (MIRT027989), hsa-mir-16-5p (MIRT031379), hsa-let-7b-5p (MIRT052150), hsa-miR-520f, hsa-miR-300, hsa-miR-15a, hsa-miR-200a, hsa-miR-605, hsa-miR-30d, hsa-miR-30a, hsa-miR-3613-3p, hsa-miR-509-3-5p, hsa-miR-34c-5p, hsa-miR-324-3p, hsa-miR-1248, hsa-miR-152, hsa-miR-548t, hsa-miR-4310, hsa-miR-145, hsa-miR-516a-3p, hsa-miR-16, hsa-miR-3668, hsa-miR-4277, hsa-miR-448, hsa-miR-16-2, hsa-miR-148b, hsa-miR-509-5p, hsa-miR-103a, hsa-miR-1265, hsa-miR-2115, hsa-miR-548c-3p, hsa-miR-148a, hsa-miR-548p, hsa-miR-513a-3p, hsa-miR-497, hsa-miR-3647-3p, hsa-miR-382, hsa-miR-30b, hsa-miR-543, hsa-let-7f-2, hsa-miR-1269, hsa-miR-3164, hsa-miR-503, hsa-miR-500a, hsa-miR-449a, hsa-miR-141, hsa-miR-424, hsa-miR-3908, hsa-miR-889, hsa-miR-2116, hsa-miR-330-3p, hsa-miR-15b, hsa-miR-181b, hsa-miR-187, hsa-miR-1237, hsa-miR-449b, hsa-miR-101, hsa-miR-381, hsa-miR-618, hsa-miR-222, hsa-miR-181a, hsa-miR-432, hsa-miR-96, hsa-miR-19b, hsa-miR-195, hsa-miR-548n, hsa-miR-485-5p, hsa-miR-217, hsa-miR-30c, hsa-miR-495, hsa-miR-137, hsa-miR-1288, hsa-miR-181c, hsa-miR-3942-5p, hsa-miR-548v, hsa-miR-487a, hsa-miR-221, hsa-miR-891 b, hsa-miR-205, hsa-miR-195, hsa-miR-4271, hsa-miR-3611, hsa-miR-516b, hsa-miR-181d, hsa-miR-154, hsa-miR-646, hsa-miR-153, hsa-miR-34a, hsa-miR-19a, hsa-miR-107, hsa-miR-30e and hsa-miR-4262 (see, e.g. Helwak et al. Cell, 153(3): 654-655, 2013; Hafner et al. Cell, 141(1): 129-141, 2010; Kishore et al, Nat Methods, 8(7):559-64, 2011; Memczak et al. Nature, 495(7441):333-8, 2013; Selbach et al. Nature, 455(7209):58-63, 2008; Chi et al. Nature. 460(7254):479-86, 2009).

In these ex vivo expansion methods, it is preferred that the stem cell is selected from HSCs, hematopoietic stem and progenitor cells (HSPCs), endothelial progenitor cells, (EPCs), mesenchymal stem cells (MSCs), cardiac stem cells (CSCs), neuronal stem cells (NSCs), and combinations thereof. According to certain aspects, the stem cell is an HSC. According to other aspects, the stem cell is a MSC. The ex vivo expansion methods can also use a population of cells comprising CAR T-cells. In these methods, the HSC and/or MSC is obtained from a mammalian, e.g., primate or human, tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, although any HSC and/or MSC-containing tissue may be used.

In another aspect of the method for ex vivo expansion of an hematopoietic stem cell (HSC) population, the expansion of the number of stem cells is by at least 2-fold, such as e.g., by at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, and including at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, or at least 20-fold or more.

In another aspect of the method for ex vivo expansion of a mesenchymal stem cell (MSC) population, the expansion of the number of stem cells is by at least 2-fold, such as e.g., by at least 2.5-fold, at least 3-fold, at least 3.5 fold, at least 4-fold, and including at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, or at least 20-fold or more.

In another aspect of the method for ex vivo expansion of CAR T-cell population, the expansion of the number of CAR T-cells is by at least 2-fold, such as e.g., by at least 2.5-fold, at least 3-fold, at least 3.5 fold, at least 4-fold, and including at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, or at least 20-fold or more.

Yet another embodiment of the present invention is an expanded, substantially undifferentiated stem cell population made by a method of the present invention, such as, e.g., the method for ex vivo expansion of a substantially undifferentiated stem cell population or the method for ex vivo expansion of an hematopoietic stem cell (HSC) population.

Yet another embodiment of the present invention is an expanded, CAR T-cell population made by a method of the present invention, such as, e.g., the method for ex vivo expansion of a CAR T-cell population.

An additional embodiment of the present invention is a method for ex vivo expansion of hematopoietic stem cells (HSCs) by at least 2-fold, wherein the expanded HSCs, are competent to reconstitute an HSC lineage upon transplantation into a mammalian subject in need thereof. This method comprises introducing a mutation into the stem cells that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader and culturing the population of HSCs in a suitable culture medium.

An additional embodiment of the present invention is a method for ex vivo expansion of mesenchymal stem cells (MSCs) by at least 2-fold, wherein the expanded MSCs, are competent to reconstitute a MSC lineage upon transplantation into a mammalian subject in need thereof. This method comprises introducing a mutation into the stem cells that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader and culturing the population of HSCs in a suitable culture medium.

An additional embodiment of the present invention is a method for ex vivo expansion of chimeric antigen receptor (CAR) T-cells by at least 2-fold, wherein the expanded CAR T-cells are competent to treat a cancer and/or blood disorder upon transplantation into a mammalian subject in need thereof. This method comprises introducing a mutation into the CAR T-cells that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader and culturing the population of CAR T-cells in a suitable culture medium.

In this aspect of the invention, “competent to reconstitute an HSC lineage” means that the expanded HSCs, when transplanted into a suitable mammalian subject, result in greater than 1% engraftment in the recipient, which engrafted cells are able to differentiate into the cell lineages necessary to have a normal functioning hematopoietic system. In this aspect of the invention, “competent to reconstitute a MSC lineage” means that the expanded MSCs, when transplanted into a suitable mammalian subject, result in greater than 1% engraftment in the recipient, which engrafted cells are able to differentiate into the cell lineages necessary to have a normal functioning hematopoietic system. In this aspect of the invention “competent to treat a cancer and/or blood disorder” means that the expanded CAR T-cells, when transplanted into a suitable mammalian subject, are capable of providing treatment of a cancer and/or blood disorder from which the mammalian subject is suffering, such as for example at least one of leukemia and lymphoma. In this method, a “suitable culture medium”, “fluid media” and “media” which are used interchangeably herein, mean physiologically balanced salt solutions that can maintain a stem cell population and/or CAR T-cell population for a required period of time, which solution may optionally be supplemented with suitable m⁶A mRNA modification pathway modulators of the present invention. Such base culture media are well known in the arts. A non-limiting example of a suitable base culture medium for HSCs is StemSpan Media (Stem Cell Technologies, Cat. No. 09600), which is supplemented with 10 ug/ml Heparin, 5× Penicillin/Streptomycin, 10 ng/ml recombinant mouse (rm) Stem Cell Factor, and 20 ng/ml rm-Thrombopoietin.

In one aspect of the invention, the ex vivo expansion of HSCs and/or MSCs and/or CAR T-cells by at least 2-fold can be performed by any of the methods that have been described herein. For example, the method may involve introducing a mutation that deletes, replaces or reduces expression of a gene expressing a m⁶A mRNA modification reader, such as Ythdf2. Further, the mutation may be introduced by any of the methods described herein, such as by exposing the stem cells and/or CAR T-cells to a Mx1-Cre targeting system that inactivates or deletes at least a portion of a gene that expresses a m⁶A mRNA modification reader. The mutation may also be introduced by incorporating shRNA into the stem cells and/or CAR-T cells to reduce expression of a gene that expresses a m⁶A mRNA modification reader. Other methods of introducing a mutation, and mutations that target other m⁶A mRNA modification readers that are described herein may also be provided.

In one aspect of this embodiment, the HSCs and/or MSCs and/or CAR T-cells are obtained from a mammalian tissue, preferably primate or human tissue, which is selected from cord blood, peripheral blood, and bone marrow. In this embodiment, the number of HSCs and/or MSCs and/or CAR T-cells is expanded by a factor of at least 2-fold, such as at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, and including at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more.

Yet another embodiment of the present invention is a kit for expanding an hematopoietic stem cell (HSC) population, mesenchymal stem cell (MSC) population, and/or CAR T-cell population for subsequent transplantation into a subject in need thereof. The kit comprises a system for introducing a mutation into the HSC, MSC and/or CAR T-cell population that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader, and instructions for use thereof. Preferably, in the kit, the system for introducing a mutation into the HSC, MSC and/or CAR-T cell population includes one or more reagents capable of introducing a mutation into the HSC, MSC and/or CAR-T cell population that results in deletion, replacement or reduced expression of a gene expressing Ythdf2. For example, the kit can include a system for introducing a mutation into the HSC, MSC and/or CAR-T cell population that comprises a Mx1-Cre targeting system that inactivates or deletes at least a portion of a gene that expresses a m⁶A mRNA modification reader. The kit can also include a system for introducing a mutation into the HSC, MSC and/or CAR T-cell population that comprises reagents for delivering a lentivirus that incorporates shRNA into the HSC, MSC and/or CAR T-cell population to reduce expression of a gene that expresses a m⁶A mRNA modification reader. The kit may further comprise other systems/methods set forth herein for introducing the mutation to modulate a m⁶A mRNA modification pathway, such as by deleting, replacing, or reducing expression of a gene expressing a m⁶A mRNA modification reader, including Ythdf2. The kit and the components therein may be packaged in any suitable manner for distribution and/or storage.

In yet another embodiment, a kit for expanding an hematopoietic stem cell population (HSC) population and/or mesenchymal stem cell (MSC) population and/or CAR-T cell population for subsequent transplantation into a subject in need thereof is provided. The kit comprises an inhibitor of a m⁶A mRNA modification reader, and instructions for use thereof, where the inhibitor may be any of the inhibitors disclosed herein, such as, e.g., an inhibitor of Ythdf2.

In one aspect of this embodiment, the kits may be able to provide an expansion of the number of stem cells and/or CAR T-cells by a factor selected from the group consisting of at least 2-fold, at least 2.5 fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more.

A further embodiment of the present invention is a method for administering an hematopoietic stem cell (HSC) to a subject in need thereof. The method comprises (a) introducing, into a sample containing an HSC population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader; (b) culturing the sample in a suitable culture media for a period of time sufficient to expand the number of HSCs in the sample to a number sufficient to transplant into the subject; and (c) administering the HSCs to the subject. In this embodiment, the method of introducing the mutation, and the mutation targeted at the m⁶A mRNA modification reader, are as previously disclosed, such as, for example, a mutation that results in deletion of a gene expressing Ythdf2, or a mutation that results in incorporation of shRNA into the HSC population that reduces expression of a gene expressing Ythdf2. Furthermore, the HSCs may be obtained from any appropriate tissue such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, and the subject may be a mammal, such as a human.

A further embodiment of the present invention is a method for administering a mesenchymal stem cell (MSC) to a subject in need thereof. The method comprises (a) introducing, into a sample containing a MSC population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader; (b) culturing the sample in a suitable culture media for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient to transplant into the subject; and (c) administering the MSCs to the subject. In this embodiment, the method of introducing the mutation, and the mutation targeted at the m⁶A mRNA modification reader, are as previously disclosed, such as, for example, a mutation that results in deletion of a gene expressing Ythdf2, or a mutation that results in incorporation of shRNA into the MSC population that reduces expression of a gene expressing Ythdf2. Furthermore, the MSCs may be obtained from any appropriate tissue such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, and the subject may be a mammal, such as a human.

A further embodiment of the present invention is a method for administering a chimeric antigen receptor (CAR) T-cell to a subject in need thereof. The method comprises (a) introducing, into a sample containing a CAR T-cell population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader; (b) culturing the sample in a suitable culture media for a period of time sufficient to expand the number of CAR T-cells in the sample to a number sufficient to transplant into the subject; and (c) administering the CAR T-cells to the subject. In this embodiment, the method of introducing the mutation, and the mutation targeted at the m⁶A mRNA modification reader, are as previously disclosed, such as, for example, a mutation that results in deletion of a gene expressing Ythdf2, or a mutation that results in incorporation of shRNA into the CAR T-cell population that reduces expression of a gene expressing Ythdf2. Furthermore, the CAR T-cells may be obtained from any appropriate tissue such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, and the subject may be a mammal, such as a human.

A further embodiment of the present invention is a method for administering an hematopoietic stem cell (HSC) to a subject in need thereof. The method comprises: (a) culturing, in a suitable culture media, a sample containing an HSC population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of HSCs in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the HSCs to the subject. In this method, the inhibitor is as previously disclosed, such as, e.g., an inhibitor of Ythdf2. Furthermore, the HSCs may be obtained from any appropriate tissue, such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, and the subject may be a mammal, such as a human.

A further embodiment of the present invention is a method for administering a mesenchymal stem cell (MSC) to a subject in need thereof. The method comprises: (a) culturing, in a suitable culture media, a sample containing an MSC population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the MSCs to the subject. In this method, the inhibitor is as previously disclosed, such as, e.g., an inhibitor of Ythdf2. Furthermore, the MSCs may be obtained from any appropriate tissue, such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, and the subject may be a mammal, such as a human.

A further embodiment of the present invention is a method for administering a chimeric antigen receptor (CAR) T-cell to a subject in need thereof. The method comprises: (a) culturing, in a suitable culture media, a sample containing an CAR T-cell population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of CAR T-cells in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the CAR T-cells to the subject. In this method, the inhibitor is as previously disclosed, such as, e.g., an inhibitor of Ythdf2. Furthermore, the CAR T-cells may be obtained from any appropriate tissue, such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, and the subject may be a mammal, such as a human.

An additional embodiment of the present invention is a method for reconstituting bone marrow in a subject in need thereof. The method comprises (a) introducing, into a sample containing an HSC population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader; (b) culturing the sample in a suitable culture media for a period of time sufficient to expand the number of HSCs in the sample to a number sufficient to transplant into the subject; and (c) administering the HSCs to the subject. In this embodiment, the method of introducing the mutation, and the mutation targeted at the m⁶A mRNA modification reader, are as previously disclosed, such as, for example, a mutation that results in deletion of a gene expressing Ythdf2, or a mutation that results in incorporation of shRNA into the HSC population that reduces expression of a gene expressing Ythdf2. Furthermore, the HSCs may be obtained from any appropriate tissue, such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, and the subject may be a mammal, such as a human.

An additional embodiment of the present invention is a method for reconstituting bone marrow in a subject in need thereof. The method comprises (a) introducing, into a sample containing a MSC population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader; (b) culturing the sample in a suitable culture media for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient to transplant into the subject; and (c) administering the MSCs to the subject. In this embodiment, the method of introducing the mutation, and the mutation targeted at the m⁶A mRNA modification reader, are as previously disclosed, such as, for example, a mutation that results in deletion of a gene expressing Ythdf2, or a mutation that results in incorporation of shRNA into the MSC population that reduces expression of a gene expressing Ythdf2. Furthermore, the MSCs may be obtained from any appropriate tissue, such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, and the subject may be a mammal, such as a human.

An additional embodiment of the present invention is a method for treating cancer and/or a blood disorder in a subject in need thereof. The method comprises (a) introducing, into a sample containing a CAR T-cell population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader; (b) culturing the sample in a suitable culture media for a period of time sufficient to expand the number of CAR T-cells in the sample to a number sufficient to transplant into the subject; and (c) administering the CAR T-cells to the subject. In this embodiment, the method of introducing the mutation, and the mutation targeted at the m⁶A mRNA modification reader, are as previously disclosed, such as, for example, a mutation that results in deletion of a gene expressing Ythdf2, or a mutation that results in incorporation of shRNA into the CAR T-cell population that reduces expression of a gene expressing Ythdf2. Furthermore, the CAR T-cells may be obtained from any appropriate tissue, such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, and the subject may be a mammal, such as a human.

In yet another embodiment, there is provided a method for reconstituting bone marrow in a subject in need thereof. This method comprises (a) culturing, in a suitable culture media, a sample containing an HSC population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of HSCs in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the HSCs to the subject. In this method, the inhibitor is as previously disclosed, such as an inhibitor of Ythdf2. Furthermore, the HSCs may be obtained from any appropriate tissue, such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, and the subject may be a mammal, such as a human.

In yet another embodiment, there is provided another method for reconstituting bone marrow in a subject in need thereof. This method comprises (a) culturing, in a suitable culture media, a sample containing an MSC population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the MSCs to the subject. In this method, the inhibitor is as previously disclosed, such as an inhibitor of Ythdf2. Furthermore, the MSCs may be obtained from any appropriate tissue, such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, and the subject may be a mammal, such as a human.

In these methods, “reconstituting bone marrow” means restoration of all or a portion of the bone marrow in a subject suffering from a disease in which normal bone marrow function has been compromised. Non-limiting examples of such diseases include blood disorders such as aplastic anemia, myelodysplastic syndromes (MDS), paroxysmal nocturnal hemoglobinuria (PNH), and blood cancers, such as leukemia. Thus, as used herein, “reconstituted” means that the transplanted HSCs and/or MSCs are able to successfully engraft in the host and differentiate into all the cell lineages typically found in or derived from bone marrow. Aspects of the methods herein may involve transplantation of HSCs and/or MSCs obtained from tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, to the subject, for the treatment of blood disorders such as leukemia and lymphoma.

In yet another embodiment, there is provided another method for treating cancer and/or a blood disorder in a subject in need thereof. This method comprises (a) culturing, in a suitable culture media, a sample containing a chimeric antigen receptor (CAR) T-cell population in the presence of an inhibitor of a m⁶A mRNA modification reader, for a period of time sufficient to expand the number of CAR T-cells in the sample to a number sufficient to transplant into the subject; (b) removing from the culture the inhibitor of the m⁶A mRNA modification reader; and (c) administering the CAR T-cells to the subject. In this method, the inhibitor is as previously disclosed, such as an inhibitor of Ythdf2. Furthermore, the T-cells that are modified to prepare the CAR T-cells may be obtained from any appropriate tissue, such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow, and the subject may be a mammal, such as a human.

In these methods, “treating cancer and/or a blood disorder in a subject” means eradicating cancer cells, alleviating symptoms, or otherwise reducing a disease state in a subject suffering from the cancer and/or blood disorder. Non-limiting examples of cancers and/or blood disorders include blood disorders such as aplastic anemia, myelodysplastic syndromes (MDS), paroxysmal nocturnal hemoglobinuria (PNH), and blood cancers, such as leukemia and lymphoma.

In these methods, “a period of time sufficient to expand the number of HSCs” means the minimum amount of time to expand the HSCs in culture to a point where there is a sufficient number of HSCs for one or more transplantations, and “a period of time sufficient to expand the number of MSCs” means the minimum amount of time to expand the MSCs in culture to a point where there is a sufficient number of MSCs for one or more transplantations. Similarly, “a period of time sufficient to expand the number of CAR T-cells” means the minimum amount of time to expand the CAR T-cells in culture to a point where there is a sufficient number of CAR T-cells for one or more transplantations. Typically, such a period of time may be at least about 10 days in culture. Under certain circumstances, it may be desirable to expand the stem cell and/or CAR T-cell, e.g., HSC and/or MSC, population beyond what is required for a single transplantation. For example, it may be desirable to expand the stem cell and/or CAR T-cell, e.g., HSC and/or MSC, population to a number sufficient for multiple transplantations, such as e.g., from about 2 to about 100 transplantations. In these circumstances, the excess cells may be preserved for later use by any conventional method, such as e.g., by cryo-preservation.

As indicated previously, “a number sufficient to transplant” means the minimum number of stem cells, e.g., HSCs and/or MSCs and/or CAR T-cells, necessary to achieve greater than 1% engraftment in a recipient. “Administering the HSCs to the subject” means conventional methods for delivering HSCs to the subject, including but not limited to, delivering the HSCs surgically and/or intravenously. “Administering the MSCs to the subject” means conventional methods for delivering MSCs to the subject, including but not limited to, delivering the MSCs surgically and/or intravenously. “Administering the CAR T-cells to the subject” means conventional methods for delivering CAR T-cells to the subject, including but not limited to, delivering the MSCs surgically and/or intravenously. In these embodiments, the tissue the HSCs and/or MSCs and/or T-cells are obtained from, and the m⁶A mRNA modification reader inhibitors are as previously disclosed.

An additional embodiment of the present invention is a method for expanding a population of hematopoietic stem cells (HSCs). This method comprises culturing a population of HSCs under conditions sufficient to result in an expansion of the HSC population by at least 2-fold, wherein the expanded population of HSCs is suitable for transplantation into a mammal in need thereof. In this embodiment the “conditions sufficient to result in an expansion of the HSC population” are those conditions that can result in expansion of HSCs in culture by, e.g., at least 2-fold, such as, e.g., by at least 2.5-fold, at least 3-fold, at least 3.5 fold, at least 4-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more. “Suitable for transplantation into a mammal” means that the number and quality of HSCs is sufficient to support greater than 1% engraftment in a mammalian recipient, such as, e.g., a primate recipient, including an human recipient, in need thereof.

An additional embodiment of the present invention is a method for expanding a population of mesenchymal stem cells (MSCs). This method comprises culturing a population of MSCs under conditions sufficient to result in an expansion of the MSC population by at least 2-fold, wherein the expanded population of MSCs is suitable for transplantation into a mammal in need thereof. In this embodiment the “conditions sufficient to result in an expansion of the MSC population” are those conditions that can result in expansion of MSCs in culture by, e.g., at least 2-fold, such as, e.g., by at least 2.5-fold, at least 3-fold, at least 3.5 fold, at least 4-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more. “Suitable for transplantation into a mammal” means that the number and quality of MSCs is sufficient to support greater than 1% engraftment in a mammalian recipient, such as, e.g., a primate recipient, including an human recipient, in need thereof.

An additional embodiment of the present invention is a method for expanding a population of chimeric antigen receptor (CAR) T-cells. This method comprises culturing a population of CAR T-cells under conditions sufficient to result in an expansion of the CAR T-cell population by at least 2-fold, wherein the expanded population of CAR T-cells is suitable for transplantation into a mammal in need thereof. In this embodiment the “conditions sufficient to result in an expansion of the CAR T-cell population” are those conditions that can result in expansion of CAR T-cells in culture by, e.g., at least 2-fold, such as, e.g., by at least 2.5-fold, at least 3-fold, at least 3.5 fold, at least 4-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more. “Suitable for transplantation into a mammal” means that the number and quality of CAR T-cells is sufficient to support greater than 1% engraftment in a mammalian recipient, such as, e.g., a primate recipient, including an human recipient, in need thereof.

Yet another embodiment of the present invention is a method for treating a subject in need of a bone marrow transplant, a peripheral blood transplant, or a cord blood transplant comprising administering to the subject a population of HSCs obtained by a method disclosed herein, particularly the methods for expanding a population of hematopoietic stem cells (HSCs). The subject may be a mammal, such as a human.

Yet another embodiment of the present invention is a method for treating a subject in need of a bone marrow transplant, a peripheral blood transplant, or a cord blood transplant comprising administering to the subject a population of MSCs obtained by a method disclosed herein, particularly the methods for expanding a population of mesenchymal stem cells (MSCs). The subject may be a mammal, such as a human.

Yet another embodiment of the present invention is a method for treating a subject suffering from cancer and/or a blood disorder, comprising administering to the subject a population of CAR T-cells obtained by a method disclosed herein, particularly the methods for expanding a population of CAR T-cells. The subject may be a mammal, such as a human.

A further embodiment of the present invention is a method for expanding a population of hematopoietic stem cells (HSCs). The method comprises (a) obtaining from a mammal a tissue sample comprising an HSC population; (b) expanding, in vitro, the HSC population from the sample, wherein (i) the HSC population expands by at least 2-fold; and (ii) the expanded HSC population has at least a 5-fold increase in total colony forming units. In one aspect of this embodiment, the HSC population expands by at least 4-fold, such as e.g., at least 5-fold, including at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more. In another aspect of this embodiment, the mammal is a primate, including a human. Preferably, the human requires a peripheral blood transplant, a cord blood transplant, or a bone marrow transplant. In a further aspect, the tissue sample is obtained from any appropriate tissue, such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow.

A further embodiment of the present invention is a method for expanding a population of mesenchylam stem cells (MSCs). The method comprises (a) obtaining from a mammal a tissue sample comprising a MSC population; (b) expanding, in vitro, the MSC population from the sample, wherein (i) the MSC population expands by at least 2-fold. In one aspect of this embodiment, the MSC population expands by at least 2.5-fold, such as at least 3-fold, at least 3.5 fold, at least 4-fold, such as e.g., at least 5-fold, including at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more. In another aspect of this embodiment, the mammal is a primate, including a human. Preferably, the human requires a peripheral blood transplant, a cord blood transplant, or a bone marrow transplant. In a further aspect, the tissue sample is obtained from any appropriate tissue, such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow.

A further embodiment of the present invention is a method for expanding a population of chimeric antigen receptor (CAR) T-cells. The method comprises (a) obtaining from a mammal a tissue sample comprising a T-cell population; (b) modifying the T-cell population with chimeric antigen receptors to provide CAR T-cell population; (c) expanding, in vitro, the CAR T-cell population from the sample, wherein (i) the CAR T-cell population expands by at least 2-fold. In one aspect of this embodiment, the CAR T-cell population expands by at least 2.5-fold, such as at least 3-fold, at least 3.5 fold, at least 4-fold, such as e.g., at least 5-fold, including at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more. In another aspect of this embodiment, the mammal is a primate, including a human. Preferably, the human is suffering from a cancer and/or a blood disorder. In a further aspect, the tissue sample is obtained from any appropriate tissue, such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow.

An additional embodiment of the present invention is a method for reconstituting an hematopoietic stem cell lineage in a recipient in need thereof. The method comprises (a) obtaining from a mammal a tissue sample comprising an HSC population; (b) expanding, in vitro, the HSC population from the sample, wherein: (i) the HSC population expands by at least 2-fold, such as for example, by at least 4-fold, including at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more, and (ii) the expanded HSC population has at least at 5-fold increase in total colony forming units; and (c) transplanting the expanded HSC population into a subject in need thereof, such as a mammal, including a primate or human. In this embodiment, the human recipient requires a peripheral blood transplant, a cord blood transplant or a bone marrow transplant. Thus, in a further aspect, the tissue sample is obtained from any appropriate tissue such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow. The sample may be obtained from an autologous or allogeneic source. Preferably, the sample is obtained from an autologous source.

An additional embodiment of the present invention is a method for reconstituting a mesenchymal stem cell (MSC) lineage in a recipient in need thereof. The method comprises (a) obtaining from a mammal a tissue sample comprising a MSC population; (b) expanding, in vitro, the MSC population from the sample, wherein: (i) the MSC population expands by at least 2-fold, such as for example, by at least 4-fold, including at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more; and (c) transplanting the expanded MSC population into a subject in need thereof, such as a mammal, including a primate or human. In this embodiment, the human recipient requires a peripheral blood transplant, a cord blood transplant or a bone marrow transplant. Thus, in a further aspect, the tissue sample is obtained from any appropriate tissue such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow. The sample may be obtained from an autologous or allogeneic source. Preferably, the sample is obtained from an autologous source.

An additional embodiment of the present invention is a method for treating a subject suffering from cancer and/or a blood disorder. The method comprises (a) obtaining from a mammal a tissue sample comprising a T-cell population; (b) modifying the T-cell population with a chimeric antigen receptor (CAR) to form a CAR T-cell population; (c) expanding, in vitro, the CAR T-cell population from the sample, wherein: (i) the CAR T-cell population expands by at least 2-fold, such as for example, by at least 4-fold, including at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more; and (c) transplanting the expanded CAR-T cell population into a subject in need thereof, such as a mammal, including a primate or human. In this embodiment, the human recipient may be suffering from, e.g., leukemia and/or lymphoma. Thus, in a further aspect, the tissue sample is obtained from any appropriate tissue such as, e.g., a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow. The sample may be obtained from an autologous or allogeneic source. Preferably, the sample is obtained from an autologous source.

In aspects of the present invention, it is preferred that the expanded HSC population comprises HSCs that have a phenotype selected from the group consisting of CD34⁻ or CD34⁺/CD38^−/low/Thy-1⁺/CD90⁺/Kit^−/lo/Lin⁻/CD133⁺VEGFR2⁺, which are markers for the most primitive and long-term undifferentiated human HSCs; CD150⁺/CD48⁻/CD244⁻, which is a marker for human HSCs and their progenitors; and/or CD150⁻/CD48⁻/CD244⁺ and CD150⁻/CD48⁺/CD244⁺, which are markers for non-self-renewing multipotent hematopoietic progenitors, and combinations thereof. (See, e.g., Mimeault, M., et al., Stem Cells: A Revolution in Therapeutics—Recent Advances in Stem Cell Biology and Their Therapeutic Applications in Regenerative Medicine and Cancer Therapies. Clin Pharmacol Ther., 82(3):252-64 (2007)). In aspects of the present invention, it is preferred that the expanded MSC population and/or MSC population subject to expansion, comprises MSCs that have a phenotype selected from the group consisting of N-cadherin+ and CD105+, and combinations thereof. That is, the MSC population of any of the embodiments described herein can comprise at least one MSC selected from the group consisting of N-cadherin+ MSCs and CD105+ MSCs. In one embodiment, the MSC population comprises N-cadherin+ MSCs.

The exact proportions of HSCs and/or MSCs having these markers in the population is not critical, so long as the expanded HSC and/or MSC population as a whole is sufficient to result in at least 1% engraftment in a recipient.

In another embodiment, the invention is a method for expanding a hematopoietic stem cell population in a mammal in need of such expansion. This method comprises administering to the mammal a therapeutically effective amount of a modulator of a N⁶-methyladenosine (m⁶A) mRNA modification pathway, for a period of time sufficient to expand the HSC population by at least 2-fold with HSCs that possess the ability to reconstitute an hematopoietic lineage in the mammal.

In another embodiment, the invention is a method for expanding a mesenchymal stem cell population in a mammal in need of such expansion. This method comprises administering to the mammal a therapeutically effective amount of a modulator of a N⁶-methyladenosine (m⁶A) mRNA modification pathway, for a period of time sufficient to expand the MSC population by at least 2-fold with MSCs that possess the ability to reconstitute a mesenchymal lineage in the mammal.

In another embodiment, the invention is a method for expanding a chimeric antigen receptor (CAR) T-cell population in a mammal in need of such expansion. This method comprises administering to the mammal a therapeutically effective amount of a modulator of a N⁶-methyladenosine (m⁶A) mRNA modification pathway, for a period of time sufficient to expand the CAR T-cell population by at least 2-fold with CAR T-cells that possess the ability to treat cancer and/or a blood disorder in the mammal.

In these methods, the modulators may be as previously disclosed herein, and/or modulation may be performed by any method disclosed herein. For example, the modulator may comprise a system for introducing a mutation into the HSC and/or MSC and/or CAR T-cell population that deletes, replaces or reduces expression of a gene expressing a N⁶-methyladenosine (m⁶A) mRNA modification reader, such as Ythdf2. As yet another example, the modulator may comprise an inhibitor of a N6-methyladenosine (m6A) mRNA modification reader, such as an inhibitor of Ythdf2. The mammal in need of expansion may be a human.

In another embodiment, the invention includes a method of isolating mesenchymal stem cells (MSCs) from a biological sample, the method comprising contacting the biological sample having a population of MSCs with one or more N-cadherin antibodies. For example, according to certain aspects, the biological sample comprises a tissue selected from the group consisting of peripheral blood, cord blood and bone marrow. Furthermore, in certain embodiments, the method of isolating the MSCs can further comprise one or more steps of expanding the population of MSCs from the biological sample, by modulating a N⁶-Methyladenosine (m⁶A) mRNA modification pathway in the population of MSCs, to expand the number of mesenchymal stem cells, such as by any of the methods described herein. In one embodiment, the population of MSCs is expanded after isolating from the biological sample. In another embodiment, the population of MSCs in the biological sample is expanded before isolation of the MSCs from the biological sample. In one embodiment, the MSC population is expanded to a sufficient quantity while maintaining a multilineage differentiation potential in the MSC population, which is sufficient for subsequent transplantation into a subject in need thereof, such that the MSCs isolated by the method can be used for such transplantation. For example, the isolated MSCs may be transplanted into a human subject. According to yet another embodiment, the MSCs may be further isolated from the biological sample by contacting with CD105 antibodies, either in addition to or as an alternative to the N-cadherin antibodies. In yet a further embodiment, the N-cadherin antibodies may be used to identify the MSCs in the biological sample, by contacting the biological sample with the N-cadherin antibodies and detecting those cells that bind to the N-cadherin antibodies.

In one embodiment, the method of isolating the MSCs comprises expanding the MSC population, by modulating the m⁶A mRNA modification pathway by introducing a mutation into the stem cells that results in modulation of a molecule in the m⁶A mRNA modification pathway or contacting the stem cell with a modulator of a molecule in the m⁶A mRNA modification pathway selected from the group consisting of a small molecule, a biologic, an antisense RNA, a small interfering RNA (siRNA), and combinations thereof, such as by any of the modulation methods described herein. For example, in one embodiment, modulating the m⁶A mRNA modification pathway comprises introducing a mutation into the stem cells to delete, replace, or reduce expression of a gene that expresses a molecule in the m⁶A mRNA modification pathway. In one embodiment, the mutation deletes, replaces or reduces expression of a gene that expresses a molecule selected from the group consisting of a m⁶A mRNA modification reader, a m⁶A mRNA modification writer, and a m⁶A mRNA eraser. In yet another embodiment, the mutation deletes, replaces or reduces expression of a gene that expresses a m⁶A mRNA modification reader. In one embodiment, the mutation deletes, replaces or reduces expression of a gene that expresses a m⁶A mRNA modification reader selected from the group consisting of Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, and elF3. In another embodiment, the mutation deletes, replaces or reduces expression of a gene that expresses Ythdf2. In one embodiment, the mutation deletes, replaces or reduces expression of a gene that expresses a m⁶A mRNA modification eraser. In one embodiment, the mutation deletes, replaces or reduces expression of a gene that expresses a m⁶A mRNA modification eraser selected from the group consisting of FTO and ALKBH5. In another embodiment, the mutation deletes, replaces or reduces expression of a gene that expresses a m⁶A mRNA modification writer. In one embodiment, the mutation deletes, replaces or reduces expression of a gene that expresses a m⁶A mRNA modification writer selected from the group consisting of METTL3, METTL14, WTAP and KIAA1429.

In another embodiment, the method of isolating MSCs comprises expanding the population of MSCS by modulating the m⁶A mRNA modification pathway, by exposing the stem cells to a Mx1-Cre targeting system that inactivates or deletes at least a portion of a gene that expresses a molecule in the m⁶A mRNA modification pathway. In one embodiment, modulating the m⁶A mRNA modification pathway comprises introducing a mutation that incorporates shRNA into the stem cells to reduce expression of a gene that expresses a molecule in the m⁶A mRNA modification pathway. For example, in one embodiment, the shRNA is introduced by exposing the stem cells to a lentivirus to deliver the shRNA. According to yet another embodiment, modulating the m⁶A mRNA modification pathway comprises down-regulating a m⁶A mRNA modification reader. In one embodiment, the m⁶A mRNA modification reader is selected from the group consisting of Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, and elF3. For example, in one embodiment, the m⁶A mRNA modification reader comprises Ythdf2.

In one embodiment, the method of isolating MSCs further comprise expanding the population of MSCs by down-regulating the m⁶A mRNA modification reader, by contacting the stem cells with an inhibitor of the m⁶A mRNA modification reader that is any of those described herein, such as any of those selected from the group consisting of: (inhibitors of HNRNPC) hsa-let-7e-5p (MIRT051596), hsa-mir-455-3p (MIRT037890), hsa-mir-30c-5p (MIRT047904), hsa-mir-186-5p (MIRT045150), hsa-mir-744-5p (MIRT037494), hsa-mir-18a-3p (MIRT040851), hsa-mir-484 (MIRT042196), hsa-mir-505-5p (MIRT037959), hsa-mir-615-3p (MIRT039991), hsa-mir-342-3p (MIRT043694), hsa-miR-3607-3p, hsa-miR-30d, hsa-miR-3916, hsa-miR-3162-5p, hsa-miR-1273d, hsa-miR-3161, hsa-miR-30a, hsa-miR-629, hsa-miR-208b, hsa-miR-489, hsa-miR-3148, hsa-miR-2113, hsa-miR-877, hsa-miR-455-5p, hsa-miR-186, hsa-miR-548o, hsa-miR-3139, hsa-miR-320a, hsa-miR-4311, hsa-miR-555, hsa-miR-3605-5p, hsa-miR-515-5p, hsa-miR-144, hsa-miR-499-5p, hsa-miR-1323, hsa-miR-548x, hsa-miR-299-5p, hsa-miR-653, hsa-miR-576-5p, hsa-miR-548p, hsa-miR-586, hsa-miR-888, hsa-miR-3647-3p, hsa-miR-484, hsa-miR-320b, hsa-miR-620, hsa-miR-30b, hsa-miR-548q, hsa-miR-29b-1, hsa-miR-570, hsa-miR-183, hsa-miR-1276, hsa-miR-208a, hsa-miR-186, hsa-miR-28-5p, hsa-miR-330-3p, hsa-miR-548am, hsa-miR-320d, hsa-miR-3175, hsa-miR-3155, hsa-miR-548aa, hsa-miR-519e, hsa-miR-1270, hsa-miR-513b, hsa-miR-599, hsa-miR-518f, hsa-miR-4301, hsa-miR-30c, hsa-miR-3135, hsa-miR-4286, hsa-miR-202, hsa-miR-4263, hsa-miR-4299, hsa-miR-606, hsa-miR-3133, hsa-miR-583, hsa-miR-3125, hsa-miR-501-5p, hsa-miR-7-1, hsa-miR-514b-3p, hsa-miR-3155b, hsa-miR-548d-3p, hsa-miR-224, hsa-miR-7-2, hsa-miR-708, hsa-miR-3199, hsa-miR-514, hsa-miR-30e; (inhibitors of HNRNPA2B1) hsa-mir-92a-3p (MIRT049721), hsa-mir-30c-5p (MIRT048009), hsa-mir-191-5p (MIRT045809), hsa-let-7f-5p (MIRT051404), hsa-mir-27b-3p (MIRT046213), hsa-mir-877-3p (MIRT037116), hsa-mir-615-3p (MIRT040278), hsa-mir-1260b (MIRT052680), hsa-mir-103a-3p (MIRT027027), hsa-mir-16-5p (MIRT031508), hsa-mir-1296-5p (MIRT036075), hsa-mir-197-3p (MIRT048098), hsa-miR-548j, hsa-miR-3678-3p, hsa-miR-607, hsa-miR-188-5p, hsa-miR-15a, hsa-miR-3653, hsa-miR-371-5p, hsa-miR-550a, hsa-miR-3622b-3p, hsa-miR-548a-5p, hsa-miR-3170, hsa-miR-3148, hsa-miR-556-3p, hsa-miR-490-3p, hsa-miR-559, hsa-miR-200c, hsa-miR-130a, hsa-miR-548y, hsa-miR-548o, hsa-miR-23c, hsa-miR-491-3p, hsa-miR-335, hsa-miR-3667-3p, hsa-miR-466, hsa-miR-23b, hsa-miR-4310, hsa-miR-127-5p, hsa-miR-548b-5p, hsa-miR-616, hsa-miR-16, hsa-miR-338-3p, hsa-miR-3200-5p, hsa-miR-362-3p, hsa-miR-448, hsa-miR-1306, hsa-miR-944, hsa-miR-3684, hsa-miR-373, hsa-miR-103a, hsa-miR-380, hsa-miR-499-5p, hsa-miR-1323, hsa-miR-323-5p, hsa-miR-3674, hsa-miR-1252, hsa-miR-33b, hsa-miR-580, hsa-miR-548c-3p, hsa-miR-103a-2, hsa-miR-548w, hsa-miR-600, hsa-miR-634, hsa-miR-586, hsa-miR-497, hsa-miR-720, hsa-miR-654-3p, hsa-miR-524-5p, hsa-miR-543, hsa-miR-548q, hsa-let-7f-2, hsa-miR-330-5p, hsa-miR-500a, hsa-miR-548I, hsa-miR-570, hsa-miR-374a, hsa-miR-1184, hsa-miR-649, hsa-miR-424, hsa-miR-3658, hsa-miR-186, hsa-miR-326, hsa-miR-548d-5p, hsa-miR-23a, hsa-miR-15b, hsa-miR-190, hsa-miR-203, hsa-miR-548h, hsa-miR-3136-5p, hsa-miR-618, hsa-miR-551b, hsa-miR-211, hsa-miR-1305, hsa-miR-513b, hsa-miR-96, hsa-miR-2117, hsa-miR-548n, hsa-miR-3910, hsa-miR-217, hsa-miR-892b, hsa-miR-502-5p, hsa-miR-548i, hsa-miR-520d-5p, hsa-miR-4299, hsa-miR-1285, hsa-miR-3133, hsa-miR-483-3p; (inhibitors of Ythdf1) hsa-miR-548g, hsa-miR-204, hsa-miR-3143, hsa-miR-521, hsa-miR-195, hsa-miR-3182, hsa-miR-3941, hsa-miR-34c-3p, hsa-miR-767-3p, hsa-miR-563, hsa-miR-548c-5p, hsa-miR-1911, hsa-miR-26b, hsa-miR-190b, hsa-miR-33a, hsa-miR-329, hsa-miR-221, hsa-miR-612, hsa-miR-3185, hsa-miR-3156-5p, hsa-miR-107, hsa-miR-664, hsa-miR-3657; (inhibitors of Ythdf2) hsa-mir-615-3p (MIRT040054), hsa-mir-106b-5p (MIRT044257), hsa-mir-1 (MIRT023842), miR-145, hsa-miR-3607-3p, hsa-miR-200a, hsa-miR-301a, hsa-miR-519a, hsa-miR-141, hsa-miR-130b, hsa-miR-181b, hsa-miR-301b, hsa-miR-3117-3p, hsa-miR-1236, hsa-miR-181a, hsa-miR-519c-3p, hsa-miR-551b, hsa-miR-519e, hsa-miR-519b-3p, hsa-miR-19b, hsa-miR-1303, hsa-miR-608, hsa-miR-145, hsa-miR-130a, hsa-miR-181c, hsa-miR-323b-3p, hsa-miR-421, hsa-miR-515-5p, hsa-miR-3666, hsa-miR-181d, hsa-miR-146a, hsa-miR-4295, hsa-miR-454, hsa-miR-3919, hsa-miR-19a, hsa-miR-543, hsa-miR-4262; (inhibitors of Ythdf3) hsa-miR-582-3p, hsa-miR-579, hsa-miR-520e, hsa-miR-520f, hsa-miR-3152-3p, hsa-miR-106a, hsa-miR-30d, hsa-miR-30a, hsa-miR-93, hsa-miR-508-5p, hsa-miR-29a, hsa-miR-3148, hsa-miR-490-5p, hsa-miR-520b, hsa-miR-20a, hsa-miR-409-3p, hsa-miR-4255, hsa-let-7i, hsa-miR-373, hsa-let-7e, hsa-miR-520c-3p, hsa-miR-3920, hsa-miR-127-5p, hsa-miR-380, hsa-miR-616, hsa-miR-4277, hsa-miR-448, hsa-miR-16-2, hsa-let-7c, hsa-miR-340, hsa-miR-373, hsa-miR-520a-3p, hsa-miR-144, hsa-miR-1265, hsa-miR-548x, hsa-miR-362-5p, hsa-miR-33b, hsa-miR-26b, hsa-miR-17, hsa-miR-569, hsa-miR-3618, hsa-miR-576-5p, hsa-miR-922, hsa-miR-302a, hsa-miR-106b, hsa-miR-888, hsa-miR-484, hsa-let-7b, hsa-miR-582-5p, hsa-let-7f, hsa-miR-30b, hsa-miR-524-5p, hsa-miR-302d, hsa-let-7d, hsa-miR-513a-5p, hsa-miR-500a, hsa-miR-570, hsa-miR-548I, hsa-miR-105, hsa-miR-374c, hsa-let-7g hsa-miR-372, hsa-miR-3658, hsa-let-7a, hsa-miR-3908, hsa-miR-302b, hsa-miR-526b, hsa-miR-190, hsa-miR-181 b, hsa-miR-433, hsa-miR-98, hsa-miR-3606, hsa-miR-595, hsa-miR-548am, hsa-miR-187, hsa-miR-561, hsa-miR-181a, hsa-miR-3155, hsa-miR-655, hsa-miR-302c, hsa-miR-195, hsa-miR-26a, hsa-miR-590-3p, hsa-miR-30c, hsa-miR-502-5p, hsa-miR-495, hsa-miR-137, hsa-miR-181c, hsa-miR-520d-5p, hsa-miR-3942-5p, hsa-miR-202, hsa-miR-302e, hsa-miR-513c, hsa-miR-885-5p, hsa-miR-520a-5p, hsa-miR-583, hsa-miR-1297, hsa-miR-7-1, hsa-miR-520d-3p, hsa-miR-3155b, hsa-miR-3182, hsa-miR-519d, hsa-miR-550a, hsa-miR-7-2, hsa-miR-181d, hsa-miR-190b, hsa-miR-1912, hsa-miR-151-3p, hsa-miR-33a, hsa-miR-525-5p, hsa-miR-20b, hsa-miR-514b-5p, hsa-miR-30e, hsa-miR-4262, hsa-miR-636; (inhibitor of elF3) hsa-mir-92b-3p (MIRT040734), hsa-mir-16-5p (MIRT031705), hsa-mir-18a-3p (MIRT040974), hsa-mir-155-5p (MIRT020771), hsa-mir-484 (MIRT042324), hsa-let-7c-5p (MIRT051776), hsa-miR-3910, hsa-miR-148b, hsa-miR-136, hsa-miR-15a, hsa-miR-488, hsa-miR-500a, hsa-miR-1297, hsa-miR-3159, hsa-miR-374c, hsa-miR-424, hsa-miR-7-1, hsa-miR-186, hsa-miR-195, hsa-miR-15b, hsa-miR-26b, hsa-miR-505, hsa-miR-1206, hsa-miR-653, hsa-miR-1283, hsa-miR-7-2, hsa-miR-196a, hsa-miR-497, hsa-miR-33a, hsa-miR-655, hsa-miR-26a hsa-miR-16, hsa-mir-151a-3p (MIRT043600), hsa-mir-92a-3p (MIRT049064), hsa-mir-615-3p (MIRT039779), hsa-mir-877-3p (MIRT036964), hsa-mir-222-3p (MIRT046746), hsa-mir-423-3p (MIRT042468), hsa-mir-324-3p (MIRT042887), hsa-mir-124-3p (MIRT022932), hsa-miR-3140-3p, hsa-miR-124, hsa-miR-198, hsa-miR-525-5p, hsa-miR-506, hsa-miR-520a-5p, hsa-miR-196a* hsa-miR-3117-3p, hsa-mir-342-5p (MIRT038210), hsa-mir-378a-5p (MIRT043981), hsa-mir-615-3p (MIRT040086), hsa-let-7b-5p (MIRT052211), hsa-mir-455-3p (MIRT037879), hsa-miR-4267, hsa-miR-590-3p, hsa-mir-106b-5p (MIRT044355), hsa-mir-320a (MIRT044466), hsa-mir-16-5p (MIRT032018), hsa-mir-155-5p (MIRT021009), hsa-miR-4302, hsa-mir-191-5p (MIRT045793), hsa-mir-1303 (MIRT035890), hsa-mir-193b-3p (MIRT016316), hsa-mir-222-3p (MIRT046640), hsa-mir-532-3p (MIRT037924), hsa-mir-18a-3p (MIRT040929), hsa-mir-92a-3p (MIRT049001), hsa-miR-582-3p, hsa-miR-4265, hsa-miR-218-2, hsa-miR-1271, hsa-miR-340, hsa-miR-221, hsa-miR-20b, hsa-miR-508-3p, hsa-miR-141, hsa-miR-4325, hsa-miR-889, hsa-miR-29a, hsa-miR-129-3p, hsa-miR-129, hsa-miR-96, hsa-miR-3163, hsa-miR-187, hsa-miR-196a, hsa-miR-222, hsa-miR-1179, hsa-miR-182, hsa-miR-9* hsa-miR-32, hsa-miR-143, hsa-miR-4296: (inhibitors of YTHDC1) hsa-mir-20a-3p (MIRT038967), hsa-mir-103a-3p (MIRT027037), hsa-mir-1 (MIRT023492), hsa-mir-19b-3p (MIRT031105), hsa-mir-100-5p (MIRT048400), hsa-mir-93-5p (MIRT027989), hsa-mir-16-5p (MIRT031379), hsa-let-7b-5p (MIRT052150), hsa-miR-520f, hsa-miR-300, hsa-miR-15a, hsa-miR-200a, hsa-miR-605, hsa-miR-30d, hsa-miR-30a, hsa-miR-3613-3p, hsa-miR-509-3-5p, hsa-miR-34c-5p, hsa-miR-324-3p, hsa-miR-1248, hsa-miR-152, hsa-miR-548t, hsa-miR-4310, hsa-miR-145, hsa-miR-516a-3p, hsa-miR-16, hsa-miR-3668, hsa-miR-4277, hsa-miR-448, hsa-miR-16-2, hsa-miR-148b, hsa-miR-509-5p, hsa-miR-103a, hsa-miR-1265, hsa-miR-2115, hsa-miR-548c-3p, hsa-miR-148a, hsa-miR-548p, hsa-miR-513a-3p, hsa-miR-497, hsa-miR-3647-3p, hsa-miR-382, hsa-miR-30b, hsa-miR-543, hsa-let-7f-2, hsa-miR-1269, hsa-miR-3164, hsa-miR-503, hsa-miR-500a, hsa-miR-449a, hsa-miR-141, hsa-miR-424, hsa-miR-3908, hsa-miR-889, hsa-miR-2116, hsa-miR-330-3p, hsa-miR-15b, hsa-miR-181 b, hsa-miR-187, hsa-miR-1237, hsa-miR-449b, hsa-miR-101, hsa-miR-381, hsa-miR-618, hsa-miR-222, hsa-miR-181a, hsa-miR-432, hsa-miR-96, hsa-miR-19b, hsa-miR-195, hsa-miR-548n, hsa-miR-485-5p, hsa-miR-217, hsa-miR-30c, hsa-miR-495, hsa-miR-137, hsa-miR-1288, hsa-miR-181c, hsa-miR-3942-5p, hsa-miR-548v, hsa-miR-487a, hsa-miR-221, hsa-miR-891b, hsa-miR-205, hsa-miR-195, hsa-miR-4271, hsa-miR-3611, hsa-miR-516b, hsa-miR-181d, hsa-miR-154, hsa-miR-646, hsa-miR-153, hsa-miR-34a, hsa-miR-19a, hsa-miR-107, hsa-miR-30e and hsa-miR-4262.

According to one embodiment, the invention comprises an isolated population of mesenchymal stem cells made by any of the processes described herein. For example, the invention in certain embodiments can comprise an expanded, isolated population of mesenchymal stem cells made by any of the expansion and/or isolation processes described herein.

In one embodiment, a kit for isolating a mesenchymal stem cell (MSC) population for subsequent transplantation into a subject in need thereof is provided. The kit comprises a system for contacting a biological sample comprising MSCs with one or more N-cadherin antibodies, and instructions for use thereof. In one embodiment, the kit further comprises a system for expanding the population of MSCs by introducing a mutation into the MSC population that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader, and instructions for use thereof. In yet another embodiment, the system for introducing a mutation into the MSC population includes one or more reagents capable of introducing a mutation into the MSC population that results in deletion, replacement or reduced expression of a gene expressing Ythdf2. In yet another embodiment, the system for introducing a mutation into the MSC population comprises a Mx1-Cre targeting system that inactivates or deletes at least a portion of a gene that expresses a m⁶A mRNA modification reader. In one embodiment, the system for introducing a mutation into the MSC population comprises reagents for delivering a lentivirus that incorporates shRNA into the MSC population to reduce expression of a gene that expresses a m⁶A mRNA modification reader.

According to another embodiment of the invention, a method for administering a mesenchymal stem cell (MSC) to a subject in need thereof is provided. The method comprises isolating MSCs from a biological sample comprising a population of MSCs, by contacting the biological sample with one or more N-cadherin antibodies, and administering the isolated MSCs to the subject. Furthermore, in one embodiment, the method further comprises introducing, into the biological sample containing the MSC population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader, and culturing the biological sample in a suitable culture media for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient to transplant into the subject. In one embodiment, wherein the mutation results in deletion a gene expressing Ythdf2. In one embodiment, the mutation results in incorporation of shRNA into the HSC population that reduces expression of a gene expressing Ythdf2. According to yet another aspect, the MSCs are obtained from a biological sample comprising a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow.

According to yet another embodiment, a method for reconstituting bone marrow in a subject in need thereof is provided. The method comprises isolating mesenchymal stem cells (MSCs) from a biological sample comprising a population of MSCs, by contacting the biological sample with one or more N-cadherin antibodies, and administering the isolated MSCs to the subject. Furthermore, according to one aspect, the method further comprises introducing, into the biological sample containing the MSC population, a mutation that results in deletion, replacement or reduced expression of a gene expressing a m⁶A mRNA modification reader, and culturing the sample in a suitable culture media for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient to transplant into the subject. In one embodiment, the MSCs are obtained from a biological sample comprising a tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow.

According to yet another embodiment, a method for treating a subject in need of a transplant, selected from the group consisting of a bone marrow transplant, a peripheral blood transplant and an umbilical cord blood transplant comprising administering to the subject a population of isolated MSCs obtained by any of the methods described herein. According to embodiment, the sample is from an autologous or allogeneic source. According to yet another embodiment, the sample is from an autologous source.

In the present invention, a “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. In terms of treatment of a mammal, a “therapeutically effective amount” of a modulator and/or expanded cells is an amount sufficient to treat, manage, palliate, ameliorate, or stabilize a condition, such as a bone marrow disease, in the mammal. A therapeutically effective amount can be administered in one or more doses.

The therapeutically effective amount is generally determined by a physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form of the drug being administered.

Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of animal, and like factors well known in the arts of medicine and veterinary medicine. In general, a suitable dose of a modulator according to the invention will be that amount of the modulator, which is the lowest dose effective to produce the desired effect. The effective dose of a modulator maybe administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.

A modulator may be administered in any desired and effective manner: as pharmaceutical compositions for oral ingestion, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, a modulator of the present invention may be administered in conjunction with other treatments. A modulator of the present invention maybe encapsulated or otherwise protected against gastric or other secretions, if desired.

While it is possible for a modulator of the invention to be administered alone, it is preferable to administer the modulator as a pharmaceutical formulation (composition). Such pharmaceutical formulations typically comprise one or more modulators as an active ingredient in admixture with one or more pharmaceutically-acceptable carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the modulator of the present invention may be formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).

Pharmaceutically acceptable carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and triglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable carrier used in a pharmaceutical composition comprising a modulator of the invention must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.

Pharmaceutical compositions comprising a modulator of the invention may, optionally, contain additional ingredients and/or materials commonly used in pharmaceutical compositions. These ingredients and materials are well known in the art and include (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monosterate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (11) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface-active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monosterate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.

Pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.

Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be prepared by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type maybe employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form.

Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.

Pharmaceutical compositions for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Pharmaceutical compositions which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active compound may be mixed under sterile conditions with a suitable pharmaceutically-acceptable carrier. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.

Pharmaceutical compositions suitable for parenteral administrations comprise one or more modulator in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.

In some cases, in order to prolong the effect of a drug containing a modulator of the present invention, it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.

The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug may be accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

The following examples are provided to further illustrate the methods and compositions of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.

EXAMPLES

The examples herein examine Ythdf2, a well-recognized m⁶A reader promoting targeted mRNA decay (Wang et al. Blood, 505: 17-120, 2014), at least partly with the purpose of investigating its role in the context of HSC maintenance. Without being limited to any particular theory, it is believed that manipulation of Ythdf2 might potentially influence the life span of a great number m⁶A-marked mRNAs, thus impacting adult HSC self-renewal versus differentiation and facilitating HSC expansion. As shown in the examples below, Ythdf2 depletion specifically expands mouse and human HSCs without skewing lineage fate. Accordingly, it is believed that Ythdf2 may play an essential role in regulating HSC self-renewal, and provide a novel approach to enhance hUCB HSCs ex vivo expansion, including in clinical applications.

The examples also show functional definition of the drug-resistant rHSC population, and a finding that rHSCs are maintained in the endosteal niche largely by N-cad⁺ cells during homeostasis and under chemotherapeutic stress. It is further shown that N-cad⁺ cells in endosteal zone are mesenchymal stem cells and contribute to rHSC maintenance.

Example A

The following protocols were used in the “A” Examples below.

Mice. Ythdf2 conditional KO mice were generated by Chuan He and Bin Shen group. Mice were housed in the animal facility at Stowers Institute for Medical Research (SIMR) and handled according to Institute and NIH guidelines. All procedures were approved by the IACUC of SIMR.

Flow cytometry and HSPC sorting. Mouse HSPCs, progenitors, and lineage cells were harvested from BM (femur and tibia) and spleen. Red blood cells were lysed using a 0.16 M ammonium chloride solution, and the cells were filtered with 70 μm strainers to generate single cell suspensions. For mouse HSC identification, cells were stained with antibodies against Sca-1 (D7), c-Kit (2B8), CD34 (RAM34), Flk2 (A2F10), CD48 (HM48-1), CD150 (TC15-12F12.2), together with lineage cocktail including CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), Mac-1 (M1/70), Gr1 (RB6-8C5), CD45R (B220, RA3-6B2), IgM (II-41) and Ter119 (TER-119). For progenitors and lineage cells, cells were stained with antibodies as previously described (Qian, P. et al. Cell Stem Cell, 18:214-228, 2016, doi:10.1016/j.stem.2015.11.001). 7-aminoactinomycin D (7-AAD) (A1310, Life technologies) was used to exclude dead cells. Human cord blood samples were acquired from the St. Louis Cord Blood Bank. Mononuclear cells were isolated with Lymphoprep™ (StemCell technologies), followed by isolation of human CD34⁺ cord blood cells by human CD34 MicroBead Kit UltraPure (Miltenyi Biotec). To quantify human HSPCs, cells were stained with antibodies against CD34 (581), CD38 (HIT2), CD45RA (HI100), CD90 (5E10), CD49f (GoH3), EPCR/CD201 (RCR-401). Cell sorting and analyses were performed on MoFlo (Dako), InFlux Cell Sorter (BD Biosciences), and/or MACSQuant (Miltenyi Biotec). Data analysis was performed using FlowJo software.

Homing assay. In vivo homing assays were performed as previously described (He et al. Methods in Molecular Biology, 1185: 279-284, 2014). Basically, whole bone marrow (WBM) cells from CD45.2 mice were labelled with 5 μM 5-(and −6)-carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (Molecular Probes) at 37° C. for 10 mins, washed three times, and 1×10⁶WBM were transplanted into lethally irradiated ptprc mice. After 18 hours, femurs and tibias were flushed, and CFDA SE+ cells were determined.

Competitive reconstitution assay. Competitive reconstitution assays were performed by intravenous transplantation of 2×10⁵, 7.5×10⁴or 2.5×10⁴donor-derived WBM cells from wt or Ythdf2 KO mice (CD45.2), together with 2×10⁵rescue cells (CD45.1) into groups of ten lethally irradiated (10 Gy) ptprc recipient mice. For secondary transplantation, primary transplant recipients were sacrificed. BM cells were dissected from femur and tibia, and then transplanted mouse-to-mouse at a dosage of 1×10⁶cells into irradiated secondary recipient mice. Baytril water was given to recipient mice three days before irradiation and continued for another two weeks after irradiation. Primary and secondary CRU frequencies were measured using ELDA software (Hu et al. Journal of Immunological Methods, 347: 70-78, 2009), in which successful engraftment was defined as the presence of a distinct CD45.2⁺ CD45.1⁻ population ≥5% and ≥1% of total hematopoietic cells in peripheral blood, respectively (Purton et al. Cell Stem Cell, 1: 263-270, 2007). Also, the secondary transplantation recipient mice that died before 16 weeks post transplantation were counted for failed engraftment.

Cell cycle and apoptosis assays. Cell cycle analysis was performed with FITC mouse anti-human Ki67 set (BD Pharmingen) according to the manufacturer's instructions. Briefly, 5×10⁶BM cells were isolated and stained with HSC antibodies as described above. Cells were fixed by 4% paraformaldehyde at 4° C. overnight or room temperature (RT) for 1 hour, and then permeabilized with 0.2% triton X-100 on ice for 15 mins. Cells were washed with PBS containing 2% FBS, and then were incubated with Ki-67 antibody at RT for 1 hour in the dark, and SYTOX Red (Invitrogen) at RT for another 5 mins, followed by flow cytometric analysis with InFlux Cell Sorter (BD Biosciences). For apoptosis analysis, Annexin V (Invitrogen) and SYTOX Red staining of 5×10⁶BM cells was performed according to the manufacturer's protocol.

m⁶A RNA-IP-seq. Two replicates of 10⁵LT-HSC, ST-HSC (LSK CD34⁺ FLK2⁻) and MPP (LSK CD34⁺ FLK2⁺) from C57BL/6J mouse were sorted into TRIzol (Invitrogen), and total RNA was isolated according to the manufacturer's instructions. RNA was fragmented to ˜100 nucleotide fragments with Ambion fragmentation reagent (2 mins incubation at 70° C.). The samples were then subjected to Turbo DNase treatment (Ambion), followed by a phenol-chloroform extraction, and resuspension in 85 μl of nuclease-free water, and 5 μl was saved as input. Then, the remaining 80 μl RNA fragments were diluted into IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5). RNA was incubated with 25 μl of protein-G magnetic beads, previously bound to 3 μg of anti-m⁶A plyclonal antibody (Synaptic Systems), for 3 hours at 4° C. in IPP buffer. Beads were washed twice with 200 μl IPP buffer, twice with 200 μl low-salt buffer (50 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5) and twice with 200 μl high-salt buffer (500 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5). Beads were then treated with 300 μl Elution Buffer (5 mM Tris-HCL pH 7.5, 1 mM EDTA pH 8.0, 0.05% SDS, 4.2 μl Proteinase K (20 mg/ml)) for 1.5 hours at 50° C., and RNA was recovered with phenol:chloroform extraction followed by ethanol precipitation. Three human CD34⁺ umbilical cord blood cells were isolated as described above and isolated total RNA with TRIzol. RNA was fragmented to ˜100 nucleotide fragments with Ambion fragmentation reagent (2 mins 50 secs incubation at 70° C.). The samples were then subjected to Turbo DNase treatment (Ambion), followed by a phenol:chloroform extraction, and resuspension in 18 μl of nuclease-free water, and 1 μl was saved as input. m⁶A RNA IP was performed with EpiMark® N6-Methyladenosine Enrichment Kit following manufacturer's instructions.

Following m⁶A preparation of RNA, quality was assessed on Agilent 2100 Bioanalyzer, and 1 ng (mouse) or 10 ng (human) RNA was used to generate RNAseq libraries according to the manufacturer's directions for the SMARTer Stranded Total RNA-Seq Kit—Pico Input Mammalian (Takara Bio Inc) using 16 cycles (mouse) or 13 cycles (human) PCR2 amplification. The method uses random priming and a template switching oligo to generate complimentary DNA, followed by the ligation of barcoded adapters; ribosomal-derived cDNA is then removed through probe-directed enzyme cleavage and subsequent enrichment of un-cleaved fragments.

The protocol was modified to retain lower molecular weight sample fragments by using a 1.2×SPRI bead concentration for PCR1 cleanup. To remove dimerized adapters, libraries underwent 160-600 bp size selection with a Pippin Prep (Sage Science) 2% gel. The resulting libraries were checked for quality and quantity using the Bioanalyzer and Qubit Fluorometer (Life Technologies). Then equal molar libraries were pooled and requantified. For mouse m⁶A-seq, libraries were sequenced as 50 bp single read on the Illumina HiSeq 2500 instrument using HiSeq Control Software 2.2.58. Following sequencing, Illumina Primary Analysis version RTA 1.18.64 and Secondary Analysis version bcl2fastq2 v2.18 were run to demultiplex reads for all libraries and generate FASTQ files. For human m⁶A-seq, libraries were sequenced as 75 bp single read on the Illumina NextSeq instrument using NextSeq Control Software 2.1.2. Following sequencing, Illumina Primary Analysis version NextSeq RTA 2.4.11 and Secondary Analysis version bcl2fastq2 v2.18 were run to demultiplex reads for all libraries and generate FASTQ files.

Plasmid construction and stable cell line generation. Mouse Ythdf2 (mYthdf2) was cloned from commercial cDNA clone (ORIGENE #MC200730) into vector pcDNA5/FRT/Flag plasmid using primers listed: mYthdf2 ORF Clone BamhI F: 5′-CGC GGA TCC TCG GCC AGC AGC CTC TTG GA-3′ (SEQ ID NO: 2) and mYthdf2 ORF Clone NotI R: 5′-ATA AGA ATG CGG CCG CCT ATT TCC CAC GAC CTT GAC GT-3′ (SEQ ID NO: 3). Then Flag-mYthdf2 was subcloned under EF1a promoter in pSicoR-EF1a-IRES-EGFP lentiviral construct (Gibson Assembly®, forward primer: 5′-GTC GAC GGT ACC GCG GGC CCA TGG ATT ACA AGG ATG ACG ACG-3′ (SEQ ID NO: 4) and reverse primer: 5′-GAG GGA GAG GGG CGG ATC CCC TAT TTC CCA CGA CCT TGA CGT-3′ (SEQ ID NO: 5)). Human Ythdf2 (hYthdf2) was cloned from plasmid provided by the Chuan He lab using primers indicated: Forward 5′-CGT TCG AAA TGT CGG CCA GCA GCC TCT-3′ (SEQ ID NO: 6); Reverse 5′-TCC CCC GGG TTA TTT CCC ACG ACC TT-3′ (SEQ ID NO: 7). Then hYthdf2 was cloned into pSicoR-EF1a-IRES-EGFP constructs under EF1a promoter by BstBI and XmaI restriction digestions and ligation. To generate Flag-mYthdf2 HPC7 stable cell line, lentiviruses were generated by transfection of pSicoR-EF1a-Flag-mYthdf2-IRES-EGFP constructs together with the psPAX2 and pMD2.G plasmids at a ratio of 10:7.5:2.5 into 293T cells using calcium phosphate transfection. The virus particles were harvested 48, 72, and 96 hours post transfection, filtered by 0.45 micrometers filter unit (Millipore), and then centrifuged at 18,000 RPM, 4° C. for 2 hours. HPC7 cells were infected with recombinant lentivirus-transducing units in the presence of 4 μg/mL polybrene (Sigma). 48 hours after infection, GFP⁺ cells were sorted and cultured for experiments.

irCLIP-seq and data analysis. For irCLIP-seq, the procedure was modified from the previously reported methods (Zarneger et al. Nature Methods, 13: 489-492, 2016); Simsek et al. Cell, 169: 1051-1065 e1018, 2017). In brief, irCLIP was performed on ˜3×10⁸Flag-Ythdf2 HPC7 cells by UV crosslinking cells at 0.4 J/cm²for 3 times. Whole-cell lysates were generated in lysis buffer (150 mM KCI, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor; 1 ml cell pellet and 2 ml lysis buffer). Pipetted up and down several times, and then the mRNP lysate was incubated on ice for 5 mins and shock-frozen at −80° C. with liquid nitrogen. The mRNP lysate was thawed on ice and centrifuged at 15,000 g for 15 mins to clear the lysate. Flag-Ythdf2 was isolated with 30 μl of protein-G magnetic beads per 1 ml lysate, previously bound to 2 μg of anti-Flag monoclonal antibody (Sigma) for 2 hours at 4° C. on rotation. The beads were collected, washed eight times with 1 ml ice-cold NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/ml RNase inhibitor) and one time with 200 μl irCLIP NT2 buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1 mM MgCl₂; 0.0005% NP-40). mRNP complex was digested with RNase 1 (Thermo Fisher #AM2294) at 0.4 U/μl in irCLIP NT2 buffer (aqueous volume of 30 μl and supplemented with 6 μl of PEG400 (16.7% final)). The nuclease reaction was incubated at 30° C. for 15 mins in an Eppendorf Thermomixer, 15 s 1,400 r.p.m., 90 s rest. Nuclease digestions were stopped by addition of 0.5 mL of ice-cold high-stringency buffer (20 mM Tris, pH 7.5; 120 mM NaCl; 25 mM KCI; 5 mM EDTA; 1% Trition-X100; 1% Na-deoxycholate). Immunoprecipitates were then quickly rinsed with 0.25 mL then with 0.05 mL of ice-cold irCLIP NT2 buffer. The irCLIP adaptor ligation and library construction followed previously reported protocol (Zarneger et al. Nature Methods, 13: 489-492, 2016).

Data were demultiplexed using FAST-iCLIP version 0.9.3 and aligned to mouse genome mm10 from UCSC using STAR (2.4.2a) with parameters “-outFilterScoreMinOverLread 0 -outFilterMatchNminOverLread 0 -outFilterMatchNmin 0”. RPM-normalized genome browser tracks were created in R (3.4.1) and plotted using the Gviz package (1.20.0). Enriched motifs were identified by taking midpoints of each binding site found in all three replicates, adding 20 bases up and downstream, and running MEME (4.11.1) with parameters “-dna -mod zoops -revcomp -minw 5-maxw 10-nmotifs 10-maxsize 1000000”. After motifs were identified, we ran tomtom (4.11.1) against transfac (1-2017) to identify known binding sites. GO enrichment analysis was performed using a hypergeometric test in R. GO terms were considered enriched if they had a BH-adjusted p-value less than 0.05. Selected terms of interest are shown in the bar plot. Bars in the bar plot indicate percentage of genes in the list being tested having the term divided by the percentage of genes in the genome having the term. Peaks found by FAST-iCLIP in all three replicates were assigned to various features in the genome. Promoters were defined as upstream 150 bases from the TSS. “trans_stop” was defined as upstream and downstream 200 bases from the transcript start site.

Cord blood transduction. Cord blood transduction was conducted as described previously (Rentas, S. et al. Nature, 532:508-511, 2016, doi:10.1038/nature17665). Briefly, fresh CD34⁺ cord blood cells or flow-sorted CD34⁺CD38⁻ cells were prestimulated for 12-18 h in StemSpan medium (StemCell Technologies) supplemented with growth factors interleukin 6(IL-6; 20 ng/ml, Peprotech), stem cell factor (SCF; 100 ng/ml, Peprotech), Flt3 ligand (FLT3-L; 100 ng/ml, Peprotech) and thrombopoietin (TPO; 20 ng/ml, Peprotech). Lentiviruses were then added in the same medium at a multiplicity of infection (MOI) of 50-200 for 24 hours. Cells were then given 2 days after transduction before in vitro or in vivo assays. Human YTHDF2 was targeted for knockdown by shRNA targeting 5′-AAGGACGTTCCCAATAGCCAA-3′ (SEQ ID NO: 8) near the N terminus of CDS, as used in a previous report (Wang, X. et al. Nature 505:117-120, 2014, doi:10.1038/nature12730). Scramble shRNA (seed sequence 5′-GCGCGATAGCGCTAATAAT-3′ (SEQ ID NO: 9)) were used as control.

Clonogenic progenitor assays. Flow-sorted GFP⁺ cord blood cells from day 10 cultured transduced cells (12,000 per ml) were resuspended in semi-solid methylcellulose medium (Methocult H4034; StemCell Technologies). Colony counts were carried out after 14 days of incubation.

Human umbilical cord blood HSPC culture. 2 days after transduction, human cord blood CD34⁺ or CD34⁺ CD38⁻ cells were collected and the GFP⁺ percentage was determined by flow cytometry. To ensure that equal numbers of GFP⁺ cells were cultured before expansion, identically cultured GFP⁻ cells were added to the one with higher GFP⁺ percentage to match the % GFP⁺ between control and hYthdf2 KD. Then cells were seeded at a density of 105 per ml in StemSpan medium (StemCell Technologies) supplemented with growth factors IL-6 (20 ng/ml), SCF (100 ng/ml), FLT3-L (100 ng/ml), TPO (20 ng/ml) and CHIR99021 (250 nM) (Stemgent) (Perry et al. Genes and Development, 25: 1928-1942, 2011).

Human HSC xenotransplantation. For human cord blood HSC ex vivo expansion analysis, 10⁵sorted CD34⁺ CD38⁻ cells were transduced with human YTHDF2 shRNA or control shRNA for 3 days and then analyzed for transduction efficiency (% GFP^−/+) and stem cell markers. On day 10, cultured cells were collected for stem cell marker analysis. For hUBC HSC primary LDA assay, CD34⁺ cells were enriched as described above and transduced with human YTHDF2 shRNA or control shRNA at 50 MOI. Media were changed at 24 hours post infection. Equal number of GFP⁺ cells were sorted out from control or YTHDF2 KD cells on 3 days post infection and cultured overnight. Three doses, 50K, 20K and 10K, of sorted GFP⁺ cells were transplanted into sublethally irradiated (3.25 Gy) NSG mice, respectively. The cut-off for HSC engraftment was an exhibition of more than 1% human CD45⁺ GFP⁺ cells out of total CD45⁺ cells in BM of primary transplantation recipients. For hUCB HSC secondary LDA assay, BM cells from highest two doses primary recipients were collected and mixed together at 10 weeks post transplantation. Three doses, 1.2×10⁷, 8×10⁶, 4×10⁶, of BM cells were transplanted into sublethally irradiated (3.25 Gy) NSG mice, respectively. The cut-off for HSC engraftment was an exhibition of more than 0.2% human CD45⁺ GFP⁺ cells out of total CD45⁺ cells in BM of secondary transplantation recipients. HSC frequency was assessed using ELDA software (Hu et al. Journal of Immunological Methods, 347: 70-78, 2009). For all human cord blood xenotransplantation experiments, female NSG mice aged 6-8 weeks were used.

m⁶A-seq data analysis. Human and mouse m⁶A-seq data were aligned to the transcriptome of hg19 and mm10. In order to identify m⁶A peaks, hg19 and mm10 transcriptome was divided into 25 nucleotide-wide tiles. The number of reads in the m⁶A IP and non-IP (control) sample was counted in each tile, and p value was calculated with Fisher exact test and adjusted for multiple testing. Tiles with significant m⁶A signal enrichment (adjust-Pval <=0.05) were merged into bigger regions. Regions smaller than 100 bp were discarded, and regions over 200 bp were divided into 100 to 200 bp sub-regions; m⁶A signal over control was calculated at each region; and regions with at least 2-fold enrichment in all replicates were identified as m⁶A peaks. m⁶A peaks distribution and m⁶A marked genes were determined by overlapping all m⁶A peaks with hg19 and mm10 RefGene annotation. m⁶A marked genes were identified by overlapping m⁶A peaks with hg19 RefGene. To filter for transcription factors, genes marked by m⁶A in all three samples were compared against human transcription factor database fantom.gsc.riken.jp/5/sstar/Browse_Transcription_Factors_hg19. GO term analysis was then performed using R package enrich GO. m⁶A marked human transcription factors were used as searching list, and all the expressed genes were used as background. Hemopoiesis related BP terms with significant enrichment were used to generate FIG. 3C.

RNA-seq. Human cord blood CD34⁺ cells were transduced with control or human YTHDF2 KD lentivirus and sorted out for GFP⁺ CD34⁺ 10 days later. Three replicates of 12,000 GFP⁺ CD34⁺ cells were sorted for each group and were used to extract total RNA. Four nanograms of high quality total RNA was used for cDNA synthesis and library preparation according to the manufacturer's directions with the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara, 634891) and Nextera XT (Illumina, FC-131-1096). Resulting short fragment libraries were checked for quality and quantity using an Agilent 2100 Bioanalyzer and Invitrogen Qubit Fluorometer. Equal molar libraries were pooled, requantified, and sequenced as 75 base pair single reads on a High Output flow cell on the Illumina NextSeq 500 instrument. Following sequencing, Illumina Primary Analysis version NextSeq RTA 2.4.11 and Secondary Analysis version bcl2fastq2 2.18 were run to demultiplex reads for all libraries and generate FASTQ files.

For RNA-seq analysis, reads were aligned to UCSC genome hg38 with Tophat version 2.0.13 with default parameters, using Ensembl 87 gene models. Read counts were generated using HTSeq-count with -m intersection-nonempty. Reads were also aligned to ERCC control sequences and counts tabulated. A scaling factor was calculated based on the median of the ERCC counts for each sample and used for normalization. Differentially expressed genes were found using the edgeR package (3.18.1) in R (3.4.1). Differentially expressed genes were required to have a BH-adjusted p-value <0.05 and a 2-fold change in expression.

RNA stability assay. 15,000 sorted LT-, ST-HSCs and MPPs were cultured in StemSpan SFEM medium (Stem Cell Technologies) supplemented with 10 μg/mL heparin (Sigma), 0.5× penicillin/streptomycin (Sigma), 10 ng/mL recombinant mouse (rm) SCF (Biovision, Inc.), and 20 ng/mL Tpo (Cell Sciences, Inc.) (Perry et al. Genes and Development, 25: 1928-1942 (2011)) at 37° C. 5% CO₂5% O₂. Sorted cells were treated with 5 μM actinomycin D (Sigma) for inhibition of mRNA transcription. Cells were harvested at 0 hour or 4 hours post treatment, and total RNA was extracted and used for RNA-seq.

m⁶A RNA Methylation Quantification.

Mouse BM Lineage negative cells from wt and Ythdf2 KO mice were enriched with mouse Lineage Cell Depletion Kit (Miltenyi Biotec), followed by total RNA extraction with TRIzol (Invitrogen). The quantification of m⁶A RNA methylation in Lin⁻ cells were performed with m⁶A RNA Methylation Quantification Kit (Abcam ab185912) following manufacturer's protocol. 200 ng total RNA were used per replicates for either group.

Qpcr Analysis.

10⁵LSK cells were sorted from wt and Ythdf2 KO mice. Total RNA were extracted with TRIzol (Invitrogen). cDNA synthesis was conducted with High-Capacity RNA-to-cDNA™ Kit (Thermo) following manufacturer's protocol. qPCR primers used are listed in Table S5, qPCR primers used to verify the expressional levels of transcription factors in wt and Ythdf2 KO HSPCs.

Western blot and intracellular staining. To validate the KO or KD efficiency in Ythdf2 KO mouse model or hUCB, 33,000 cKit⁺ cells or 120,000 GFP⁺ cells were sorted from BM or transfected hUCB samples, respectively. Hela cells transduced to overexpress human YTHDF2 were used validate overexpression efficiency as shown in FIG. 14B. Immunoblotting was performed with anti-YTHDF2 rabbit polyclonal antibody (MBL, RN123PW) and β-actin mouse monoclonal antibody (NOVUS, NB600-501). Secondary antibodies used were IRDye 8000 W Goat anti-Mouse IgG and IRDye 8000 W Goat anti-Rabbit IgG antibodies (LI-COR). For intracellular staining, BM cells from wt and Ythdf2 KO mice were stained with HSC markers as above, then fixed with the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's instructions. Fixed and permeabilized cells were immunostained with anti YTHDF2 antibody (MBL RN123PW), anti TAL1 antibody (Santacruz sc-393287), anti GATA2 antibody (Santacruz sc-267), anti RUNX1 antibody (Santacruz sc-365644), anti STAT5 antibody (Santacruz sc-74442) and detected by Alexa-488 donkey anti-rabbit IgG antibody (Invitrogen).

Single cell immunostaining. 10,000 LSKs from wt and Ythdf2 KO mice were sorted onto Poly-L-lysine coating slides, which were placed in a moisture chamber and incubated at 4° C. for 30 mins to allow cells settling onto the slides. Cells were fixed with chilled methanol at RT for 10 mins, blocked with universal blocking reagent (BioGenex) at RT for 30 mins, and stained with mouse TAL1 antibody (Santa Cruz, SC393287) or mouse IgG control (Abcam) at 4° C. overnight. Cells were then stained with Alexa Fluor 488 donkey anti-mouse IgG (Thermo Fisher Scientific) at 4° C. for 30 mins. Images were taken on a PerkinElmer Ultraview spinning disk system with Yokagawa CS-X1 disk. All emission was collected onto a C9100-23 Hamamatsu EM-CCD using Velocity software (PerkinElmer). For Z-stacks, the step size was set at 400 nm. Staining intensity per image was quantified by ImageJ program.

FISH in conjugation with fluorescent immunostaining. Sorted LSKs were spun onto microscope glass slide (Fisher Scientific Cat. No. 12-544-4) using a Cytospin™ 4 Cytocentrifuge at 800 rpm for 1 min with medium acceleration (Thermo Scientific, cat. no. A78300003), followed by an immediate immersion into 4% PFA (diluted from 16% (wt/vol) aqueous solution, Electron Microscopy Sciences, cat. no. 15710). Cells were fixed at RT (25±2° C.) for 30 mins. RNA in situ hybridization was performed using RNAscope multiplex fluorescent detection kit according to the manufacturer's instructions (Advanced Cell Diagnostics) with a couple of modifications: Antigen retrieval was unnecessary, and digestion was performed with 1:15 diluted proteinase Ill solution for 10 mins at RT. RNAscope probes targeting mouse Tal1 and Gata2 were designed and produced by ACDbio. After the in situ hybridization was completed, slides were rinsed twice with PBST and directly processed with background blocking (Background buster solution, Innovex, cat. no. NB306) and primary antibody incubation. Anti-YTHDF2 (MBL, 1:500) and anti-Dcp1a (Santa Cruz, SC100706, 1:200) antibodies were diluted with antibody diluent reagent buffer (Life technologies, cat. no. 003118) and incubated at 4° C. overnight. Donkey anti-rabbit Alexa Fluor 488 (Invitrogen, 1:500) and donkey anti-mouse Alexa Fluor 633 (Invitrogen, 1:500) were used for protein target multiplexing.

Example A-1

Ythdf2 KO Leads to Increase in Phenotypic HSCs in Primary Mice.

To investigate the effects of Ythdf2 on phenotypic HSCs, Crispr-Cas9 technology was utilized to generate Ythdf2^f/fconditional knockout mice, and then crossed with Mx1-Cre mice to specifically reduce Ythdf2 expression in hematopoietic cells (hereafter Ythdf2 KO mice) (FIG. 1A and B). BM HSPCs showed no discernible difference at the absence of pl:pC (FIG. 7A). Four weeks after pl:pC injections, a significant increase was observed in both frequency and absolute number of long-term HSCs (Lin⁻ Sca1⁺ cKit⁺ (LSK) CD34⁻ Flk2⁻; LT-HSCs) and short-term HSCs (LSK CD34⁺ Flk2⁻; ST-HSCs), but not multipotent progenitors (LSK CD34⁺ Flk2⁺; MPPs) in Ythdf2 KO mice compared to littermate wild type (wt) mice (FIG. 1C to 1E). It was found that the frequency and absolute cell number of Long Term HSCs (LT-HSCs) and ST-HSCs increased by over 2-fold while MPP exhibited milder response (FIGS. 1C and 1E). Although Ythdf2 KO led to increased BM cellularity, the absolute number of committed progenitors, including common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythrocyte progenitors (MEPs) and common lymphoid progenitors (CLPs), as well as mature lineage cells, erythrocytes, myeloid cells, B cells and T cells, showed no significant difference between Ythdf2 KO and wt mice (FIG. 1F to H). Cell cycle analysis revealed no discernible change of quiescence in HSCs or MPPs after Ythdf2 KO (FIG. 7B). Notably, the percentage of apoptotic cells in Ythdf2 KO LT-, ST-HSCs and MPPs significantly reduced compared to wt controls (FIG. 7C). To further identify any potential HSC defects in Ythdf2 KO mice, the number of HSCs, committed progenitors, and mature lineages in the spleen were examined, and no significant differences were found between wt and Ythdf2 KO mice (FIGS. 7D to 7H). In summary, Ythdf2 KO in primary mice specifically increases HSC numbers with no bias or defects in either progenitor or lineage cells.

Example A-2

Ythdf2 KO Expands Functional HSCs in Mice.

To determine whether Ythdf2 KO expands functional HSCs, a short-term homing assay was initially performed by transplanting 1×10⁶carboxyfluorescein diacetate succinimidyl ester (CFDA SE)-labelled BM cells from KO mice or their control littermates into lethally irradiated recipient mice, and no significant difference was found in their homing capacity between mutant and wt controls (FIG. 7I). Limited dilution, competitive repopulation unit assay (LDA) was then executed by transplanting 2×10⁵, 7.5×10⁴or 2.5×10⁴donor BM cells (CD45.2), together with 2×10⁵recipient BM cells derived from the ptprc mutant strain (CD45.1), into lethally irradiated recipient mice (FIG. 2A). Consistent with an increased number of phenotypic HSCs in Ythdf2 KO mice, it was found that competitive repopulating units (CRUs) increased 2.2-fold in Ythdf2 KO HSCs compared to controls (FIG. 2B). In the 2×10⁵group, compared to controls, and a significant increase was observed in the overall repopulation rate from Ythdf2 KO donor cells at 16 weeks post transplantation (FIG. 2C). Moreover, recipients of Ythdf2 KO BM cells, compared to that of controls, exhibited markedly higher frequency and absolute number of donor derived LT-HSCs and ST-HSCs, but not MPPs in BM (FIGS. 2D and 2E). Furthermore, it was found that donor derived committed progenitors and mature lineages in BM from transplantation recipients of mutant and wt cells showed no significant changes (FIGS. 2F and 2G). To determine the long-term repopulation ability of HSCs from Ythdf2 KO mice, the secondary transplantation with BM cells derived from primary recipients was conducted. Notably, it was found that compared to controls, CRUs from Ythdf2 KO cells revealed a 3.5-fold increase (FIG. 2H) and exhibited no signs of leukemia in both BM and spleen at 16 weeks after secondary transplantation (FIGS. 9A to 9F). Furthermore, in FIGS. 2I and 2J, a limiting dilution assay was conducted by transplanting total bone marrow (BM) from WT or Ythdf2 KO mice with 3 different dosages. Four weeks post transplantation, it was observed that the engraftment of donor cells increased in the mice transplanted with 200,000 Ythdf2 KO BM cells, as compared to that with WT BM cells. Furthermore, this increase did not generate lineage bias in the transplantation recipients.

We also investigated the long-term effect of Ythdf2 KO on hematopoiesis under homeostasis condition by examining the stem cells, progenitor cells, and lineages in both BM and spleen at over 5 months post pl:pC injections (FIG. 9A). Although we observed a modest increase in LT-HSCs in BM from Ythdf2 KO mice compared to that of controls (FIG. 9C), there were no discernible differences between Ythdf2 KO and control mice in progenitors and lineage cells from either BM or spleen (FIGS. 9D to 9J). These observations indicate that long-term effect of Ythdf2 KO in vivo neither skews lineage differentiation nor facilitates aberrant proliferation, which is in line with previous reports that Ythdf2 is not required for leukemogenesis. To verify the frequency of functional HSCs in the BM at 5 moths post pl:pC induction, we transplanted 7.5×10⁴BM cells from wt and Ythdf2 KO mice with competent cells into lethally irradiated recipients. We found that Ythdf2 KO led to significantly higher engraftment in recipients comparing to wt controls, suggesting that Ythdf2 KO has long-term capability on mouse HSC expansion in vivo (FIG. 9K). Taken together, these data reveal that Ythdf2 KO results in specific and significant mouse HSC expansion in vivo without affecting lineage commitment.

Example A-3

Ythdf2 Regulates HSC Self-Renewal Gene Expression by m⁶A-Mediated mRNA Decay.

To explore the underlying mechanisms of how Ythdf2 KO expands HSCs, mapping was performed of the m⁶A methylome by methylated RNA immunoprecipitation combined with high-throughput sequencing (MeRIP-seq or m⁶A-seq) in LT-HSCs, ST-HSCs, and MPPs sorted from adult C57BL/6J mice (Meyer et al. Cell, 149: 1635-1646, 2012; Schwartz et al. Cell, 155: 1409-1421, 2013; Dominissini et al. Nature, 485: 201-206, 2012). m⁶A peaks were selected by identifying significantly enriched overlapping peaks from two independent replicates. Consistent with previous studies (Meyer et al. Cell, 149: 1635-1646, 2012); Dominissini et al. Nature, 485: 201-206, 2012), it was found that m⁶A peaks were abundant in mRNA open reading frame (ORF), in 3′ untranslated regions (UTRs), and around the stop codon in all three HSPC populations. Transcripts of moderately expressed genes were more likely to be methylated (FIGS. 10A to 10C). Intriguingly, it was found that m⁶A modifications were enriched in the mRNAs of transcription factors, such as Gata2, Etv6, Stat5 and Tal1, which have been documented to be critical for HSC self-renewal and stem cell state maintenance (Wang et al. Blood, 113: 4856-5865, 2009; Ebina et al. The EMBO Journal, 34: 694-709, 2015; Orkin et al. Cell, 132: 631-644, 2008: de Pater et al. The Journal of Experimental Medicine, 210: 2843-2850, 2013; Hock et al. Genes and Development, 18: 2336-2341, 2004; Lim et al. The Journal of Clinical Investigation 122: 3705-3717, 2012; Reynaud et al. Blood, 106: 2318-2328, 2005; and Kato et al. The Journal of Experimental Medicine, 202: 169-179, 2005), suggesting the m⁶A modification could play critical roles in the regulation of HSCs (Table S1, Key transcription factors critical for HSC self-renewal and maintenance are labeled by m⁶A in HSPCs). Given the accumulating evidence that m⁶A mRNA methylation regulates stem cell fate determination by facilitating the decay of mRNAs coding for transcription factors and genes in key signaling pathways involved in self-renewal and differentiation (Batista et al. Cell Stem Cell, 15: 707-719, 2014; (Geula et al. Science 347: 1001-1006, 2015; Yoon et al. Cell, 2017; Zhang et al. Nature, 549: 273-276, 2017; Zhao et al. Nature, 542: 475-478, 2017; Li et al. Cancer Cell, 31: 127-141, 2017; Li et al. Nature, 548: 338-342, 2017) the mRNA degradation rates were next measured in LT-, ST-HSCs, and MPPs by monitoring mRNA levels after transcription inhibition with actinomycin D. It was found that degradation rates of methylated mRNAs were significantly faster than unmethylated mRNAs in ST-HSCs, and MPPs (FIG. 10D). As Ythdf2 is a well-recognized m⁶A “reader” that mediates mRNA decay (Wang et al. Nature, 505: 117-120, 2014), the targets of Ythdf2 were further determined by performing infrared UV-crosslinking immunoprecipitation sequencing (irCLIP-seq) in the mouse multipotent hematopoietic precursor cell line HPC-7 (Pinto do et al. The EMBO Journal, 17: 5744-5756, 1998; Zarnegar et al. Nature Methods, 13: 489-492, 2016) (FIG. 3A; FIGS. 11A to 11C). The results showed that 57.8% of Ythdf2 target mRNAs contained m⁶A peaks (FIG. 11D). Ythdf2 binding sites were enriched with the conserved m⁶A motif and exhibited the characteristic of m⁶A distribution features (FIGS. 3B and 3C). Gene ontology (GO) analysis of Ythdf2 target transcripts revealed enrichment of genes related to hematopoietic or lymphoid organ development, suggesting the involvement of Ythdf2 in the regulation of hematopoiesis (FIG. 3D). Notably, it was found that Ythdf2 bound to transcription factor mRNAs, such as that of Tal1 and Gata2, on sites largely overlapping with m⁶A peaks (FIG. 3E; FIG. 5E and Table S2, Ythdf2 targeted mRNAs from three irCLIP-seq replicates). Significant change was not observed in the total RNA mass in LSK Flk2⁻ cells from wt and Ythdf2 KO mice. Though m⁶A modification only constitute 0.1-0.4% of adenosine nucleotide in mammal cells, it was found that Ythdf2 KO led to increased level of m⁶A content in total RNA from BM Lin⁻ cells, suggesting that Ythdf2 specifically regulates the stability of m⁶A-marked mRNAs (FIGS. 12A and 12B). Consistently, qPCR analysis of total mRNA revealed increased levels of Tal1, Gata2, Runx1 and Stat5a, whose mRNAs have shown to be modified by m⁶A, in Ythdf2 KO LSK cells compared to wt controls (FIG. 3F). Single cell immunofluorescence staining and intracellular flow cytometry further revealed that Ythdf2 KO HSPCs exhibited significant increases in the intensities of m6A-labeled transcription factors involved in stem cell self-renewal, such as TAL1, GATA2, RUNX1 and STAT5, indicative of a suppressive role of Ythdf2 in HSC self-renewal (FIG. 3G; FIG. 12C). A previous study has shown that Ythdf2 regulates RNA metabolism through localizing the bound mRNAs to mRNA decay sites (Wang et al. Nature, 505: 117-120, 2014). To further explore the mechanism of Ythdf2 in regulating HSC self-renewal, fluorescence in situ hybridization (FISH) was performed of Tal1 mRNA and fluorescence immunostaining of Ythdf2 and Dcp1a, a marker of mRNA decay sites (Sheth et al. Science, 30:805-808, 2003; Kedersha et al. Methods in Enzymology, 431: 61-81, 2007), and their relative spatial distribution was analyzed in sorted wt and Ythdf2 KO HSPCs. Co-localization of Tal1 mRNA, Dcp1a and Ythdf2 was observed in wt cells while substantially reduced in Ythdf2 KO controls (FIGS. 3H and 3I). Furthermore, similar observation was confirmed by co-staining Gata2 mRNA FISH with Ythdf2 and Dcp1a in wt or Ythdf2 KO HSPCs (FIGS. 12D and 12E). To determine whether the increased transcription factors, such as Tal1, expression accounts for the HSC expansion in Ythdf2 KO mice, rescue experiments were performed using short hairpin (sh) RNA-mediated Tal1 knock down (KD) in wt and Ythdf2 KO LSK cells, followed by transplanting into lethally irradiated recipients. Depletion of Tal1 in HSPCs significantly impaired the reconstitution capacity of wt cells as reported previously and also rescued the increased engraftment of Ythdf2 KO cells (FIG. 12F). Overall, these data indicate that Ythdf2 regulates HSC self-renewal by enabling the degradation of mRNAs encoding transcription factors essential for stem-cell renewal.

Example A-4

Dissecting the Role of Ythdf2 in Human UCB HSPCs by m⁶A-Seq and RNA-Seq.

The limited number of HSCs in a single human umbilical cord blood unit has been an obstacle for clinical applications, such as HSC transplantation (Walasek et al. Annals of the New York Academy of Sciences, 1266: 138-150, 2012). The observation that Ythdf2 KO resulted in an increase of phenotypic and functional mouse HSCs prompted a test whether YTHDF2 knockdown (KD) could facilitate human UCB HSC expansion. First, m⁶A-seq with CD34⁺ cells isolated from 3 individual hUCB samples was performed (FIG. 13A). m⁶A modifications predominantly occurred in mRNAs (˜95%), preferential in mRNA ORF regions, 3′UTRs, and near the stop codon, as expected (˜90%) (FIGS. 4A and 4B; FIG. 13B). m⁶A landscapes in mouse and hUCB HSPCs were compared, and it was found that 2,239 genes were commonly m⁶A tagged (FIG. 4C). These commonly m⁶A-tagged transcripts were enriched for genes related to hematopoiesis and stem cell maintenance (FIG. 4D). Due to the enrichment of m⁶A labeling in the mRNAs of transcription factors responsible for mouse HSC self-renewal, the m⁶A-marked transcription factor transcripts in hUCB CD34⁺ cells were next characterized by performing GO term analysis. Among the 722 identified m⁶A-labeled transcription factor mRNAs, major GO terms were related to cell fate commitment and stem cell maintenance (FIG. 13C). For example, HOXB4, overexpression of which has been reported to expand human and mouse HSCs ex vivo (Amsellem et al. Nature Medicine, 9: 1423-1427, 2003; Antonchuck et al. Cell, 109: 39-45, 2002), was marked by m⁶A in hUCB CD34⁺ cells (FIG. 4E). Other transcription factors required for HSC self-renewal and critical to induce HSCs from other cell types (Ebina et al. The EMBO Journal, 34: 694-709, 2015; Galan-Caridad et al. Cell, 129: 345-357, 2007, such as Zfx, RUNX1 and FOSB, were also m⁶A-tagged in hUCB CD34⁺ cells (FIGS. 13F and 13G and see also Supplementary Table S3 of article “Supression of m6A Reader Ythdf2 Prmotd Hematopoeitic Stem Cell Expansion” by Li et al, Cell Research 28, 904-917 (2018), which article (and including the Supplementary Tables thereof) is hereby incorporated by reference herein in its entirety, Genes marked by m⁶A in human UCB CD34⁺ HSPCs from individual samples). To further dissect the role of YTHDF2 in hUCB HSPCs, RNA-seq was performed with control or YTHDF2 KD hUCB CD34⁺ cells (FIG. 4F; FIGS. 13D and 13E). Remarkably, transcripts marked by m⁶A, including HOXB4 and other HSC self-renewal related transcription factors, showed significant increases of input mRNA reads in the YTHDF2 KD cells compared to the control, without noticeable changes for non-m⁶A labelled genes (FIGS. 4G to 4I; FIGS. 13F and 13G). These results support the role of YTHDF2 in regulating hUCB HSC self-renewal through RNA degradation.

Example A-5

Expansion of hUCB HSCs by YTHDF2 KD.

To further explore whether suppression of YTHDF2 can expand human HSCs, short hairpin (sh) RNA-induced YTHDF2 KD in hUCB HSPCs was performed as above (FIG. 4F). After 7 days ex vivo culture, lentiviral knockdown of YTHDF2 resulted in an average 14.3-fold and 13.6-fold increase, respectively, in the frequency and absolute number of CD34⁺ CD38⁻ CD45RA⁻ EPCR⁺ phenotypic HSCs and a 5.1-fold increase in CFUs relative to control cells, especially the most primitive CFU-granulocyte erythrocyte monocyte megakaryocyte (GEMM) colony type and burst forming unit-erythroid (BFU-E), reflecting higher expression level of key transcription factors for hematopoiesis, such as TAL1, in YTHDF2 KD hUCB cells (FIGS. 5A to 5D; FIG. 14A). Interestingly, the apoptotic rate was significantly reduced in YTHDF2 KD hUCB HSPCs compared to control cells, similar to the trend of HSCs in Ythdf2 KO mouse (FIG. 5E). Also, FIG. 5F shows that the Ythdf2 knockdown (KD) HSCs exhibited up to a 10-fold increase compared to control HSCs 10 days post transduction. Next, the effect of overexpression (OE) YTHDF2 on HSPC function was explored. Overexpression of YTHDF2 reduced clonogenic potential of hUCB HSPCs by 2.2 fold, suggesting YTHDF2 negatively regulates HSC maintenance ex vivo (FIGS. 14B and 14C).

To determine whether YTHDF2 KD can expand human HSCs in vivo, LDA was performed by transplanting GFP⁺ cells sorted from hUCB CD34⁺ cells infected with control and YTHDF2 shRNA at day 4 post infection (FIG. 6A). At 10 weeks post transplantation, we analyzed BM cells were analyzed from recipient NOD/SCID //2rg^null(NSG) mice and measured the functional HSC frequency after in vivo expansion. Notably, compared to control group, recipients of YTHDF2 KD cells displayed a 9-fold increase in human hematopoietic cell (hCD45⁺ GFP⁺) engraftment in BM without changes in the proportions of each lineage (FIGS. 6B and 6CH; FIGS. 15A and 15B). YTHDF2 KD significantly increased the percentage of myeloid, megakaryocyte and erythrocyte in the BM of primary recipients (FIG. 15C). Accordingly, it was found that the HSC frequency in YTHDF2 KD cells was increased 4.4-fold relative to that in control cells (FIG. 6D). It was confirmed the long-term capability of YTHDF2 KD hUCB cells to be reconstituted and undergo self-renewal; 12 weeks after transplantation of BM from primary recipients into sublethally irradiated secondary NSG recipient mice, human hematopoietic cell chimerism in BM were higher in the YTHDF2 KD group, as compared to that in the control group (FIGS. 6E, 6F; FIG. 15D). CRUs from YTHDF2 KD cells revealed an 8-fold increase relative to that in control cells (FIG. 6G). These data demonstrate that YTHDF2 KD remarkably facilitates the expansion of both phenotypic and functional hUCB HSCs ex vivo.

Example A-6

The examples herein demonstrate that conditional deletion of Ythdf2, m6A reader, lead to expansion of phenotypic and functional HSCs without lineage bias. To investigate if there is concomitant increase in mesenchymal stem cells in vivo, Ytdhf2^f/fif mice were crossed with Mx1-Cre mice to conditionally delete Ythdf2 expression from mesenchymal cells. Nine-months post pl:pc injection bone marrow cells were isolated from Ythdf2^KOand their wild type littermates. Single-cell suspension of total bone marrow cells were immuno-stained with flow antibodies. Hematopoietic (CD45, Ter119) and endothelial cells (CD31) were excluded using specific markers. Mesenchymal stem cell present within the total bone marrow stromal cells were further purified by inclusion of N-Cadherin and CD105 antibodies. Mesenchymal stem cells marking both N-Cadherin⁺ and CD105⁺ were quantitated. Conditional deletion of Ythdf2 led to 3.6-fold expansion in frequency of mesenchymal stem cells (FIG. 16). Our study clearly demonstrates that loss of Ythdf2 can expand bone marrow mesenchymal stem cells in vivo.

DISCUSSION

Although recent studies explore the biological functions of mRNA m⁶A modifications (Zheng et al. Molecular Cell, 49: 18-29, 2013; Zhou et al. Nature, 526: 591-594, 2015; Alarcon et al. Cell, 162: 1299-1308, 2015; Zhang et al. Cancer Cell, 31: 591-606 e596, 2017; Lence et al. Nature, 540: 242-247, 2016; Haussmann et al. Nature, 540: 301-302, 2016; Chen et al. Cell Stem Cell, 16: 289-301, 2015; Alarcon et al. Nature, 519: 482-485, 2015; Xiao et al. Molecular Cell, 61: 507-519, 2016; Wojtas et al. Molecular Cell, 68: 374-387 e312, 2017; Ivanovna et al. Molecular Cell, 67: 1059-1067 e1054, 2017; Fustin et al. Cell, 155: 793-806, 2013; Slobodin et al. Cell, 169: 326-337 e312, 2017; Schwartz et al. Cell, 159: 148-162, 2014; Pendleton et al. Cell, 169: 824-835 e814, 2017; Shi et al. Cell Research, 27: 315-328, 2017; Huang et al. Nature Cell Biology, 20: 285-295, 2018; Bertero et al. Nature, 2018; Liu et al. Nature, 518: 560-564, 2015), embodiments herein identify Ythdf2 as an important regulator of human and mouse HSC self-renewal by coupling the post-transcriptional m⁶A modification to the degradation of mRNAs encoding key transcription factors for self-renewal. Repression of Ythdf2 in mouse HSPCs and hUCB HSCs can lead to increased expression of multiple key TFs critical for self-renewal, thereby facilitating ex vivo expansion of both phenotypic and functional HSCs without noticeable lineage bias and leukemia potential. In addition, stem cell niches, to some extent, may contribute to Ythdf2 suppression-mediated mouse HSC expansion as Mx1-cre can be activated in mesenchyme stromal cells. It would be intriguing to study the function of Ythdf2 on mesenchymal stem cells (MSCs) and how repression of Ythdf2 in both HSCs and MSCs may synergistically expand HSCs in vivo.

Given the broad and complicated impact of m⁶A writer complex Mettl3 and Mettl14 on mRNA splicing, translation, and pri-miRNA processing (Barbieri et al. Nature, 2017; Alarcon et al. Nature, 519: 482-485, 2015; Liu et al. Nature, 518: 560-564, 2015), Mettl3 or Mettl14 depletion results in distinct outcomes in normal stem cells and leukemia. Recent studies have demonstrated that Mettl3 and Mettl14 play essential roles in leukemia development and leukemia stem cell maintenance (Vu et al. Nature Medicine, 2017; Barbieri et al. Nature, 2017; Weng et al. Cell Stem Cell, 22: 191-205 e199, 2018). In contrast, Ythdf2 is believed to be mainly involved in m⁶A-mediated mRNA decay (Batista et al. Cell Stem Cell, 15: 707-719, 2014; Yoon et al. Cell, 2017; Zhang et al. Nature, 549: 273-276, 2017; Wang et al. Nature, 505: 117-120, 2014). According to certain embodiments herein, it is believed that manipulating Ythdf2 may extend the half-life of specific m⁶A-marked mRNAs encoding TFs critical for stem cell self-renewal without affecting other aspects of mRNA processing. The Examples herein show that Ythdf2 depletion in HSCs neither skews the lineage commitment nor induces hematological malignancies, reducing the risk of leukemogenesis with expanded HSCs. Furthermore, stem cell self-renewal is a complexed process comprised of cell division, survival, prevention of differentiation and stemness retention. The observation that Ythdf2-deficient HSCs exhibited lower apoptotic rate indicates embodiments of methods herein also benefit another feature of stem cell self-renewal.

A major limitation in using hUCB HSC transplantation is the insufficient number of HSCs in one hUCB unit. Albeit previous studies have revealed that Dlk1, SR1, Musashi2 and UM171 can expand hUCB HSCs by targeting Notch, AHR signaling or other unknown pathway (Boitano et al. Science, 329: 1345-1348, 2010; Fares et al. Science, 345: 1509-1512, 2014; Rentas et al. Nature, 532: 508-511, 2016; Chou et al. Experimental Hematologyl, 41: 479-490 e474, 2013). Accordingly, embodiments herein provide a novel and potent way to target multiple key TFs critical for HSC self-renewal and to enhance the expansion of HSCs. For example, reducing Ythdf2 level and function during in vitro culture via small chemicals or AAV-mediated KD may allow the Ythdf2 level and function to restore after transplantation in vivo, and thus not affect normal HSC maintenance and function in human patients. Furthermore, in certain embodiments, methods described herein may be combined with other methods to facilitate the expansion of not only human HSCs, but also other stem cells, rendering an approach for stem cell-based therapies.

Example B

The following protocols were used in the “B” Examples below.

Animals. C57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/J(iDTR), B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J(R26RtdT), Tg(Cspg4-DsRed.T1)1Akik/J, Cxcl12tm2.1Sjm/J, KitItm2.1Sjm/J (SCF^f/f), Cxcl12tm1.1Sjm/J (CXCL12^f/f) mice were obtained from the Jackson Laboratory. N-cad-CreER^T, and N-cad-TdT mice were generated by Applied StemCell, Inc. To induce N-cad-CreER^T; R26-tdT mouse, tamoxifen (Sigma) was injected intraperitoneally at 2 mg per injection for 3 days. To induce N-cad-CreER^T; R26-tdT at embryonic stage, a single dose of 1.5 mg of TMX was injected intraperitoneally (IP) into the E12.5 pregnant dam. Cesarean section was performed at E19.5 and the neonatal mice were transferred to foster mice. To induce N-Cad⁺ cells ablation in N-cad-CreER^T;iDTR mice, DT (Sigma) was injected intraperitoneally every other day at a dose of 50 ng per g body as indicated. 5FU (Sigma-Aldrich) was injected once in the tail vein at 150 μg per g body weight. After 5FU injection, mice were analyzed as described in the text. All mouse strains used in this study had a C57BL/6J genetic background. Animals were randomly included in the experiments according to genotyping results. Animal experiments were conducted in a blinded fashion with respect to the investigator. The numbers of animals used per experiment are stated in the figure legends. All mice used in this study were housed in the animal facility at the Stowers Institute for Medical Research (SIMR) and were handled according to SIMR and National Institutes of Health (NIH) guidelines. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the SIMR.

Flow cytometry. For phenotype analysis, hematopoietic cells were harvested from bone marrow (femur and tibia). Red blood cells were lysed using a 0.16 M ammonium chloride solution. For cell surface phenotyping, a lineage cocktail (Lin, phycoerythrin (PE)-Cy5) was used, including anti-CD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-Mac-1 (M1/70), anti-Gr1 (RB6-8C5), anti-B220 (RA3-6B2), anti-IgM (II/41) and anti-TER119 (TER-119) (100 ng antibody cocktail per million bone marrow cells, eBioscience). Monoclonal antibodies to SCA1 (D7, eBioscience), c-KIT (2B8, eBioscience), FLK2 (A2F10, eBioscience), CD34 (RAM34, eBioscience), CD48 (HM48-1, eBioscience), CD150 (TC15-12F12.2, BioLegend) and CD49b (HMa2, Biolegend) (all used as 50 ng per million bone marrow cells) were also used where indicated. For lineage analysis of peripheral blood, monoclonal antibodies to CD45.1 (A20, eBioscience), CD45.2 (104, eBioscience), CD3, B220, Mac-1 and Gr1 were used. 7-aminoactinomycin D (7-AAD) (A1310, Life technologies) was used to exclude dead cells. For stromal niche cell analysis, CD45 (30-F11, eBioscience), CD31 (390, eBioscience), PDGFRα-biotin (APB5, eBioscience), LepR-Bio (R&D), CD51 (clone RM7-V, Biolegend). Samples stained with biotin conjugated antibodies were washed with staining medium, then incubated with streptavidin brilliant violet 421TM (Biolegend, 1:500). Cell sorting and analysis were performed using MoFlo (Dako), InFlux Cell Sorter (BD Biosciences), MACSQuant (Miltenyi Biotec) or CyAn ADP (Dako) instruments. Data analysis was performed using FlowJo software.

Whole-mount sternum HSC immunostaining. Sternal bones were collected and transected with a surgical blade into 3-4 fragments. The fragments were bisected sagittally to expose bone marrow cavity, fixed in 4% PFA. blocked/permeabilized in PBS containing 20% normal goat serum and 0.5% Triton X100, and stained with primary antibodies for 3 days. The tissues were incubated with secondary antibodies for 2 hours (Bruns et al., 2014; Kunisaki et al., 2013). Fluorescence imaging was performed on a spinning-disk confocal microscope (UltraVIEW; PerkinElmer), including an inverted microscope (Axiovert 200 M; Carl Zeiss Microimaging, Jena, Germany), attached to a spinning-disk confocal system (CSU-X1; Yokogawa Corporation of America) and Orca-R2 camera (Hamamatsu) with Volocity acquisition software (PerkinElmer) and a 20×/0.8 Plan-Apochromat objective (Carl Zeiss). Images were collected as a series of optical sections, with a step size of 4 μm. Images were collected in a tile pattern (overlap 10%) sufficient to cover the entire sample. Channels were collected sequentially. (Blue dye) was excited using 405 nm light (50 mW diode laser, OEM) and (red dye) was excited using 561 nm light (50 mW solid state laser, OEM), and each was collected using a multibandpass emission filter with 415 nm-775 nm 580 nm-650 nm bands. (Green dye) was excited using 488 nm light (50 mW solid state laser, OEM) and collected using a multibandpass emission filter of 500 nm-550 nm, and (far red dye) was excited using 640 nm light (50 mW solid state laser, OEM) and collected using a multibandpass emission filter with 455 nm-515 nm and 660 nm-750 nm. Exposure times and laser powers were adjusted to compensate for variations in staining.

Second Harmonic Generation (SHG) imaging was performed immediately following fluorescence imaging. SHG images were collected on a LSM-780 laser scanning confocal microscope (Carl Zeiss) equipped with a QUASAR detection unit, a 10×0.45 Plan-Apochromat objective (Carl Zeiss), and Zen 2012 acquisition software (v8.1.3, Carl Zeiss). Images were collected as a series of optical sections, with a step size of 8 μm and a pixel size (1.32 μm/pixel) an integer multiple of four times the fluorescence pixel size. SHG images were taken with a laser light wavelength of 900 nm and collected at 371-420 nm. As a reference for image alignment, images of Cd150-PE were taken using the 561 nm line of a DPSS laser (Melles Griot) and collected at 566-735 nm concurrently with the SHG images. Images were collected in a tile pattern (no overlap) sufficient to cover the entire sample. Tiles were stitched into a complete 3D image using Zen software.

Images were analyzed using Fiji software (1.51 g National Institutes of Health). To align SHG and fluorescence images, fluorescence images were first background subtracted, and image tiles were stitched into a complete 3D image using the Grid/Collection stitching plugin (reference bioinformatics.oxfordjournals.org/content/25/11/1463.abstract). Given that the transfer of the sample from the fluorescence microscope to the SHG microscope involved a small amount of sample rotation, it was necessary to realign the SHG and fluorescence images in 3 dimensions. Alignment of SHG and fluorescence image sets was carried out using a custom plugin available at research.stowers.org/imagejplugins. Firstly, a minimum of 8 common landmarks were identified by visual inspection of both the SHG and fluorescence data sets. Next the Kabsch algorithm was used to find the best scaled rotation to transform the fluorescence coordinates to the SHG image coordinates. Finally, each 3D voxel in the fluorescence image was transformed to the corresponding SHG position and trilinear interpolation was used the find the SHG intensity at that position to create the realigned composite image.

Image analysis was conducted by researchers unfamiliar with the hypotheses of the study. HSCs were identified by eye. Cells were considered negative for staining if the shape of the cell could not be discerned by eye, or if the shape of the cell formed a dark region in a field of positive signal. Distance measurements to niche components were made using Fiji and Microsoft Excel software. The locations of the HSC and nearest point of each of the three niche components were marked using point ROIs in Fiji and locations were transferred to excel, where distances in 3D were then calculated using the 3D Pythagorean Theorem. To calculate distances for randomly distributed HSCs, random point ROIs were generated using a custom plugin in FIJI in the same images analyzed for observed HSCs. The randomly generated points were considered simulated HSCs if they appeared in a reasonable HSC location (as assessed by the presence of Lin⁺ cells in the surrounding regions). Niche distance measurements for simulated HSCs were then made in the same manner as for observed HSCs.

The statistical significance of differences in the distribution of distances was assessed by Kolmogorov-Smirnov analysis using Origin software. Statistical significance of changes in percentages of HSCs at 5 μm were assessed using a Student's T test in Microsoft Excel. Changes were considered significant if P<0.05.

Final images shown in figures are maximum projections that have been background subtracted and contrast adjusted for clarity.

Transplantation and Repopulation assay. 100 sorted pHSCs or rHSC cells together with 1.0×10⁵CD45.1 rescue bone marrow cells were transplanted into lethally irradiated (10 Gy) CD45.1 recipients. 2.0×10⁵CD45.2 BM cells from N-cad-CreER^T;iDTR and control mice together with 2.0×10⁵CD45.1 rescue bone marrow cells were transplanted into lethally irradiated (10 Gy) CD45.1 recipients. Every 4 weeks post transplantation, peripheral blood was collected from the submandibular vein. Hematopoietic repopulation was measured from donor-derived blood cells (CD45.2).

RNA sequencing and analysis. cDNA was generated from 1000 purified cells using SMARTer Ultra Low Input RNA kit (Clonetech) and library was generated by the Nextera XT DNA Library Preparation Kit (Illumina), followed by sequencing on an Illumina HiSeq2500 for 50 bp single reads. Raw reads were demultiplexed into Fastq format allowing up to one mismatch using Illumina bcl2fastq2 v2.18. Reads were aligned to UCSC genome mm10 with TopHat v2.0.13, default parameters. FPKM values were generated using Cufflinks v2.2.1 with “-u—max-bundle-frags 100000000”. Read counts were generated using HTSeq-count with “-m intersection-nonempty”. Three or four replicates were sequenced for each population. Data are accessible at NCBI GEO: GSE104887

CFU-F assay and in vitro differentiation. Cells were sorted directly into culture at a 96-well plate. The cultures were incubated at 37° C. in a humidified atmosphere with 5% 02 and 10% CO₂for 7-10 days. Colonies were stained by CellTracker™ Green CMFDA (Life technologies) and image was acquired by Celigo Imaging Cytometer (Nexcelom). For in vitro differentiation, clonally expanded Ncad-CreER^Tdriven Tomato⁺ BM/Bone stromal cells were isolated from CFU-F cultures by digesting with 0.25% Trypsin/EDTA, split into 3 aliquots, and seeded into separate cultures permissive for differentiation: StemPro Osteogenesis kit Gibco A10072-01, Adipogenesis kit A10070-01 and Chondrogenesis differentiation kit A10071-01. The osteoblastic differentiation was assessed by VECTOR Red Alkaline Phosphatase; the adipogenic differentiation was detected by Oil Red O (Sigma); and the chondrogenic differentiation was detected by Toluidine blue (Sigma, 0.1 g T Blue/100 mL distilled water).

Bone Sectioning, Immunostaining and Imaging.

Freshly isolated femurs were fixed in 4% paraformaldehyde overnight, followed by 1 to 3 days decalcification in 10% EDTA. For paraffin section, bone samples were processed with Sakura Tissue Tek VIP 5 Tissue Processor (Sakura America, Torrance, CA), and paraffin sections were cut in 5 μm thickness. Sections were deparaffinized with xylene, followed by Alcian Blue/Hematoxylin/Orange G staining. For frozen section, bone samples were processed with the CryoJane tape-transfer system. Sections were blocked with Power Block™ Universal Blocking Reagent for 30 minutes to 1 hour and then stained overnight with rabbit-anti-Aggrecan (Millipore, 1:300), rabbit-anti-Perilipin (Cell Signaling, 1:300) and goat-anti-Osteopontin (R&D, 1:300). Donkey-anti-goat Alexa Fluor 488 and Donkey-anti-goat Alexa Fluor 647 were used as secondary antibodies (all from Invitrogen, 1:300). Antibodies were diluted with Antibody Diluent Solution (Invitrogen 00-3218). Slides were mounted with FLUORO-GEL (Electron Microscopy Science 1798510), and images were acquired with an Olympus slide scanner.

Femoral groove surgery. Mice were anesthetized with 2.5% isoflurane, and buprenorphine was administered for analgesia. The skin of the right leg was shaved and scrubbed with alcohol and iodine. A small incision was made in the skin, lateral to the knee joint. After sliding the skin medially for visualization, an internal incision was made medial to the patella, extending into the quadriceps muscle and along the patella tendon to release the tissue. The patella was subluxated laterally, and the distal femur was exposed. The subchondral bone was perforated using a microsaw to penetrate the articular cartilage at the knee joint. The extensor mechanism (quadriceps, patella tendon and patella) was returned to its original anatomical location. The internal incision was sutured with absorbable suture, and the skin sutured with non-absorbable suture.

Statistics.

Values are shown as the mean±s.e.m. All statistical analyses were generated using GraphPad Prism 5 (GraphPad Software). Student's t test was used for comparisons between two groups. Statistical significance was defined as p<0.05.

Example B-1

Functionally distinguished reserve and primed HSCs in mouse bone marrow. To explore the reserve HSC (hereafter rHSC(s)) subpopulation, a cell surface marker was adapted, CD49b (Integrin α2), which can distinguish intermediate-term from permanently long-term HSCs (LT-HSCs) (Benveniste et al., 2010; Qian et al., 2015; Wagers and Weissman, 2005; Yang et al., 2005). Intriguingly, it was found a CD48⁻ CD49b⁻subpopulation which exists only in conventional LT-HSCs (CD34⁻Flk2⁻Lineage⁻ Sca-1⁺c-Kit⁺ (LSK) cells) but not in short-term HSCs (ST-HSCs; CD34⁺FLK2⁻LSK) or multipotent progenitor cells (MPPs; CD34⁺FLK2⁺LSK). It was proposed that the CD48⁻ CD49b⁻ LT-HSCs subpopulation enriches rHSCs and that the CD48⁻CD49b⁺ LT-HSCs subpopulation enriches primed HSCs (hereafter pHSC(s)) (FIG. 17A) and tested with a repopulation assay. It was found that both rHSCs and pHSCs supported hematopoiesis in lethally irradiated mice for up to 40 weeks after transplantation without significant difference (FIG. 17B), consistent with a previous report (Benveniste et al., 2010). However, transplanted pHSCs had very low efficiency in generating rHSCs (95.4% reduction) as well as ST-HSCs and MPPs (˜78% reduction for both populations) in recipients compared to transplanted rHSCs, suggesting that rHSCs hierarchically precede pHSCs (FIG. 17C). Using the Scl-tTA-induced H2B-GFP label-retaining model, it was found that rHSCs significantly enriched more H2B-GFP^highcells compared to pHSCs (P=0.0039), indicating that rHSCs have slower cell cycle compared to pHSCs (FIG. 17D). Molecularly, it was found that rHSCs had higher expression of Gadd45 g, Cdkn1c (encoding p57), and Foxo1 that are all involved in maintaining Go phase of HSCs, and lower expression of cell cycle activators such as Myc, Pcna, Ccng2, and Cdk4. There was no difference in Ki67 expression between rHSCs and pHSCs, indicating that Ki67 expression alone is insufficient to distinguish the two HSC subpopulations (FIG. 17E).

Because the functional definition for rHSC is drug-resistance, rHSCs or pHSCs were transplanted into recipient mice and the mice were challenged with 5FU at 4 weeks post transplantation. As shown in FIG. 17F, rHSCs were insensitive to 5FU treatment; however, pHSCs dramatically reduced their reconstitution ability (44% reduction at 20 weeks post transplantation). Taken together, the data indicated that CD48⁻CD49b⁻ LT-HSC indeed enriched rHSCs that were resistant to chemotherapeutic treatment, whereas CD48⁻CD49b⁺ LT-HSC enriched pHSCs that were sensitive to chemotherapy; furthermore, the former gave rise to the latter but not vice versa in the transplantation assay.

The direct consequences of acute 5FU challenge on rHSCs and pHSCs were further analyzed. As shown in FIG. 17G, at 3 days post 5FU, around 92% of pHSCs were eliminated and only rHSCs survived, suggesting that rHSCs must have specifically turned on their DNA repair system to overcome the chemotherapeutic stress. To test this hypothesis, the transcriptome profiling of DNA damage response genes in rHSCs, pHSCs and rHSCs post 5FU treatment was analyzed. It was observed that rHSCs maintained a lower expression of genes involving the DNA damage repair system compared to pHSCs during homeostasis (FIG. 17H), but that most DNA repair pathways, such as DNA mismatch repair (MMR), nucleotide excision repair (NER), base excision repair (BER), and homologous recombination, (HR) were significantly activated in rHSCs under 5FU challenge (FIG. 17I). Furthermore, in parallel, a great number of stress response genes (Rodina et al., 2016), which primarily belong to Hsp90 and Hsp70 family, were also upregulated (1.8±0.17-fold and 1.4±0.1-fold respectively) (FIG. 17J), which partially explained how rHSCs survived and reconstituted the hematopoietic system under chemotherapeutic stress.

Taken together, the coexistence of pHSCs and rHSCs were functionally demonstrated in BM. Even with their quiescent state and active DNA-repairing pathways, pHSCs were still sensitive to chemotherapy, whereas rHSCs activated their DNA damage repair and stress response genes to survive chemotherapeutic stress and give rise to pHSCs; thus, rHSCs play a critical role in supporting hematopoietic regeneration under severe stress.

Example B-2

Drug-Resistant rHSCs Predominantly Localize in the Endosteal Region of Bone Marrow

Whether extrinsic mechanisms from the BM niche contributed to rHSC maintenance during hemostasis and under chemotherapeutic stress was further examined. To this end, whole mount HSC staining was performed, which simultaneously detected the relative distribution of rHSCs and pHSCs to bones (achieved by second-harmonic generation, SHG), megakaryocytes (MKs) or vessels within ˜75 μm thickness bone cavity (FIG. 18A-B). Our quantification data showed that 43.4% rHSCs and 31.0% pHSCs were located within 10 μm distance from vessels, and that 22.6% rHSCs and 24.6% pHSCs were located within 10 μm from MKs (FIG. 18C-D), consistent with previous reports that the bulk of the HSC population resides in perivascular and sinusoid zones (Acar et al., 2015; Bruns et al., 2014; Chen et al., 2016; Zhao et al., 2014). Interestingly, it was noticed that 16.4% rHSCs compared to only 3.69% pHSCs located within 10 μm from the bone surface (FIG. 2E). These data showed that both rHSCs and pHSCs were unbiasedly distributed to vessels and MKs, but that rHSCs were located significantly closer to endosteal bone surface compared to pHSCs (P=0.00182).

To test whether the endosteal region preserved rHSCs during chemotherapeutic stress, the distribution of rHSCs was examined at day 3 post 5FU treatment when pHSCs were eliminated (FIG. 18B). Interestingly, we found that ˜55% of surviving rHSCs were preserved by the endosteal niche upon acute 5FU stress, which is a ˜3.5-fold enrichment compared to homeostasis (FIG. 18E). However, there was no significant difference in frequency of surviving rHSCs observed near vessels or MKs (FIG. 18C-D). Consistently, it was observed that only 4.24% of pHSCs survived acute 5FU stress as shown in FIG. 17G. Next, the dynamic process of BM damage and the subsequent recovery after 5FU stress was studied by examining the BrdU-labeled surviving and proliferation cells. It was noticed that at day 2 post 5FU treatment, there was a large loss of the BrdU-labeled cells, indicating an active apoptosis induced by 5FU. At day 3 post 5FU treatment, it was observed that surviving BrdU⁺ cells (mostly single cells) were mainly detected adjacent to bone lining cells in the endosteal region (FIG. 24A-D). At day 3.5, it was observed that pairs of BrdU⁺ cells appeared at the endosteal surface, indicating activation and division of surviving cells. Starting and continuing at days 4 and 5, the number of proliferating cells gradually increased, and these cells were very often detected as clusters close to either vessels or potential adipocyte structures (˜55% at day 4 and ˜65% at day 6) (FIG. 24E-I). This observation suggested that bone surface was the niche where cells post 5FU initially survived. These cells were then activated and gave rise to daughter cells, the latter of which underwent expansion mainly in vessels or adjacent to megakaryocytes (Zhao et al., 2014). It was further confirmed that surviving 5FU-rHSCs were indeed detected often as single cells adjacent to the bone surface, and proliferating HSCs often associated with MKs or near the vessels. Surviving rHSCs (green, CD150+ Lin− CD49b−) at day 3 post 5FU treatment were often detected as single cells adjacent to the bone surface (white, SHG), and proliferating HSCs were often associated with MKs (CD150+ Lin+) or near the vessels (red, CD31+) (FIG. 29).

N-cad⁺ pre-osteoblastic cells in bone surface have been found resistant, whereas Osx⁺ osteoblasts have been found to be sensitive to 5FU treatment and N-cad⁺ stromal cells have been found to give rise to Osx⁺ osteoblasts during recovery post 5FU treatment (Sugimura et al., 2012). A recent study showed dramatic depletion of LepR⁺ stromal cells in central marrow due to cell death 1 day following irradiation (Zhou et al., 2017). To track the early changes in vascular and endosteal niches, an apoptotic assay was performed at day 1 post 5FU treatment. It was found that the apoptotic CD31⁺VE-cadherin⁺ cells greatly increased 1 day post 5FU (FIG. 18F), consistent with previous reports of disrupted blood vessel structure post 5FU (Dominici et al., 2009; Sugimura et al., 2012). To study the endosteal niche, the N-cad-tdTomato (N-cad-TdT) mouse line was established in which the Tomato⁺ cells report N-cad expression in both central marrow and bone surface (FIG. 24J). A marked increase of apoptotic N-Cad driven Tomato⁺ cells in the central marrow was noticed, whereas the Tomato⁺ cells in endosteal zone remained stable 1 day post 5FU (FIG. 18G). At day 3 post 5FU treatment when hematopoietic regeneration started, N-Cad driven Tomato⁺ cells in central marrow as well as in bone surface increased by 2.2-fold and 1.7-fold respectively (FIG. 18H, FIG. 24K). The number of CD31⁺ vessel cells also increased but showed a dilated and damaged architecture (FIG. 18I). Taken together, previous reports and this data showed that vessels and associated stromal cells in the perivascular niche, including LepR⁺ and N-cad⁺ cells, suffered immediate damage and were sensitive to 5FU stress, whereas N-cad⁺ endosteal stromal cells remained stable.

The data partially explain previous findings that most HSCs are predominantly distributed in perivascular and sinusoidal zones (Acar et al., 2015; Chen et al., 2016; Kunisaki et al., 2013). pHSCs which account for 51.3% of the quiescent HSC population are near perivascular and sinusoid zones in homeostasis. Under stress, however, rHSCs reside closer to the bone surface survive chemotherapeutic stress. Collectively, the data indicate that the endosteal niche plays a critical role in protecting rHSCs from chemotherapy.

Example B-3

N-Cad⁺ Niche Cells Maintain Functional HSCs Including rHSCs in Bone Marrow

Though N-cad⁺ stromal cells were the first identified HSC niche cells, and though N-cad⁺ stromal cells at the endosteal niche were resistant while Osx⁺ osteoblasts were sensitive to chemotherapy, a direct evidence for N-cad⁺ functionally supporting HSCs was still missing due to lack of proper genetic mouse lines. According to aspects herein, the N-cad-CreER^Tline was generated (FIG. 25A) and showed that N-cad⁺ stromal cells gave rise to both Col2.3-GFP⁺ osteoblastic cells and perivascular cells (FIG. 25B). A previous study showed that depleting the mature Col2.3-GFP⁺ osteoblasts did not affect overall engraftment of BM cells (Ding et al., 2012; Greenbaum et al., 2013), but only caused impaired regenerative capacity of a subset of LT-HSCs (Bowers et al., 2015). Though N-cad⁺ stromal cells developmentally proceed Osx⁺ osteoprogenitor cells, which further give rise to mature Col2.3⁺ osteoblasts, a functional contribution to HSC maintenance by N-cad⁺ stromal cells is unknown. To investigate the HSC niche role of N-cad⁺ cells, N-cad-CreER^Tinduced DTR (encoding diphtheria toxin receptor) line (N-cad-CreER^T;iDTR) was generated, in which N-cad⁺ niche cells were rendered sensitive to diphtheria toxin (DT). Three tamoxifen (TMX) injections were administered and followed with intraperitoneal injections of DT (one injection every other day) to the N-cad-CreER^T;iDTR mice and analyzed them on the first day after the last injection (FIG. 19A). The efficient ablation was observed of N-cad⁺ stromal cells in N-cad-CreER^T;iDTR; R26-tdT compared to N-cad-CreER^T; R26-tdT mice treated concurrently with DT (FIG. 19B). Whether N-cad⁺ cell ablation would affect HSCs in vivo was further examined. After ablating N-cad⁺ cells, no significant change was observed of cellularity in BM compared to controls (FIG. 19C). However, the numbers of HSCs were dramatically reduced: rHSCs (65.0% reduction), pHSCs (60.0% reduction), ST-HSCs (59.6% reduction), and MPPs (29.3% reduction) (FIG. 19D). This indicated that N-cad⁺ niche cells contributed to the most primitive, including reserve, HSC maintenance.

A transplantation assay was also performed to test the functional HSC numbers in N-cad⁺ stromal cell ablated mice. It was found that bone marrow cells from N-cad⁺ stromal cell ablated mice gave significantly lower levels of donor cell reconstitution (28.3% reduction at 20 weeks) (FIG. 19E) with reduced myeloid cell production (24.5% to 17.1%) (FIG. 19F). To further investigate whether N-cad⁺ niche cells contributed to maintaining the long-term self-renewal of HSCs, a secondary transplantation was conducted at 20 weeks post the primary transplantation. It was observed that BM cells from the N-cad⁺ stromal cell ablated mice had deeper reduction of donor cell reconstitution capacity in the secondary transplantation (40.5% reduction at 16 weeks) (FIG. 19G), although they were capable of multilineage reconstitution (FIG. 19H).

Furthermore, we found that conditional knockout of Cxcl12 from N-cad⁺ stromal cells significantly reduced pHSCs (48% reduction) and ST-HSCs (28.8% reduction), but no significant reduction was observed in rHSCs. There was a slight albeit insignificant increase of MPPs. (FIG. 19I). Conditional deletion of SCF from N-cad⁺ stromal cells significantly reduced rHSCs (60% reduction), pHSCs (38.6% reduction), and ST-HSCs (53.7% reduction), but with a slight increase in MPPs (FIG. 19J). Overall, we provided the first functional evidence that N-cad⁺ niche cells contributed to HSC maintenance, including rHSCs, via producing maintenance factors.

Example B-4

Transcriptome Analysis for Hematopoietic Cells and their BM Niche Cells

To understand the molecular mechanisms governing how different niche cells contribute to HSC subpopulation regulation, a transcriptome profiling analysis was performed on 4 types of hematopoietic stem and progenitor cells (HSPCs) during homeostasis and on rHSCs at day 3 post 5FU, as well as on 10 types of BM niche cells. The BM niche cells were harvested from different niche zones of endosteum (B) and central bone marrow (M) (FIG. 26A-B). The pearson distance tree and principal component analysis (PCA) data showed that rHSCs and pHSCs shared a unique transcriptome profiling compared to ST-HSCs and MPPs (FIG. 20A). Interestingly, rHSCs post 5FU treatment appeared to be closer to pHSCs (FIG. 20B), suggesting that surviving rHSCs were primed for activation to support subsequent hematopoietic regeneration post chemotherapy.

It was found that the rHSCs enriched most of the published HSC specific markers, such as Slamf1 (CD150), H19, Ctnnal1 (α-Catulin), Fgd5, vWF, Tek(Tie2), Procr(Epcr), Hoxb5 and Meg3 (or Gtl2) (Acar et al., 2015; Chen et al., 2016; Qian et al., 2015; Sanjuan-Pla et al., 2013; Venkatraman et al., 2013). Particularly, vWF in rHSCs were 6.1-fold higher than in pHSCs, suggesting that rHSCs enriched most of the vWF⁺ HSCs which reside at the apex of the HSC hierarchy (Sanjuan-Pla et al., 2013). Consistently, progenitor signature genes such as CD34, CD48, and Flt3(Flk2) and Itga2 (CD49b) had low expression in rHSCs (Acar et al., 2015; Chen et al., 2016; Gazit et al., 2014). Interestingly, rHSCs post 5FU had high expression levels of CXCR4, Ctnnal1 (α-Catulin)(Park et al., 2002), Esam (Endothelial cell-selective adhesion molecule) and CD150 (encoding Slamf1, signaling lymphocytic activated molecule), consistent with their functions associated with the converted primed state (FIG. 20E).

In niche cell analysis, it was found the N-cad driven tdTomato (N-cad-TdT) (M) had a very similar transcriptome profile compared to other niche cells with mesenchymal stem cell (MSC) potential such as LepR, Cxcl12-RFP, and Nestin-GFP cells in both pearson distance tree (FIG. 20C) and PCA analysis (FIG. 20D), suggesting that N-cad⁺ cells might have MSC potential and similar function in regulating HSCs. Interestingly, NG2-RFP cells had a very similar transcriptome profile to Col2.3-GFP⁺ osteoblasts (FIG. 20C-D). It was also found that NG2-RFP cells were predominantly restricted to endosteum in the epiphysis or diaphysis, though they were also detected in the peri-arterial region (FIG. 26C).

It was found that Pecam (CD31), CDH5 (VE-cadherin) were enriched in Pecam-GFP⁺ endothelial cells. PF4 and Adgre1 were enriched in MKs and macrophages (Macs). Cspg4 (NG2) was enriched in NG2-RFP cells. Nestin-GFP cells did not have endogenous Nestin (Nes) expression, consistent with previous reports (Ding et al.,2012; Greenbaum et al., 2013). Interestingly, it was found that several marker genes such as KitI (SCF), LepR, Cdh2 (N-cadherin, N-CAD), Cxcl12, Pdgfrα were broadly expressed in perivascular niche cells (FIG. 20F). N-cad-TdT(B) cells isolated from the endosteal zone also had high levels of SCF, Cxcl12, LepR and Pdgfrα expression compared to other cells from the endosteal zone, such as Col2.3-GFP and NG2-RFP cells (FIG. 20F). Our GO term analysis showed that Col2.3-GFP, Pecam-GFP, MK and Mac cells highly enriched RNA metabolism, protein metabolism, other metabolism pathways and translation activities, indicating that these cells were in a relatively active functional state compared to N-cad-TdT cells and other perivascular zone cells which were in a relatively lower metabolic state. Endothelial cells and perivascular cells had immune system process and stress response activities (FIG. 20G). Because N-cad⁺ cells had transcriptome profiles similar to other MSCs such as LepR⁺, Cxcl12-RFP, and Nestin-GFP cells, we next analyzed and compared stromal development related genes from different niche cells (FIG. 20H). It was found that Col2.3-GFP cells enriched osteoblast gene Col1a and progenitor cell gene Ly6a (SCA1) (Yang et al., 2014). N-Cad-TdT(B) cells from the endosteal region enriched chondrocyte genes Spock1, Col2a1 and Col1a2 and bone development genes such as Tnc. Interestingly both N-Cad-TdT(B) and N-Cad-TdT(M) enriched most of the mesenchymal stem and progenitor cell (MSPC) genes such as Prrx1, Pdgfr6, Pdgfrα, Sp7, Sox9 and Grem1. Marrow harvested NG2-RFP cells also had higher levels of MSPC gene expression, such as Alcam, Sp7, Mcam, Sox9 and Gli1, but with high level osteoblast and chondrocyte related genes such as Col1a1, Col2a1, and Col1a2. It was also found that among all perivascular niche cells, LepR⁺ cells exclusively enriched Gla as well as several osteoblast and chondrocyte markers such as Atf4 and Tnc. Cxcl12-RFP cells significantly enriched MSPC gene Itgav. Furthermore, Grem1 was enriched in N-Cad-TdT(B) and in all perivascular niche cells but not in NG2-RFP cells.

Consistent with earlier data, Col2.3-GFP cells were enriched for more mature osteo-lineage genes (Dmp1, Col1a1, Spp1, Bglap). Nestin-GFP, Cxcl12-RFP and LepR were consistent with their known role in enriching MSC genes (Prrx1, Pdgfrα, Itgb1, Grem1 (FIG. 20H). Interestingly, apart from expressing MSC genes, N-Cad-TdT(M) and N-Cad-TdT(B) were enriched for chondrogenic (Sox9, Col2a1 and Tnc), adipogenic (Cebpα, Pparγ, Adipoq) and osteogenic (Col1a1, Runx2) genes, suggesting a tri-potential nature of N-Cad⁺ stromal cells. Surprisingly, NG2-RFP was enriched for both bone development genes (Dmp1, Bglap, Sp7, Runx2, Col1a1) and chondrogenic genes (Sox9, Col2a1 and Tnc), suggesting an osteo-chondrogenic role (FIG. 26D-E). These data strongly indicated the heterogeneity of the MSPCs in BM.

Example B-5

N-Cad-CreER^TInduced Reporter Cells Largely Overlap with LepR⁺ and Cxcl12⁺ Stem/Stromal Cells

To confirm the transcriptional analysis, N-cad in vivo lineage tracing was performed using the TdT or ZsG reporter, and found that, at day 3 post induction, N-cad-CreER^Tlineage traced cells partially overlapped with Cxcl12-RFP and Nestin-GFP cells (FIG. 20I). Moreover, 98.3% and 97.9% N-cad-CreER^Tderived cells were positive for LepR and Pdgfrα expression respectively (FIG. 20J). This result further supported that N-cad⁺ stromal cells have MSC potential as suggested by the transcriptome analysis. To analyze the lineage potential of N-cad⁺ stromal cells, colony-forming unit-fibroblasts (CFU-F) assay was performed to test their proliferating capacity. It was found that the majority of reported niche cells, whether from endosteal zone or from perivascular zone, had one out of fewer than 10 cells with CFU-F activity, apart from Nestin-GFP⁺ cells, which had only one out of 17.6 cells with CFU-F activity. N-cad⁺ cells from bone had one cell with CFU-F activity out of 8.79 cells. N-cad-CreER^Tderived bone cells had 1 in 10.7 cells with CFU-F activity, and N-cad-CreER^Tderived marrow cells had 1 in 7.37 cells. (FIG. 20K). Most CFU-F colonies maintained tdTomato signals (FIG. 21A-B), suggesting that N-cad⁺ cells were the main source of MSPCs with CFU-F activity.

Example B-6

N-Cad⁺ Stromal Cells Give Rise to Osteoblasts, Chondrocytes, and Adipocytes In Vitro and In Vivo

To test the MSC potential of N-cad⁺ cells, in vitro differentiation assay was performed by splitting cells obtained from individual CFU-F colonies formed by N-cad⁺ cells into three aliquots and sub-cloned them into cultures permissive for bone, fat, or cartilage cell differentiation. It was found that Tomato⁺ cells underwent multilineage differentiation, giving rising to Alkaline phosphate-positive osteoblastic cells, Oil-red-O-positive adipocytes, and Aggrecan-stained and Toluidine-blue-positive chondrocytes (FIG. 21C-E).

To characterize in vivo function of N-cad⁺ stromal cells, the dynamic anatomical distribution of N-cad derived cells was analyzed after one dose of TMX treatment (FIG. 21F, G). Interestingly, detected N-cad derived cells were detected in metaphysis of trabecular as early as 6 hours post TMX, but only very few cells were seen in central marrow, suggesting that a large portion of N-cad⁺ cells originated from the endosteal region (FIG. 21H). It was also observed that the number of N-cad derived cells increased by 2.2±0.2-fold in trabecular bone region from 1 to 2 weeks post TMX treatment and remained stable afterwards, in cortical bone region, the cell number increased by 2.95±0.2-fold at 2 weeks post TMX, but then declined at 6 weeks post TMX, suggesting that N-cad⁺ cells in trabecular bone region were more quiescent compared to cortical bone region. Moreover, N-cad derived cells continuously increased in central marrow up to 6 weeks, suggesting that N-cad derived cells might be proliferating and differentiating within this area (FIG. 21I).

Next, an in vivo lineage tracing assay was performed and it was observed that N-cad⁺ cells generated Col2.3-GFP⁺ osteoblasts (Dacic et al., 2001) in a time dependent manner (FIG. 22A, FIG. 27A-B). N-cad-derived osteoblasts were detected in the peri-trabecular region as early as 6 hours post TMX induction (FIG. 22B) and in the compact bone region at 14 hours post TMX induction (FIG. 22C). Further analysis showed that at 6 hours post TMX, immature N-cad⁺ Col2.3⁻ cells (1.1%±0.2%) were 10-times more enriched in trabecular bone region compared to cortical bone region (0.11%±0.04%) (FIG. 22D, E). Consistently, more immature N-cad⁺Col2.3⁻ cells were enriched in trabecular bone region compared to compact bone region at 24 hours and 2 weeks post TMX induction (FIG. 22F). At 4 weeks post TMX induction, a large portion of Col2.3-GFP cells (72%±6%) were derived from N-cad⁺ cells (FIG. 22G).

It was further observed that at 4 weeks post TMX injection, N-cad⁺ stromal cells generated adipocytes in the trabecular bone region, particularly the endosteal cells, (FIG. 22H), and this was further confirmed by BODIPY lipid probe staining (FIG. 22I). Interestingly, the frequency of N-cad⁺ derived adipocytes increased quickly initially and declined later on, as evidenced by the quantification that 39.3%±4.5%, 77.1%±6.7% and 17.2%±3% of N-cad⁺ derived cells were observed at 6, 14 and 24 hours respectively after TMX induction, suggesting that adipocytes might have a frequent turnover rate (FIG. 22J-K, FIG. 27C-E). Collectively, the data demonstrated that N-cad⁺ stromal cells generated both osteoblast and adipocyte lineages in vivo during homeostasis in adult mice.

Furthermore, it was found that Ncad+ MSCs were enriched >10-fold as compared to other markers. For example, as shown in FIG. 30, just 10K Ncad+ hMSCs produced even higher human thrombopoietin (THPO or TPO) than total hMSCs in vivo. Similarly, Ncad− hMSCs exhibited a function similar to the total hMSCs. Accordingly, isolating MSC with N-cadherin antibodies can provide for enrichment of the MSC population.

Example B-7

N-Cad⁺ Stromal Cells Give Rise to Chondrocytes During Development and Post Injury

Chondrogenesis is active in fetal development and rarely active in adulthood (Raghunath et al., 2005; Sophia Fox et al., 2009). In mice with TMX induction at postnatal day 2 (P2), no Tomato expression was detected among Aggrecan⁺ chondrocytes in the femur of N-cad-CreER^T; R26-tdT mice (FIG. 28A) despite Tomato⁺ osteoblasts and perichondrocytes being adjacent to the growth plate. After N-cad-CreER^T; R26-tdT mice were induced with TMX at 1 or 2 weeks when the growth plate develops, Tomato⁺ chondrocytes in cartilages of femur or tibia remained undetectable (data not shown). TMX induction was started to N-cad-CreER^T; R26-tdT mice at embryonic stage E12.5, when MSCs were undergoing condensation and chondrocyte differentiation in the fetal bone (FIG. 23A). It was found that the majority of undifferentiated N-cad derived tdT⁺ cells were located peripherally, with differentiated N-cad derived Tomato⁺ chondrocytes located in the central region of the rib at E14.5 embryos (FIG. 23B). Importantly, in the trabecular region of femur at P2, N-cad derived chondrocytes were detected in both the columnar zone where the secondary ossification center forms, and in the superficial zone, which is imperative to resist the sheer force for deeper layer protection (FIG. 23C, D). Consistently, N-cad derived adipocytes were detected at 2 months and up until 10 months after birth (FIG. 23E, FIG. 28B), suggesting that N-cad⁺ cells contributed to early and long-term adipogenesis during development. However, N-cad⁺ cells derived Osteopontin⁺ osteoblast and osteocyte were detected at 2 months after birth but not at early P2 (FIG. 23F, FIG. 28C). These data demonstrated that embryonic N-cad-derived cells gave rise to all three osteo-adipogenic-chondrogenic lineages, and only N-cad⁺ at embryonic stage could efficiently form chondrocytes.

Whether N-cad⁺ cell could generate chondrocytes in adult mice post injury was next investigated. Cartilage perforation was performed in N-cad-CreER^T; R26-tdT mice that received TMX induction at E12.5 (FIG. 23G). At 2 to 3 weeks post cartilage damage, we found that N-cad derived chondrocytes were clustered in the callus at the damage site with a 2-fold increase compared to undamaged control mice (FIG. 23H-1). The femorotibial joint was swollen due to articular cartilage damage in mice post-surgery (FIG. 28D). Alcian blue positive cells with a typical chondrocyte feature were dramatically increased in the post injury region (FIG. 23J), indicating that N-cad⁺ MSCs quickly regenerated chondrocytes in response to injury.

Taken together, the data proved that N-cad-derived cells induced at the fetal stage (E12.5) could give rise to chondrocyte progenitor in adults and support chondrocyte regeneration in response to injury.

Discussion

rHSCs vs. pHSCs

The heterogeneity of HSCs has been widely studied. HSCs can be maintained in either active, quiescent or deeply quiescent states. It is well known that the quiescence characterization of HSCs is functionally related to their long-term self-renewal potential (Foudi et al., 2008; Wilson et al., 2008). However, how the quiescent HSC population overcomes the consequence of myeloablation in vivo is an unanswered question. In spite of their quiescence, the majority of HSCs cannot survive chemotherapeutic stress such as 5FU (Longley et al., 2003). According to aspects herein, it was found that a small portion of the HSC subpopulation could survive 5FU treatment in primary mice and were resistant to 5FU treatment in transplantation model. Thus, this HSC subpopulation is defined as rHSCs, while other quiescent but chemotherapeutic sensitive HSC subpopulations are defined as pHSCs (Li and Clevers, 2010). Although both HSC subpopulations supported long-term hematopoiesis in transplantation experiments, pHSCs rarely gave rise to rHSCs, whereas the latter were able to give rise to pHSCs. Mechanistically, it was found that rHSCs have an attenuated DNA repair system compared to pHSCs during homeostasis, but rHSCs can quickly activate their DNA repair pathways and the stress-response program to survive chemotherapeutic stress, and support subsequent hematopoiesis to overcome the consequence of myeloablation.

Niche Matters in Term of Conferring Resistance to Chemotherapy

To explore an extrinsic mechanism underlying the chemo-resistance of rHSCs, an idea was tested that they were preserved in a specific microenvironment in BM. Using whole mount HSC staining (Kunisaki et al., 2013), it was first observed that the bulk of HSCs were associated with vessels and MKs as previously reported. However, it was surprisingly found that the rHSCs were predominantly associated with the endosteal niche compared to pHSCs during homeostasis and post 5FU treatment. This indicated that the endosteal niche could form a distinct BM microenvironment to protect rHSCs from chemotherapeutic stress. Consistently, upon chemotherapeutic stress, most rHSCs were protected in the endosteal niche that enriches chemo-resistant N-cad⁺ stromal cells, whereas the vessel and perivascular cells were sensitive to 5FU treatment, accounting for a large loss of pHSCs induced by chemotherapy. The whole mount HSC staining was done in sternum which has abundant bone branches and extensions inside the marrow. This feature makes it very similar to the trabecular bone region in the femur. The transplantation assay showing that depletion of N-cad⁺ niche cells affected HSC maintenance, including rHSCs, supports this notion. HSC quiescence also correlated with their low metabolic state, which may be considered analogous to ‘sleeping’, and was termed as HSC dormancy or hibernation (Takubo et al., 2013; Wilson et al., 2008; Yamazaki et al., 2011). However, it was further showed according to aspects herein that quiescence was not the only mechanism underlying drug resistance; instead, both the intrinsic stress-response program and the extrinsic niche protection contributed to drug resistance.

Identity and Function of N-Cad Stromal Cell in HSC Maintenance in the BM Niche

N-cad⁺ stromal cells were the first identified HSC niche cells (Zhang et al., 2003) and confirmed by subsequent studies (Arai et al., 2004; Sugiyama et al., 2006). N-cad⁺ cells were initially proposed as osteoblastic progenitor cells based on their endosteal location. According to apects herein, by using two reporter lines, it was found that N-cad⁺ stromal cells were distributed in both endosteal region and perivascular sites. More intriguingly, it was found that the majority of N-cad⁺ cells overlapped with LepR⁺ cells and Pdgfrα⁺ cells. Transcriptional analysis showed that N-cad⁺ cells, LepR⁺ cells, Cxcl12-RFP (CAR) cells and Nestin-GFP cells had a very similar gene expression pattern. The data strongly indicated that the long-standing controversy of HSC niche concepts might very likely be due to different cell markers being used rather than to their cellular identities.

To determine the identity of N-Cad⁺ stromal cells, their regional distribution was visualized relative to other known niche cells in the bone marrow using different niche reporter mice. N-Cad⁺ stromal cells generated 72%±6% Col2.3-GFP⁺ cells; however, 9.3%±4.7% N-cad⁺Col2.3GFP⁻ cells were also detected in the endosteal region. These immature cells accounted for the primitive MSCs, which could explain the insufficient efficiency of Col2.3-Cre genetic model in HSC niche function studies (Ding and Morrison, 2013; Ding et al., 2012; Greenbaum et al., 2013). Though both N-Cad-TdT⁺ and Nestin-GFP⁺ were enriched in the trabecular region, N-Cad-TdT⁺ was concentrated in the trabecular region where engraftment of transplanted HSCs was detected as previously reported (Nilsson et al., 2001; Xie et al., 2009) and survived after stress as observed here. All these data indicated that although N-cad⁺ cells share a similar transcriptome profile with other MSCs, their anatomic distribution may indicate their unique HSC niche function; indeed, it was shown that the N-cad⁺ endosteal niche cell plays a critical role in preserving rHSCs.

By using an inducible DTR system, it was found that ablation of N-cad⁺ cells eliminated both pHSCs and rHSCs in BM. This could be explained by the anatomical distribution of N-cad⁺ niche cell in both endosteal and perivascular zones. Furthermore, it was shown that N-cad expression could be detected in a subset of HSCs; however, the N-cad-TdT reporter mouse lines did not support this observation (data not shown). This could be partially explained by the inconsistency between their protein and transcription levels, because another mouse line of N-cad-mCherry (fusion at protein level) indeed had a small subset of HSCs (CD49b⁻CD34⁻Flt2⁻LSK) showing a low level of N-cad expression (primary observation). The functional transplantation data showed that ablation of N-cad⁺ niche cells resulted in the reduced HSCs including rHSCs. By deletion of Cxcl12 and SCF from N-cad⁺ cells, it was found N-cad⁺ stromal cells contributed to HSC maintenance and regulation by producing these two factors. Overall, aspects herein demonstrate that N-cad⁺ stromal cells function as MSCs and support primitive HSC maintenance, especially under stress.

Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer, but the limited number of HSCs in a single hUCB unit can restrict its widespread use. Although extensive efforts have developed multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it has been unknown whether simultaneously manipulating a large number of targets essential for stem cell self-renewal could be achievable. Recent studies have emerged that N⁶-methyladenosine (m⁶A) modulates expression of a group of mRNAs critical for stem cell fate determination by influencing their stability. Among several m⁶A readers, Ythdf2 is well recognized to promote the targeted mRNA decay. However, the physiological functions of Ythdf2 on adult stem cells are still elusive. Embodiments herein demonstrate that conditional knockout (KO) mouse Ythdf2 increased phenotypic and functional HSC numbers, but neither skewed lineage differentiation nor led to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to over 10-fold increase in ex vivo expansion of hUCB HSCs, 5-fold increase in colony-forming units (CFUs), and more than 8-fold increase in functional hUCB HSCs in the 2 rounds of limiting dilution transplantation assay. Mechanistically, m⁶A mapping of RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed m⁶A enrichment on mRNAs encoding transcription factors critical for stem cell self-renewal. These m⁶A-marked mRNAs were recognized by Ythdf2 and underwent mRNA decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, leading to an increase in protein levels and facilitating HSC expansion which can be rescued by knockdown the mRNA, such as Tal1 mRNA. Therefore, embodiments show the function of Ythdf2 in adult stem cells maintenance and identify an important role of Ythdf2 in regulating HSC ex vivo expansion via the mechanism of controlling the stability of multiple mRNAs critical for HSC self-renewal, thus having a strong potential for future clinical applications.

Furthermore, regulation of hematopoietic stem cells (HSCs) by the bone marrow (BM) niches has been substantially studied, however, whether and how HSC subpopulations are distinctively regulated by different BM niches remain largely unclear. Here, reserve HSCs (rHSCs) have been functionally distinguished from primed HSCs (pHSCs) and their respective BM niches have been further examined. It has been found that both pHSCs and rHSCs could support long-term hematopoiesis under homeostasis; however, pHSCs were sensitive to chemotherapy, whereas rHSCs survived chemotherapy and supported subsequent regeneration after myeloablation. The whole-mount HSC distribution study revealed that rHSCs were preferentially maintained in the endosteal region that enriches N-cadherin⁺ bone-lining cells during homeostasis and post-chemotherapy. pHSCs were predominantly associated with blood vessels which were vulnerable to chemotherapy compared to bone. Transcriptome profiling and in vivo lineage tracing results showed N-cadherin⁺ stromal cells to be functional mesenchymal stem cells, which gave rise to osteoblasts, adipocytes, and chondrocytes during development and regeneration. Finally, it was demonstrated that ablation of N-cadherin⁺ niche cells or deletion of either Scf or Cxcl12 from N-cadherin⁺ niche cells affected HSC number and maintenance.

Example C

Expansion of CAR-T Cells Using shRNA

The effect of manipulating Ythdf2 on the expansion of CAR-T cells is being assessed using lentivirus driven human Ythdf2 shRNAs. Successful cloning of YTHDF2 shRNA in a CAR-T lentivector has occurred. The lentivirus has been used to infect human CAR-T cells, and the expansion of the human CAR-T cells is in progress. The expansion is expected to take days to weeks for results. It is believed that significantly enhanced expansion will be demonstrated in the lentivirus-infected CAR-T cell population as compared to a CAR-T control population.

All sequencing data, including the m⁶A-seq, irCLIP-seq and RNA-seq datasets, are available through the Gene Expression Ombibus (GEO) under accession GSE107957.

Original Data Repository at www.stowers.org/research/publications/LIBPB-1248.

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The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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TABLE S1

Table S1: Key transcription factors critical for HSC self-

renewal and maintenance are labeled by m6A in HSPCs.

Gata2

Gfi1

Pbx1

Lmo2

Etv6

Erg

Runx1

Tal1

Hoxa9

Meis1

Kmt2a

Kmt2b

Kmt2c

Kmt2d

Bmi1

Sox4

Evi1

Stat3

Stat5a

Hoxb4

Zfx

Foxo3

Foxp1

TABLE S2

Ythdf2 targeted mRNAs from three irCLIP-seq replicates.

GeneID
Name
Biotype
Description

ENSMUSG
Atp6v1h
protein_coding
ATPase, H+ transporting, lysosomal V1 subunit H

00000033793

[Source:MGI Symbol;Acc:MGI:1914864]

ENSMUSG
Arfgef1
protein_coding
ADP-ribosylation factor guanine nucleotide-exchange

00000067851

factor 1(brefeldin A-inhibited) [Source:MGI

Symbol;Acc:MGI:2442988]

ENSMUSG
Ncoa2
protein_coding
nuclear receptor coactivator 2 [Source:MGI

00000005886

Symbol;Acc:MGI:1276533]

ENSMUSG
Tram1
protein_coding
translocating chain-associating membrane protein 1

00000025935

[Source:MGI Symbol;Acc:MGI:1919515]

ENSMUSG
Mcm3
protein_coding
minichromosome maintenance deficient 3 (S.

00000041859

cerevisiae) [Source:MGI Symbol;Acc:MGI:101845]

ENSMUSG
Bai3
protein_coding
brain-specific angiogenesis inhibitor 3 [Source:MGI

00000033569

Symbol;Acc:MGI:2441837]

ENSMUSG
Phf3
protein_coding
PHD finger protein 3 [Source:MGI

00000048874

Symbol;Acc:MGI:2446126]

ENSMUSG
Bag2
protein_coding
BCL2-associated athanogene 2 [Source:MGI

00000042215

Symbol;Acc:MGI:1891254]

ENSMUSG
Fam168b
protein_coding
family with sequence similarity 168, member B

00000037503

[Source:MGI Symbol;Acc:MGI:2448487]

ENSMUSG
Uggt1
protein_coding
UDP-glucose glycoprotein glucosyltransferase 1

00000037470

[Source:MGI Symbol;Acc:MGI:2443162]

ENSMUSG
Sema4c
protein_coding
sema domain, immunoglobulin domain (Ig),

00000026121

transmembrane domain (TM) and short cytoplasmic

domain, (semaphorin) 4C [Source:MGI

Symbol;Acc:MGI:109252]

ENSMUSG
Inpp4a
protein_coding
inositol polyphosphate-4-phosphatase, type I

00000026113

[Source:MGI Symbol;Acc:MGI:1931123]

ENSMUSG
Lipt1
protein_coding
lipoyltransferase 1 [Source:MGI

00000037216

Symbol;Acc:MGI:3645211]

ENSMUSG
Eif5b
protein_coding
eukaryotic translation initiation factor 5B

00000026083

[Source:MGI Symbol;Acc:MGI:2441772]

ENSMUSG
Aff3
protein_coding
AF4/FMR2 family, member 3 [Source:MGI

00000037138

Symbol;Acc:MGI:106927]

ENSMUSG
Map4k4
protein_coding
mitogen-activated protein kinase kinase kinase kinase

00000026074

4 [Source:MGI Symbol;Acc:MGI:1349394]

ENSMUSG
Tgfbrap1
protein_coding
transforming growth factor, beta receptor associated

00000070939

protein 1 [Source:MGI Symbol;Acc:MGI:2447427]

ENSMUSG
Nck2
protein_coding
non-catalytic region of tyrosine kinase adaptor

00000066877

protein 2 [Source:MGI Symbol;Acc:MGI:1306821]

ENSMUSG
1700019D03Rik
protein_coding
RIKEN cDNA 1700019D03 gene [Source:MGI

00000043629

Symbol;Acc:MGI:1914330]

ENSMUSG
Stk17b
protein_coding
serine/threonine kinase 17b (apoptosis-inducing)

00000026094

[Source:MGI Symbol;Acc:MGI:2138162]

ENSMUSG
Hecw2
protein_coding
HECT, C2 and WW domain containing E3 ubiquitin

00000042807

protein ligase 2 [Source:MGI

Symbol;Acc:MGI:2685817]

ENSMUSG
Ankrd44
protein_coding
ankyrin repeat domain 44 [Source:MGI

00000052331

Symbol;Acc:MGI:3045243]

ENSMUSG
Sf3b1
protein_coding
splicing factor 3b, subunit 1 [Source:MGI

00000025982

Symbol;Acc:MGI:1932339]

ENSMUSG
Hspd1
protein_coding
heat shock protein 1 (chaperonin) [Source:MGI

00000025980

Symbol;Acc:MGI:96242]

ENSMUSG
Mars2
protein_coding
methionine-tRNA synthetase 2 (mitochondrial)

00000046994

[Source:MGI Symbol;Acc:MGI:2444136]

ENSMUSG
Satb2
protein_coding
special AT-rich sequence binding protein 2

00000038331

[Source:MGI Symbol;Acc:MGI:2679336]

ENSMUSG
Bzw1
protein_coding
basic leucine zipper and W2 domains 1 [Source:MGI

00000051223

Symbol;Acc:MGI:1914132]

ENSMUSG
Cflar
protein_coding
CASP8 and FADD-like apoptosis regulator

00000026031

[Source:MGI Symbol;Acc:MGI:1336166]

ENSMUSG
Trak2
protein_coding
trafficking protein, kinesin binding 2 [Source:MGI

00000026028

Symbol;Acc:MGI:1918077]

ENSMUSG
Nop58
protein_coding
NOP58 ribonucleoprotein homolog (yeast)

00000026020

[Source:MGI Symbol;Acc:MGI:1933184]

ENSMUSG
Abi2
protein_coding
abl-interactor 2 [Source:MGI

00000026782

Symbol;Acc:MGI:106913]

ENSMUSG
Pard3b
protein_coding
par-3 partitioning defective 3 homolog B (C. elegans)

00000052062

[Source:MGI Symbol;Acc:MGI:1919301]

ENSMUSG
Eef1b2
protein_coding
eukaryotic translation elongation factor 1 beta 2

00000025967

[Source:MGI Symbol;Acc:MGI:1929520]

ENSMUSG
Klf7
protein_coding
Kruppel-like factor 7 (ubiquitous) [Source:MGI

00000025959

Symbol;Acc:MGI:1935151]

ENSMUSG
Erbb4
protein_coding
v-erb-a erythroblastic leukemia viral oncogene

00000062209

homolog 4 (avian) [Source:MGI

Symbol;Acc:MGI:104771]

ENSMUSG
Abca12
protein_coding
ATP-binding cassette, sub-family A (ABC1), member

00000050296

12 [Source:MGI Symbol;Acc:MGI:2676312]

ENSMUSG
Atic
protein_coding
5-aminoimidazole-4-carboxamide ribonucleotide

00000026192

formyltransferase/IMP cyclohydrolase [Source:MGI

Symbol;Acc:MGI:1351352]

ENSMUSG
Tns1
protein_coding
tensin 1 [Source:MGI Symbol;Acc:MGI:104552]

00000055322

ENSMUSG
Arpc2
protein_coding
actin related protein 2/3 complex, subunit 2

00000006304

[Source:MGI Symbol;Acc:MGI:1923959]

ENSMUSG
Aamp
protein_coding
angio-associated migratory protein [Source:MGI

00000006299

Symbol;Acc:MGI:107809]

ENSMUSG
Ctdsp1
protein_coding
CTD (carboxy-terminal domain, RNA polymerase II,

00000026176

polypeptide A) small phosphatase 1 [Source:MGI

Symbol;Acc:MGI:2654470]

ENSMUSG
Usp37
protein_coding
ubiquitin specific peptidase 37 [Source:MGI

00000033364

Symbol;Acc:MGI:2442483]

ENSMUSG
Rqcd1
protein_coding
rcd1 (required for cell differentiation) homolog 1 (S.

00000026174

pombe) [Source:MGI Symbol;Acc:MGI:1928902]

ENSMUSG
Zfp142
protein_coding
zinc finger protein 142 [Source:MGI

00000026135

Symbol;Acc:MGI:1924514]

ENSMUSG
Ttl14
protein_coding
tubulin tyrosine ligase-like family, member 4

00000033257

[Source:MGI Symbol;Acc:MGI:1914784]

ENSMUSG
Fam134a
protein_coding
family with sequence similarity 134, member A

00000049339

[Source:MGI Symbol;Acc:MGI:2388278]

ENSMUSG
Obsl1
protein_coding
obscurin-like 1 [Source:MGI

00000026211

Symbol;Acc:MGI:2138628]

ENSMUSG
Farsb
protein_coding
phenylalanyl-tRNA synthetase, beta subunit

00000026245

[Source:MGI Symbol;Acc:MGI:1346035]

ENSMUSG
Cul3
protein_coding
cullin 3 [Source:MGI Symbol;Acc:MGI:1347360]

00000004364

ENSMUSG
Dock10
protein_coding
dedicator of cytokinesis 10 [Source:MGI

00000038608

Symbol;Acc:MGI:2146320]

ENSMUSG
Trip12
protein_coding
thyroid hormone receptor interactor 12 [Source:MGI

00000026219

Symbol;Acc:MGI:1309481]

ENSMUSG
Cab39
protein_coding
calcium binding protein 39 [Source:MGI

00000036707

Symbol;Acc:MGI:107438]

ENSMUSG
Itm2c
protein_coding
integral membrane protein 2C [Source:MGI

00000026223

Symbol;Acc:MGI:1927594]

ENSMUSG
Psmd1
protein_coding
proteasome (prosome, macropain) 26S subunit, non-

00000026229

ATPase, 1 [Source:MGI Symbol;Acc:MGI:1917497]

ENSMUSG
Ncl
protein_coding
nucleolin [Source:MGI Symbol;Acc:MGI:97286]

00000026234

ENSMUSG
Ptma
protein_coding
prothymosin alpha [Source:MGI

00000026238

Symbol;Acc:MGI:97803]

ENSMUSG
Gigyf2
protein_coding
GRB10 interacting GYF protein 2 [Source:MGI

00000048000

Symbol;Acc:MGI:2138584]

ENSMUSG
Inpp5d
protein_coding
inositol polyphosphate-5-phosphatase D [Source:MGI

00000026288

Symbol;Acc:MGI:107357]

ENSMUSG
Atg16l1
protein_coding
autophagy-related 16-like 1 (yeast) [Source:MGI

00000026289

Symbol;Acc:MGI:1924290]

ENSMUSG
Agap1
protein_coding
ArfGAP with GTPase domain, ankyrin repeat and PH

00000055013

domain 1 [Source:MGI Symbol;Acc:MGI:2653690]

ENSMUSG
Gbx2
protein_coding
gastrulation brain homeobox 2 [Source:MGI

00000034486

Symbol;Acc:MGI:95668]

ENSMUSG
D130058E05Rik
protein_coding
RIKEN cDNA D130058E05 gene [Source:MGI

00000092627

Symbol;Acc:MGI:2444614]

ENSMUSG
Iqca
protein_coding
IQ motif containing with AAA domain [Source:MGI

00000026301

Symbol;Acc:MGI:1922168]

ENSMUSG
Cops8
protein_coding
COP9 (constitutive photomorphogenic) homolog,

00000034432

subunit 8 (Arabidopsis thaliana) [Source:MGI

Symbol;Acc:MGI:1915363]

ENSMUSG
Lrrfip1
protein_coding
leucine rich repeat (in FLII) interacting protein 1

00000026305

[Source:MGI Symbol;Acc:MGI:1342770]

ENSMUSG
Rnpepl1
protein_coding
arginyl aminopeptidase (aminopeptidase B)-like 1

00000026269

[Source:MGI Symbol;Acc:MGI:1914170]

ENSMUSG
Ppp1r7
protein_coding
protein phosphatase 1, regulatory (inhibitor) subunit

00000026275

7 [Source:MGI Symbol;Acc:MGI:1913635]

ENSMUSG
Hdlbp
protein_coding
high density lipoprotein (HDL) binding protein

00000034088

[Source:MGI Symbol;Acc:MGI:99256]

ENSMUSG
2-Sep
protein_coding
septin 2 [Source:MGI Symbol;Acc:MGI:97298]

00000026276

ENSMUSG
Dtymk
protein_coding
deoxythymidylate kinase [Source:MGI

00000026281

Symbol;Acc:MGI:108396]

ENSMUSG
Ppip5k2
protein_coding
diphosphoinositol pentakisphosphate kinase 2

00000040648

[Source:MGI Symbol;Acc:MGI:2142810]

ENSMUSG
Zcchc2
protein_coding
zinc finger, CCHC domain containing 2 [Source:MGI

00000038866

Symbol;Acc:MGI:2444114]

ENSMUSG
Phlpp1
protein_coding
PH domain and leucine rich repeat protein

00000044340

phosphatase 1 [Source:MGI

Symbol;Acc:MGI:2138327]

ENSMUSG
Bcl2
protein_coding
B cell leukemia/lymphoma 2 [Source:MGI

00000057329

Symbol;Acc:MGI:88138]

ENSMUSG
Cntnap5a
protein_coding
contactin associated protein-like 5A [Source:MGI

00000070695

Symbol;Acc:MGI:3643623]

ENSMUSG
Clasp1
protein_coding
CLIP associating protein 1 [Source:MGI

00000064302

Symbol;Acc:MGI:1923957]

ENSMUSG
Steap3
protein_coding
STEAP family member 3 [Source:MGI

00000026389

Symbol;Acc:MGI:1915678]

ENSMUSG
Dpp10
protein_coding
dipeptidylpeptidase 10 [Source:MGI

00000036815

Symbol;Acc:MGI:2442409]

ENSMUSG
Actr3
protein_coding
ARP3 actin-related protein 3 homolog (yeast)

00000026341

[Source:MGI Symbol;Acc:MGI:1921367]

ENSMUSG
Gpr39
protein_coding
G protein-coupled receptor 39 [Source:MGI

00000026343

Symbol;Acc:MGI:1918361]

ENSMUSG
Nckap5
protein_coding
NCK-associated protein 5 [Source:MGI

00000049690

Symbol;Acc:MGI:2686394]

ENSMUSG
Mgat5
protein_coding
mannoside acetylglucosaminyltransferase 5

00000036155

[Source:MGI Symbol;Acc:MGI:894701]

ENSMUSG
R3hdm1
protein_coding
R3H domain 1 (binds single-stranded nucleic acids)

00000056211

[Source:MGI Symbol;Acc:MGI:2448514]

ENSMUSG
Mcm6
protein_coding
minichromosome maintenance deficient 6 (MIS5

00000026355

homolog, S. pombe) (S. cerevisiae) [Source:MGI

Symbol;Acc:MGI:1298227]

ENSMUSG
Dars
protein_coding
aspartyl-tRNA synthetase [Source:MGI

00000026356

Symbol;Acc:MGI:2442544]

ENSMUSG
Thsd7b
protein_coding
thrombospondin, type I, domain containing 7B

00000042581

[Source:MGI Symbol;Acc:MGI:2443925]

ENSMUSG
Dyrk3
protein_coding
dual-specificity tyrosine-(Y)-phosphorylation

00000016526

regulated kinase 3 [Source:MGI

Symbol;Acc:MGI:1330300]

ENSMUSG
Eif2d
protein_coding
eukaryotic translation initiation factor 2D

00000026427

[Source:MGI Symbol;Acc:MGI:109342]

ENSMUSG
Rassf5
protein_coding
Ras association (RalGDS/AF-6) domain family member

00000026430

5 [Source:MGI Symbol;Acc:MGI:1926375]

ENSMUSG
Nucks1
protein_coding
nuclear casein kinase and cyclin-dependent kinase

00000026434

substrate 1 [Source:MGI Symbol;Acc:MGI:1934811]

ENSMUSG
Elk4
protein_coding
ELK4, member of ETS oncogene family [Source:MGI

00000026436

Symbol;Acc:MGI:102853]

ENSMUSG
Mdm4
protein_coding
transformed mouse 3T3 cell double minute 4

00000054387

[Source:MGI Symbol;Acc:MGI:107934]

ENSMUSG
Ppp1r15b
protein_coding
protein phosphatase 1, regulatory (inhibitor) subunit

00000046062

15b [Source:MGI Symbol;Acc:MGI:2444211]

ENSMUSG
Zc3h11a
protein_coding
zinc finger CCCH type containing 11A [Source:MGI

00000026464

Symbol;Acc:MGI:1917829]

ENSMUSG
Atp2b4
protein_coding
ATPase, Ca++ transporting, plasma membrane 4

00000026463

[Source:MGI Symbol;Acc:MGI:88111]

ENSMUSG
Kdm5b
protein_coding
lysine (K)-specific demethylase 5B [Source:MGI

00000042207

Symbol;Acc:MGI:1922855]

ENSMUSG
Ptpn7
protein_coding
protein tyrosine phosphatase, non-receptor type 7

00000031506

[Source:MGI Symbol;Acc:MGI:2156893]

ENSMUSG
Ipo9
protein_coding
importin 9 [Source:MGI Symbol;Acc:MGI:1918944]

00000041879

ENSMUSG
Nav1
protein_coding
neuron navigator 1 [Source:MGI

00000009418

Symbol;Acc:MGI:2183683]

ENSMUSG
Csrp1
protein_coding
cysteine and glycine-rich protein 1 [Source:MGI

00000026421

Symbol;Acc:MGI:88549]

ENSMUSG
Phlda3
protein_coding
pleckstrin homology-like domain, family A, member 3

00000041801

[Source:MGI Symbol;Acc:MGI:1351485]

ENSMUSG
Kif21b
protein_coding
kinesin family member 21B [Source:MGI

00000041642

Symbol;Acc:MGI:109234]

ENSMUSG
2310006M14Rik
protein_coding
RIKEN cDNA 2310006M14 gene [Source:MGI

00000087230

Symbol;Acc:MGI:1923672]

ENSMUSG
Ptprc
protein_coding
protein tyrosine phosphatase, receptor type, C

00000026395

[Source:MGI Symbol;Acc:MGI:97810]

ENSMUSG
Aspm
protein_coding
asp (abnormal spindle)-like, microcephaly associated

00000033952

(Drosophila) [Source:MGI Symbol;Acc:MGI:1334448]

ENSMUSG
Cfhr2
protein_coding
complement factor H-related 2 [Source:MGI

00000033898

Symbol;Acc:MGI:3611575]

ENSMUSG
Rgs2
protein_coding
regulator of G-protein signaling 2 [Source:MGI

00000026360

Symbol;Acc:MGI:1098271]

ENSMUSG
Rgs1
protein_coding
regulator of G-protein signaling 1 [Source:MGI

00000026358

Symbol;Acc:MGI:1354694]

ENSMUSG
Rgs18
protein_coding
regulator of G-protein signaling 18 [Source:MGI

00000026357

Symbol;Acc:MGI:1927498]

ENSMUSG
Fam5c
protein_coding
family with sequence similarity 5, member C

00000035131

[Source:MGI Symbol;Acc:MGI:2443035]

ENSMUSG
Pla2g4a
protein_coding
phospholipase A2, group IVA (cytosolic, calcium-

00000056220

dependent) [Source:MGI Symbol;Acc:MGI:1195256]

ENSMUSG
Tpr
protein_coding
translocated promoter region [Source:MGI

00000006005

Symbol;Acc:MGI:1922066]

ENSMUSG
Hmcn1
protein_coding
hemicentin 1 [Source:MGI Symbol;Acc:MGI:2685047]

00000066842

ENSMUSG
Ivns1abp
protein_coding
influenza virus NS1A binding protein [Source:MGI

00000023150

Symbol;Acc:MGI:2152389]

ENSMUSG
Rnf2
protein_coding
ring finger protein 2 [Source:MGI

00000026484

Symbol;Acc:MGI:1101759]

ENSMUSG
Fam129a
protein_coding
family with sequence similarity 129, member A

00000026483

[Source:MGI Symbol;Acc:MGI:2137237]

ENSMUSG
Edem3
protein_coding
ER degradation enhancer, mannosidase alpha-like 3

00000043019

[Source:MGI Symbol;Acc:MGI:1914217]

ENSMUSG
Rgl1
protein_coding
ral guanine nucleotide dissociation stimulator,-like 1

00000026482

[Source:MGI Symbol;Acc:MGI:107484]

ENSMUSG
Smg7
protein_coding
Smg-7 homolog, nonsense mediated mRNA decay

00000042772

factor (C. elegans) [Source:MGI

Symbol;Acc:MGI:2682334]

ENSMUSG
Dhx9
protein_coding
DEAH (Asp-Glu-Ala-His) box polypeptide 9

00000042699

[Source:MGI Symbol;Acc:MGI:108177]

ENSMUSG
Rgs8
protein_coding
regulator of G-protein signaling 8 [Source:MGI

00000042671

Symbol;Acc:MGI:108408]

ENSMUSG
Rgs16
protein_coding
regulator of G-protein signaling 16 [Source:MGI

00000026475

Symbol;Acc:MGI:108407]

ENSMUSG
Glul
protein_coding
glutamate-ammonia ligase (glutamine synthetase)

00000026473

[Source:MGI Symbol;Acc:MGI:95739]

ENSMUSG
A930039A15Rik
protein_coding
RIKEN cDNA A930039A15 gene [Source:MGI

00000078493

Symbol;Acc:MGI:3641728]

ENSMUSG
Gm11074
protein_coding
predicted gene 11074 [Source:MGI

00000079255

Symbol;Acc:MGI:3779301]

ENSMUSG
Cep350
protein_coding
centrosomal protein 350 [Source:MGI

00000033671

Symbol;Acc:MGI:1921331]

ENSMUSG
Tor1aip2
protein_coding
torsin A interacting protein 2 [Source:MGI

00000050565

Symbol;Acc:MGI:3582695]

ENSMUSG
Torlaip2
protein_coding
torsin A interacting protein 2 [Source:MGI

00000079252

Symbol;Acc:MGI:3582695]

ENSMUSG
Nphs2
protein_coding
nephrosis 2 homolog, podocin (human) [Source:MGI

00000026602

Symbol;Acc:MGI:2157018]

ENSMUSG
Soat1
protein_coding
sterol O-acyltransferase 1 [Source:MGI

00000026600

Symbol;Acc:MGI:104665]

ENSMUSG
Abl2
protein_coding
v-abl Abelson murine leukemia viral oncogene

00000026596

homolog 2 (arg, Abelson-related gene) [Source:MGI

Symbol;Acc:MGI:87860]

ENSMUSG
Fam20b
protein_coding
family with sequence similarity 20, member B

00000033557

[Source:MGI Symbol;Acc:MGI:2443990]

ENSMUSG
Ralgps2
protein_coding
Ral GEF with PH domain and SH3 binding motif 2

00000026594

[Source:MGI Symbol;Acc:MGI:1925505]

ENSMUSG
Rfwd2
protein_coding
ring finger and WD repeat domain 2 [Source:MGI

00000040782

Symbol;Acc:MGI:1347046]

ENSMUSG
Rc3h1
protein_coding
RING CCCH (C3H) domains 1 [Source:MGI

00000040423

Symbol;Acc:MGI:2685397]

ENSMUSG
Prdx6
protein_coding
peroxiredoxin 6 [Source:MGI

00000026701

Symbol;Acc:MGI:894320]

ENSMUSG
4930558K02Rik
protein_coding
RIKEN cDNA 4930558K02 gene [Source:MGI

00000086277

Symbol;Acc:MGI:1922618]

ENSMUSG
Pigc
protein_coding
phosphatidylinositol glycan anchor biosynthesis, class

00000026698

C [Source:MGI Symbol;Acc:MGI:1914542]

ENSMUSG
Prrc2c
protein_coding
proline-rich coiled-coil 2C [Source:MGI

00000040225

Symbol;Acc:MGI:1913754]

ENSMUSG
Kifap3
protein_coding
kinesin-associated protein 3 [Source:MGI

00000026585

Symbol;Acc:MGI:107566]

ENSMUSG
BC055324
protein_coding
cDNA sequence BC055324 [Source:MGI

00000041406

Symbol;Acc:MGI:3590554]

ENSMUSG
Atp1b1
protein_coding
ATPase, Na+/K+ transporting, beta 1 polypeptide

00000026576

[Source:MGI Symbol;Acc:MGI:88108]

ENSMUSG
Rcsd1
protein_coding
RCSD domain containing 1 [Source:MGI

00000040723

Symbol;Acc:MGI:2676394]

ENSMUSG
Cd247
protein_coding
CD247 antigen [Source:MGI Symbol;Acc:MGI:88334]

00000005763

ENSMUSG
Pou2f1
protein_coding
POU domain, class 2, transcription factor 1

00000026565

[Source:MGI Symbol;Acc:MGI:101898]

ENSMUSG
Uck2
protein_coding
uridine-cytidine kinase 2 [Source:MGI

00000026558

Symbol;Acc:MGI:1931744]

ENSMUSG
Aldh9a1
protein_coding
aldehyde dehydrogenase 9, subfamily A1 [Source:MGI

00000026687

Symbol;Acc:MGI:1861622]

ENSMUSG
Pbx1
protein_coding
pre B cell leukemia homeobox 1 [Source:MGI

00000052534

Symbol;Acc:MGI:97495]

ENSMUSG
Uhmk1
protein_coding
U2AF homology motif (UHM) kinase 1 [Source:MGI

00000026667

Symbol;Acc:MGI:1341908]

ENSMUSG
Fcgr3
protein_coding
Fc receptor, IgG, low affinity III [Source:MGI

00000059498

Symbol;Acc:MGI:95500]

ENSMUSG
Ndufs2
protein_coding
NADH dehydrogenase (ubiquinone) Fe-S protein 2

00000013593

[Source:MGI Symbol;Acc:MGI:2385112]

ENSMUSG
Pvrl4
protein_coding
poliovirus receptor-related 4 [Source:MGI

00000006411

Symbol;Acc:MGI:1918990]

ENSMUSG
Arhgap30
protein_coding
Rho GTPase activating protein 30 [Source:MGI

00000048865

Symbol;Acc:MGI:2684948]

ENSMUSG
Copa
protein_coding
coatomer protein complex subunit alpha [Source:MGI

00000026553

Symbol;Acc:MGI:1334462]

ENSMUSG
Dcaf8
protein_coding
DDB1 and CUL4 associated factor 8 [Source:MGI

00000026554

Symbol;Acc:MGI:91860]

ENSMUSG
Tagln2
protein_coding
transgelin 2 [Source:MGI Symbol;Acc:MGI:1312985]

00000026547

ENSMUSG
Cep170
protein_coding
centrosomal protein 170 [Source:MGI

00000057335

Symbol;Acc:MGI:1918348]

ENSMUSG
Sdccag8
protein_coding
serologically defined colon cancer antigen 8

00000026504

[Source:MGI Symbol;Acc:MGI:1924066]

ENSMUSG
Pppde1
protein_coding
PPPDE peptidase domain containing 1 [Source:MGI

00000026502

Symbol;Acc:MGI:1926075]

ENSMUSG
Hnrnpu
protein_coding
heterogeneous nuclear ribonucleoprotein U

00000039630

[Source:MGI Symbol;Acc:MGI:1858195]

ENSMUSG
Smyd3
protein_coding
SET and MYND domain containing 3 [Source:MGI

00000055067

Symbol;Acc:MGI:1916976]

ENSMUSG
Cnst
protein_coding
consortin, connexin sorting protein [Source:MGI

00000038949

Symbol;Acc:MGI:2445141]

ENSMUSG
Ahctf1
protein_coding
AT hook containing transcription factor 1 [Source:MGI

00000026491

Symbol;Acc:MGI:1915033]

ENSMUSG
Itpkb
protein_coding
inositol 1,4,5-trisphosphate 3-kinase B [Source:MGI

00000038855

Symbol;Acc:MGI:109235]

ENSMUSG
Parp1
protein_coding
poly (ADP-ribose) polymerase family, member 1

00000026496

[Source:MGI Symbol;Acc:MGI:1340806]

ENSMUSG
H3f3a
protein_coding
H3 histone, family 3A [Source:MGI

00000060743

Symbol;Acc:MGI:1097686]

ENSMUSG
BC031781
protein_coding
cDNA sequence BC031781 [Source:MGI

00000038806

Symbol;Acc:MGI:2384788]

ENSMUSG
Tmem63a
protein_coding
transmembrane protein 63a [Source:MGI

00000026519

Symbol;Acc:MGI:2384789]

ENSMUSG
Wdr26
protein_coding
WD repeat domain 26 [Source:MGI

00000038733

Symbol;Acc:MGI:1923825]

ENSMUSG
Lbr
protein_coding
lamin B receptor [Source:MGI

00000004880

Symbol;Acc:MGI:2138281]

ENSMUSG
Trp53bp2
protein_coding
transformation related protein 53 binding protein 2

00000026510

[Source:MGI Symbol;Acc:MGI:2138319]

ENSMUSG
Rab3gap2
protein_coding
RAB3 GTPase activating protein subunit 2

00000039318

[Source:MGI Symbol;Acc:MGI:1916043]

ENSMUSG
Eprs
protein_coding
glutamyl-prolyl-tRNA synthetase [Source:MGI

00000026615

Symbol;Acc:MGI:97838]

ENSMUSG
Ush2a
protein_coding
Usher syndrome 2A (autosomal recessive, mild)

00000026609

homolog (human) [Source:MGI

Symbol;Acc:MGI:1341292]

ENSMUSG
Kctd3
protein_coding
potassium channel tetramerisation domain

00000026608

containing 3 [Source:MGI Symbol;Acc:MGI:2444629]

ENSMUSG
Vash2
protein_coding
vasohibin 2 [Source:MGI Symbol;Acc:MGI:2444826]

00000037568

ENSMUSG
Ppp2r5a
protein_coding
protein phosphatase 2, regulatory subunit B (B56),

00000026626

alpha isoform [Source:MGI Symbol;Acc:MGI:2388479]

ENSMUSG
Kcnh1
protein_coding
potassium voltage-gated channel, subfamily H (eag-

00000058248

related), member 1 [Source:MGI

Symbol;Acc:MGI:1341721]

ENSMUSG
Hhat
protein_coding
hedgehog acyltransferase [Source:MGI

00000037375

Symbol;Acc:MGI:2444681]

ENSMUSG
Cd34
protein_coding
CD34 antigen [Source:MGI Symbol;Acc:MGI:88329]

00000016494

ENSMUSG
Fam171a1
protein_coding
family with sequence similarity 171, member A1

00000050530

[Source:MGI Symbol;Acc:MGI:2442917]

ENSMUSG
Olah
protein_coding
oleoyl-ACP hydrolase [Source:MGI

00000026645

Symbol;Acc:MGI:2139018]

ENSMUSG
Hspa14
protein_coding
heat shock protein 14 [Source:MGI

00000051396

Symbol;Acc:MGI:1354164]

ENSMUSG
Fam107b
protein_coding
family with sequence similarity 107, member B

00000026655

[Source:MGI Symbol;Acc:MGI:1913790]

ENSMUSG
Frmd4a
protein_coding
FERM domain containing 4A [Source:MGI

00000026657

Symbol;Acc:MGI:1919850]

ENSMUSG
Sephs1
protein_coding
selenophosphate synthetase 1 [Source:MGI

00000026662

Symbol;Acc:MGI:1923580]

ENSMUSG
Usp6nl
protein_coding
USP6 N-terminal like [Source:MGI

00000039046

Symbol;Acc:MGI:2138893]

ENSMUSG
Celf2
protein_coding
CUGBP, Elav-like family member 2 [Source:MGI

00000002107

Symbol;Acc:MGI:1338822]

ENSMUSG
Prkcq
protein_coding
protein kinase C, theta [Source:MGI

00000026778

Symbol;Acc:MGI:97601]

ENSMUSG
Rsu1
protein_coding
Ras suppressor protein 1 [Source:MGI

00000026727

Symbol;Acc:MGI:103040]

ENSMUSG
Vim
protein_coding
vimentin [Source:MGI Symbol;Acc:MGI:98932]

00000026728

ENSMUSG
Cacnb2
protein_coding
calcium channel, voltage-dependent, beta 2 subunit

00000057914

[Source:MGI Symbol;Acc:MGI:894644]

ENSMUSG
Plxdc2
protein_coding
plexin domain containing 2 [Source:MGI

00000026748

Symbol;Acc:MGI:1914698]

ENSMUSG
Etl4
protein_coding
enhancer trap locus 4 [Source:MGI

00000036617

Symbol;Acc:MGI:95454]

ENSMUSG
Arhgap21
protein_coding
Rho GTPase activating protein 21 [Source:MGI

00000036591

Symbol;Acc:MGI:1918685]

ENSMUSG
Apbb1ip
protein_coding
amyloid beta (A4) precursor protein-binding, family B,

00000026786

member 1 interacting protein [Source:MGI

Symbol;Acc:MGI:1861354]

ENSMUSG
Psd4
protein_coding
pleckstrin and Sec7 domain containing 4 [Source:MGI

00000026979

Symbol;Acc:MGI:2674093]

ENSMUSG
Zmynd19
protein_coding
zinc finger, MYND domain containing 19 [Source:MGI

00000026974

Symbol;Acc:MGI:1914437]

ENSMUSG
Tubb4b
protein_coding
tubulin, beta 4B class IVB [Source:MGI

00000036752

Symbol;Acc:MGI:1915472]

ENSMUSG
Tprn
protein_coding
taperin [Source:MGI Symbol;Acc:MGI:2139535]

00000048707

ENSMUSG
Anapc2
protein_coding
anaphase promoting complex subunit 2 [Source:MGI

00000026965

Symbol;Acc:MGI:2139135]

ENSMUSG
2010317E24Rik
protein_coding
RIKEN cDNA 2010317E24 gene [Source:MGI

00000026955

Symbol;Acc:MGI:1919330]

ENSMUSG
Mamdc4
protein_coding
MAM domain containing 4 [Source:MGI

00000026941

Symbol;Acc:MGI:2685841]

ENSMUSG
Phpt1
protein_coding
phosphohistidine phosphatase 1 [Source:MGI

00000036504

Symbol;Acc:MGI:1922704]

ENSMUSG
B230208H17Rik
protein_coding
RIKEN cDNA B230208H17 gene [Source:MGI

00000015087

Symbol;Acc:MGI:2442633]

ENSMUSG
Med22
protein_coding
mediator complex subunit 22 [Source:MGI

00000015776

Symbol;Acc:MGI:98446]

ENSMUSG
Rpl7a
protein_coding
ribosomal protein L7A [Source:MGI

00000062647

Symbol;Acc:MGI:1353472]

ENSMUSG
Adamts13
protein_coding
a disintegrin-like and metallopeptidase (reprolysin

00000014852

type) with thrombospondin type 1 motif, 13

[Source:MGI Symbol;Acc:MGI:2685556]

ENSMUSG
Brd3
protein_coding
bromodomain containing 3 [Source:MGI

00000026918

Symbol;Acc:MGI:1914632]

ENSMUSG
Rxra
protein_coding
retinoid X receptor alpha [Source:MGI

00000015846

Symbol;Acc:MGI:98214]

ENSMUSG
Mrps2
protein_coding
mitochondrial ribosomal protein S2 [Source:MGI

00000035772

Symbol;Acc:MGI:2153089]

ENSMUSG
Ak8
protein_coding
adenylate kinase 8 [Source:MGI

00000026807

Symbol;Acc:MGI:1916120]

ENSMUSG
Gtf3c4
protein_coding
general transcription factor IIIC, polypeptide 4

00000035666

[Source:MGI Symbol;Acc:MGI:2138937]

ENSMUSG
Setx
protein_coding
senataxin [Source:MGI Symbol;Acc:MGI:2443480]

00000043535

ENSMUSG
Ntng2
protein_coding
netrin G2 [Source:MGI Symbol;Acc:MGI:2159341]

00000035513

ENSMUSG
Rapgef1
protein_coding
Rap guanine nucleotide exchange factor (GEF) 1

00000039844

[Source:MGI Symbol;Acc:MGI:104580]

ENSMUSG
Spna2
protein_coding
spectrin alpha 2 [Source:MGI Symbol;Acc:MGI:98386]

00000057738

ENSMUSG
Wdr34
protein_coding
WD repeat domain 34 [Source:MGI

00000039715

Symbol;Acc:MGI:1919070]

ENSMUSG
Set
protein_coding
SET nuclear oncogene [Source:MGI

00000054766

Symbol;Acc:MGI:1860267]

ENSMUSG
Ppp2r4
protein_coding
protein phosphatase 2A, regulatory subunit B (PR 53)

00000039515

[Source:MGI Symbol;Acc:MGI:1346006]

ENSMUSG
Tor1a
protein_coding
torsin family 1, member A (torsin A) [Source:MGI

00000026849

Symbol;Acc:MGI:1353568]

ENSMUSG
Fnbp1
protein_coding
formin binding protein 1 [Source:MGI

00000075415

Symbol;Acc:MGI:109606]

ENSMUSG
Abl1
protein_coding
c-abl oncogene 1, non-receptor tyrosine kinase

00000026842

[Source:MGI Symbol;Acc:MGI:87859]

ENSMUSG
Nup214
protein_coding
nucleoporin 214 [Source:MGI

00000001855

Symbol;Acc:MGI:1095411]

ENSMUSG
Prrc2b
protein_coding
proline-rich coiled-coil 2B [Source:MGI

00000039262

Symbol;Acc:MGI:1923304]

ENSMUSG
Eng
protein_coding
endoglin [Source:MGI Symbol;Acc:MGI:95392]

00000026814

ENSMUSG
Cdk9
protein_coding
cyclin-dependent kinase 9 (CDC2-related kinase)

00000009555

[Source:MGI Symbol;Acc:MGI:1328368]

ENSMUSG
Rpl12
protein_coding
ribosomal protein L12 [Source:MGI

00000038900

Symbol;Acc:MGI:98002]

ENSMUSG
Ralgps1
protein_coding
Ral GEF with PH domain and SH3 binding motif 1

00000038831

[Source:MGI Symbol;Acc:MGI:1922008]

ENSMUSG
Lmx1b
protein_coding
LIM homeobox transcription factor 1 beta

00000038765

[Source:MGI Symbol;Acc:MGI:1100513]

ENSMUSG
Fam125b
protein_coding
family with sequence similarity 125, member B

00000038740

[Source:MGI Symbol;Acc:MGI:1919793]

ENSMUSG
Pbx3
protein_coding
pre B cell leukemia homeobox 3 [Source:MGI

00000038718

Symbol;Acc:MGI:97496]

ENSMUSG
Hspa5
protein_coding
heat shock protein 5 [Source:MGI

00000026864

Symbol;Acc:MGI:95835]

ENSMUSG
Psmd5
protein_coding
proteasome (prosome, macropain) 26S subunit, non-

00000026869

ATPase, 5 [Source:MGI Symbol;Acc:MGI:1914248]

ENSMUSG
Ggta1
protein_coding
glycoprotein galactosyltransferase alpha 1, 3

00000035778

[Source:MGI Symbol;Acc:MGI:95704]

ENSMUSG
Dab2ip
protein_coding
disabled homolog 2 (Drosophila) interacting protein

00000026883

[Source:MGI Symbol;Acc:MGI:1916851]

ENSMUSG
Dennd1a
protein_coding
DENN/MADD domain containing 1A [Source:MGI

00000035392

Symbol;Acc:MGI:2442794]

ENSMUSG
Lhx2
protein_coding
LIM homeobox protein 2 [Source:MGI

00000000247

Symbol;Acc:MGI:96785]

ENSMUSG
Lrp1b
protein_coding
low density lipoprotein-related protein 1B (deleted in

00000049252

tumors) [Source:MGI Symbol;Acc:MGI:2151136]

ENSMUSG
Arhgap15
protein_coding
Rho GTPase activating protein 15 [Source:MGI

00000049744

Symbol;Acc:MGI:1923367]

ENSMUSG
Zeb2
protein_coding
zinc finger E-box binding homeobox 2 [Source:MGI

00000026872

Symbol;Acc:MGI:1344407]

ENSMUSG
Mbd5
protein_coding
methyl-CpG binding domain protein 5 [Source:MGI

00000036792

Symbol;Acc:MGI:2138934]

ENSMUSG
Kif5c
protein_coding
kinesin family member 5C [Source:MGI

00000026764

Symbol;Acc:MGI:1098269]

ENSMUSG
Rif1
protein_coding
Rap1 interacting factor 1 homolog (yeast)

00000036202

[Source:MGI Symbol;Acc:MGI:1098622]

ENSMUSG
Gpd2
protein_coding
glycerol phosphate dehydrogenase 2, mitochondrial

00000026827

[Source:MGI Symbol;Acc:MGI:99778]

ENSMUSG
7-Mar
protein_coding
membrane-associated ring finger (C3HC4) 7

00000026977

[Source:MGI Symbol;Acc:MGI:1931053]

ENSMUSG
Psmd14
protein_coding
proteasome (prosome, macropain) 26S subunit, non-

00000026914

ATPase, 14 [Source:MGI Symbol;Acc:MGI:1913284]

ENSMUSG
Tbr1
protein_coding
T-box brain gene 1 [Source:MGI

00000035033

Symbol;Acc:MGI:107404]

ENSMUSG
Dhrs9
protein_coding
dehydrogenase/reductase (SDR family) member 9

00000027068

[Source:MGI Symbol;Acc:MGI:2442798]

ENSMUSG
Ssb
protein_coding
Sjogren syndrome antigen B [Source:MGI

00000068882

Symbol;Acc:MGI:98423]

ENSMUSG
Tlk1
protein_coding
tousled-like kinase 1 [Source:MGI

00000041997

Symbol;Acc:MGI:2441683]

ENSMUSG
Itga6
protein_coding
integrin alpha 6 [Source:MGI Symbol;Acc:MGI:96605]

00000027111

ENSMUSG
Rapgef4
protein_coding
Rap guanine nucleotide exchange factor (GEF) 4

00000049044

[Source:MGI Symbol;Acc:MGI:1917723]

ENSMUSG
Sp3
protein_coding
trans-acting transcription factor 3 [Source:MGI

00000027109

Symbol;Acc:MGI:1277166]

ENSMUSG
Sp9
protein_coding
trans-acting transcription factor 9 [Source:MGI

00000068859

Symbol;Acc:MGI:3574660]

ENSMUSG
Wipf1
protein_coding
WAS/WASL interacting protein family, member 1

00000075284

[Source:MGI Symbol;Acc:MGI:2178801]

ENSMUSG
Atf2
protein_coding
activating transcription factor 2 [Source:MGI

00000027104

Symbol;Acc:MGI:109349]

ENSMUSG
Atp5g3
protein_coding
ATP synthase, H+ transporting, mitochondrial F0

00000018770

complex, subunit C3 (subunit 9) [Source:MGI

Symbol;Acc:MGI:2442035]

ENSMUSG
Hoxd9
protein_coding
homeobox D9 [Source:MGI Symbol;Acc:MGI:96210]

00000043342

ENSMUSG
Nfe2l2
protein_coding
nuclear factor, erythroid derived 2, like 2 [Source:MGI

00000015839

Symbol;Acc:MGI:108420]

ENSMUSG
Ttn
protein_coding
titin [Source:MGI Symbol;Acc:MGI:98864]

00000051747

ENSMUSG
Cwc22
protein_coding
CWC22 spliceosome-associated protein homolog (S.

00000027014

cerevisiae) [Source:MGI Symbol;Acc:MGI:2136773]

ENSMUSG
Ssfa2
protein_coding
sperm specific antigen 2 [Source:MGI

00000027007

Symbol;Acc:MGI:1917849]

ENSMUSG
Nckap1
protein_coding
NCK-associated protein 1 [Source:MGI

00000027002

Symbol;Acc:MGI:1355333]

ENSMUSG
Zc3h15
protein_coding
zinc finger CCCH-type containing 15 [Source:MGI

00000027091

Symbol;Acc:MGI:1919747]

ENSMUSG
Calcrl
protein_coding
calcitonin receptor-like [Source:MGI

00000059588

Symbol;Acc:MGI:1926944]

ENSMUSG
Ctnnd1
protein_coding
catenin (cadherin associated protein), delta 1

00000034101

[Source:MGI Symbol;Acc:MGI:105100]

ENSMUSG
Ptprj
protein_coding
protein tyrosine phosphatase, receptor type, J

00000025314

[Source:MGI Symbol;Acc:MGI:104574]

ENSMUSG
Psmc3
protein_coding
proteasome (prosome, macropain) 26S subunit,

00000002102

ATPase 3 [Source:MGI Symbol;Acc:MGI:1098754]

ENSMUSG
Sfpi1
protein_coding
SFFV proviral integration 1 [Source:MGI

00000002111

Symbol;Acc:MGI:98282]

ENSMUSG
Ckap5
protein_coding
cytoskeleton associated protein 5 [Source:MGI

00000040549

Symbol;Acc:MGI:1923036]

ENSMUSG
Ambra1
protein_coding
autophagy/beclin 1 regulator 1 [Source:MGI

00000040506

Symbol;Acc:MGI:2443564]

ENSMUSG
Dgkz
protein_coding
diacylglycerol kinase zeta [Source:MGI

00000040479

Symbol;Acc:MGI:1278339]

ENSMUSG
Phf21a
protein_coding
PHD finger protein 21A [Source:MGI

00000058318

Symbol;Acc:MGI:2384756]

ENSMUSG
Slc35c1
protein_coding
solute carrier family 35, member C1 [Source:MGI

00000049922

Symbol;Acc:MGI:2443301]

ENSMUSG
Prdm11
protein_coding
PR domain containing 11 [Source:MGI

00000075028

Symbol;Acc:MGI:2685553]

ENSMUSG
Hsd17b12
protein_coding
hydroxysteroid (17-beta) dehydrogenase 12

00000027195

[Source:MGI Symbol;Acc:MGI:1926967]

ENSMUSG
Ldlrad3
protein_coding
low density lipoprotein receptor class A domain

00000048058

containing 3 [Source:MGI Symbol;Acc:MGI:2138856]

ENSMUSG
Cd44
protein_coding
CD44 antigen [Source:MGI Symbol;Acc:MGI:88338]

00000005087

ENSMUSG
Caprin1
protein_coding
cell cycle associated protein 1 [Source:MGI

00000027184

Symbol;Acc:MGI:1858234]

ENSMUSG
D430041D05Rik
protein_coding
RIKEN cDNA D430041D05 gene [Source:MGI

00000068373

Symbol;Acc:MGI:2181743]

ENSMUSG
Hipk3
protein_coding
homeodomain interacting protein kinase 3

00000027177

[Source:MGI Symbol;Acc:MGI:1314882]

ENSMUSG
Ryr3
protein_coding
ryanodine receptor 3 [Source:MGI

00000057378

Symbol;Acc:MGI:99684]

ENSMUSG
Arhgap11a
protein_coding
Rho GTPase activating protein 11A [Source:MGI

00000041219

Symbol;Acc:MGI:2444300]

ENSMUSG
Aqr
protein_coding
aquarius [Source:MGI Symbol;Acc:MGI:1276102]

00000040383

ENSMUSG
Spred1
protein_coding
sprouty protein with EVH-1 domain 1, related

00000027351

sequence [Source:MGI Symbol;Acc:MGI:2150016]

ENSMUSG
Vps18
protein_coding
vacuolar protein sorting 18 (yeast) [Source:MGI

00000034216

Symbol;Acc:MGI:2443626]

ENSMUSG
Rtf1
protein_coding
Rtf1, Paf1/RNA polymerase Il complex component,

00000027304

homolog (S. cerevisiae) [Source:MGI

Symbol;Acc:MGI:1309480]

ENSMUSG
Mga
protein_coding
MAX gene associated [Source:MGI

00000033943

Symbol;Acc:MGI:1352483]

ENSMUSG
Zfp106
protein_coding
zinc finger protein 106 [Source:MGI

00000027288

Symbol;Acc:MGI:1270153]

ENSMUSG
Tubgcp4
protein_coding
tubulin, gamma complex associated protein 4

00000027263

[Source:MGI Symbol;Acc:MGI:1196293]

ENSMUSG
Catsper2
protein_coding
cation channel, sperm associated 2 [Source:MGI

00000033486

Symbol;Acc:MGI:2387404]

ENSMUSG
Pdia3
protein_coding
protein disulfide isomerase associated 3 [Source:MGI

00000027248

Symbol;Acc:MGI:95834]

ENSMUSG
Dut
protein_coding
deoxyuridine triphosphatase [Source:MGI

00000027203

Symbol;Acc:MGI:1346051]

ENSMUSG
Trpm7
protein_coding
transient receptor potential cation channel, subfamily

00000027365

M, member 7 [Source:MGI Symbol;Acc:MGI:1929996]

ENSMUSG
Snrnp200
protein_coding
small nuclear ribonucleoprotein 200 (U5) [Source:MGI

00000003660

Symbol;Acc:MGI:2444401]

ENSMUSG
Tmem127
protein_coding
transmembrane protein 127 [Source:MGI

00000034850

Symbol;Acc:MGI:1916720]

ENSMUSG
Stard7
protein_coding
START domain containing 7 [Source:MGI

00000027367

Symbol;Acc:MGI:2139090]

ENSMUSG
Anapc1
protein_coding
anaphase promoting complex subunit 1 [Source:MGI

00000014355

Symbol;Acc:MGI:103097]

ENSMUSG
Ttl
protein_coding
tubulin tyrosine ligase [Source:MGI

00000027394

Symbol;Acc:MGI:1916987]

ENSMUSG
Slc20a1
protein_coding
solute carrier family 20, member 1 [Source:MGI

00000027397

Symbol;Acc:MGI:108392]

ENSMUSG
Sirpa
protein_coding
signal-regulatory protein alpha [Source:MGI

00000037902

Symbol;Acc:MGI:108563]

ENSMUSG
Stk35
protein_coding
serine/threonine kinase 35 [Source:MGI

00000037885

Symbol;Acc:MGI:1914583]

ENSMUSG
Snrpb
protein_coding
small nuclear ribonucleoprotein B [Source:MGI

00000027404

Symbol;Acc:MGI:98342]

ENSMUSG
Nop56
protein_coding
NOP56 ribonucleoprotein homolog (yeast)

00000027405

[Source:MGI Symbol;Acc:MGI:1914384]

ENSMUSG
Ptpra
protein_coding
protein tyrosine phosphatase, receptor type, A

00000027303

[Source:MGI Symbol;Acc:MGI:97808]

ENSMUSG
Spef1
protein_coding
sperm flagellar 1 [Source:MGI

00000027329

Symbol;Acc:MGI:3513546]

ENSMUSG
Cenpb
protein_coding
centromere protein B [Source:MGI

00000068267

Symbol;Acc:MGI:88376]

ENSMUSG
Plcb4
protein_coding
phospholipase C, beta 4 [Source:MGI

00000039943

Symbol;Acc:MGI:107464]

ENSMUSG
Macrod2
protein_coding
MACRO domain containing 2 [Source:MGI

00000068205

Symbol;Acc:MGI:1920149]

ENSMUSG
Snx5
protein_coding
sorting nexin 5 [Source:MGI

00000027423

Symbol;Acc:MGI:1916428]

ENSMUSG
Csrp2bp
protein_coding
cysteine and glycine-rich protein 2 binding protein

00000027425

[Source:MGI Symbol;Acc:MGI:1917264]

ENSMUSG
Sec23b
protein_coding
SEC23B (S. cerevisiae) [Source:MGI

00000027429

Symbol;Acc:MGI:1350925]

ENSMUSG
Slc24a3
protein_coding
solute carrier family 24 (sodium/potassium/calcium

00000063873

exchanger), member 3 [Source:MGI

Symbol;Acc:MGI:2137513]

ENSMUSG
Ralgapa2
protein_coding
Ral GTPase activating protein, alpha subunit 2

00000037110

(catalytic) [Source:MGI Symbol;Acc:MGI:3036245]

ENSMUSG
Cd93
protein_coding
CD93 antigen [Source:MGI Symbol;Acc:MGI:106664]

00000027435

ENSMUSG
Tmem90b
protein_coding
transmembrane protein 90B [Source:MGI

00000074736

Symbol;Acc:MGI:3702158]

ENSMUSG
Pygb
protein_coding
brain glycogen phosphorylase [Source:MGI

00000033059

Symbol;Acc:MGI:97828]

ENSMUSG
Fam110a
protein_coding
family with sequence similarity 110, member A

00000027459

[Source:MGI Symbol;Acc:MGI:1921097]

ENSMUSG
Scrt2
protein_coding
scratch homolog 2, zinc finger protein (Drosophila)

00000060257

[Source:MGI Symbol;Acc:MGI:2139287]

ENSMUSG
Csnk2a1
protein_coding
casein kinase 2, alpha 1 polypeptide [Source:MGI

00000074698

Symbol;Acc:MGI:88543]

ENSMUSG
Bcl2l1
protein_coding
BCL2-like 1 [Source:MGI Symbol;Acc:MGI:88139]

00000007659

ENSMUSG
Tpx2
protein_coding
TPX2, microtubule-associated protein homolog

00000027469

(Xenopus laevis) [Source:MGI

Symbol;Acc:MGI:1919369]

ENSMUSG
Dusp 15
protein_coding
dual specificity phosphatase-like 15 [Source:MGI

00000042662

Symbol;Acc:MGI:1934928]

ENSMUSG
Plagl2
protein_coding
pleiomorphic adenoma gene-like 2 [Source:MGI

00000051413

Symbol;Acc:MGI:1933165]

ENSMUSG
Asxl1
protein_coding
additional sex combs like 1 (Drosophila) [Source:MGI

00000042548

Symbol;Acc:MGI:2684063]

ENSMUSG
Dnmt3b
protein_coding
DNA methyltransferase 3B [Source:MGI

00000027478

Symbol;Acc:MGI:1261819]

ENSMUSG
Cdk5rap1
protein_coding
CDK5 regulatory subunit associated protein 1

00000027487

[Source:MGI Symbol;Acc:MGI:1914221]

ENSMUSG
Chmp4b
protein_coding
charged multivesicular body protein 4B [Source:MGI

00000038467

Symbol;Acc:MGI:1922858]

ENSMUSG
Raly
protein_coding
hnRNP-associated with lethal yellow [Source:MGI

00000027593

Symbol;Acc:MGI:97850]

ENSMUSG
a
protein_coding
nonagouti [Source:MGI Symbol;Acc:MGI:87853]

00000027596

ENSMUSG
Ncoa6
protein_coding
nuclear receptor coactivator 6 [Source:MGI

00000038369

Symbol;Acc:MGI:1929915]

ENSMUSG
Trpc4ap
protein_coding
transient receptor potential cation channel, subfamily

00000038324

C, member 4 associated protein [Source:MGI

Symbol;Acc:MGI:1930751]

ENSMUSG
Cpne1
protein_coding
copine I [Source:MGI Symbol;Acc:MGI:2386621]

00000074643

ENSMUSG
Nfs1
protein_coding
nitrogen fixation gene 1 (S. cerevisiae) [Source:MGI

00000027618

Symbol;Acc:MGI:1316706]

ENSMUSG
Rbm12
protein_coding
RNA binding motif protein 12 [Source:MGI

00000089824

Symbol;Acc:MGI:1922960]

ENSMUSG
Tgif2
protein_coding
TGFB-induced factor homeobox 2 [Source:MGI

00000062175

Symbol;Acc:MGI:1915299]

ENSMUSG
4922505G16Rik
protein_coding
RIKEN cDNA 4922505G16 gene [Source:MGI

00000074627

Symbol;Acc:MGI:3603828]

ENSMUSG
Rpn2
protein_coding
ribophorin II [Source:MGI Symbol;Acc:MGI:98085]

00000027642

ENSMUSG
Rprd1b
protein_coding
regulation of nuclear pre-mRNA domain containing

00000027651

1B [Source:MGI Symbol;Acc:MGI:1917720]

ENSMUSG
Actr5
protein_coding
ARP5 actin-related protein 5 homolog (yeast)

00000037761

[Source:MGI Symbol;Acc:MGI:1924748]

ENSMUSG
Dhx35
protein_coding
DEAH (Asp-Glu-Ala-His) box polypeptide 35

00000027655

[Source:MGI Symbol;Acc:MGI:1918965]

ENSMUSG
Top1
protein_coding
topoisomerase (DNA) I [Source:MGI

00000070544

Symbol;Acc:MGI:98788]

ENSMUSG
Plcg1
protein_coding
phospholipase C, gamma 1 [Source:MGI

00000016933

Symbol;Acc:MGI:97615]

ENSMUSG
Chd6
protein_coding
chromodomain helicase DNA binding protein 6

00000057133

[Source:MGI Symbol;Acc:MGI:1918639]

ENSMUSG
Srsf6
protein_coding
serine/arginine-rich splicing factor 6 [Source:MGI

00000016921

Symbol;Acc:MGI:1915246]

ENSMUSG
Jph2
protein_coding
junctophilin 2 [Source:MGI Symbol;Acc:MGI:1891496]

00000017817

ENSMUSG
Pkig
protein_coding
protein kinase inhibitor, gamma [Source:MGI

00000035268

Symbol;Acc:MGI:1343086]

ENSMUSG
Ada
protein_coding
adenosine deaminase [Source:MGI

00000017697

Symbol;Acc:MGI:87916]

ENSMUSG
Ywhab
protein_coding
tyrosine 3-monooxygenase/tryptophan 5-

00000018326

monooxygenase activation protein, beta polypeptide

[Source:MGI Symbol;Acc:MGI:1891917]

ENSMUSG
Pcif1
protein_coding
PDX1 C-terminal inhibiting factor 1 [Source:MGI

00000039849

Symbol;Acc:MGI:2443858]

ENSMUSG
Ncoa5
protein_coding
nuclear receptor coactivator 5 [Source:MGI

00000039804

Symbol;Acc:MGI:2385165]

ENSMUSG
Eya2
protein_coding
eyes absent 2 homolog (Drosophila) [Source:MGI

00000017897

Symbol;Acc:MGI:109341]

ENSMUSG
Zmynd8
protein_coding
zinc finger, MYND-type containing 8 [Source:MGI

00000039671

Symbol;Acc:MGI:1918025]

ENSMUSG
Ncoa3
protein_coding
nuclear receptor coactivator 3 [Source:MGI

00000027678

Symbol;Acc:MGI:1276535]

ENSMUSG
Ddx27
protein_coding
DEAD (Asp-Glu-Ala-Asp) box polypeptide 27

00000017999

[Source:MGI Symbol;Acc:MGI:2385884]

ENSMUSG
1500012F01Rik
protein_coding
RIKEN cDNA 1500012F01 gene [Source:MGI

00000074578

Symbol;Acc:MGI:1916199]

ENSMUSG
Spata2
protein_coding
spermatogenesis associated 2 [Source:MGI

00000047030

Symbol;Acc:MGI:2146885]

ENSMUSG
Ptpn1
protein_coding
protein tyrosine phosphatase, non-receptor type 1

00000027540

[Source:MGI Symbol;Acc:MGI:97805]

ENSMUSG
Adnp
protein_coding
activity-dependent neuroprotective protein

00000051149

[Source:MGI Symbol;Acc:MGI:1338758]

ENSMUSG
Tshz2
protein_coding
teashirt zinc finger family member 2 [Source:MGI

00000047907

Symbol;Acc:MGI:2153084]

ENSMUSG
Zfp217
protein_coding
zinc finger protein 217 [Source:MGI

00000052056

Symbol;Acc:MGI:2685408]

ENSMUSG
Dok5
protein_coding
docking protein 5 [Source:MGI

00000027560

Symbol;Acc:MGI:1924079]

ENSMUSG
Rae1
protein_coding
RAE1 RNA export 1 homolog (S. pombe) [Source:MGI

00000027509

Symbol;Acc:MGI:1913929]

ENSMUSG
1700021F07Rik
protein_coding
RIKEN cDNA 1700021F07 gene [Source:MGI

00000027518

Symbol;Acc:MGI:1919471]

ENSMUSG
Gnas
protein_coding
GNAS (guanine nucleotide binding protein, alpha

00000027523

stimulating) complex locus [Source:MGI

Symbol;Acc:MGI:95777]

ENSMUSG
AL593857.1
protein_coding

00000090625

ENSMUSG
C330013J
protein_coding
RIKEN cDNA C330013J21 gene [Source:MGI

00000074529
21Rik

Symbol;Acc:MGI:1925953]

ENSMUSG
Slco4a1
protein_coding
solute carrier organic anion transporter family,

00000038963

member 4a1 [Source:MGI Symbol;Acc:MGI:1351866]

ENSMUSG
Dido1
protein_coding
death inducer-obliterator 1 [Source:MGI

00000038914

Symbol;Acc:MGI:1344352]

ENSMUSG
Ythdf1
protein_coding
YTH domain family 1 [Source:MGI

00000038848

Symbol;Acc:MGI:1917431]

ENSMUSG
Zfp704
protein_coding
zinc finger protein 704 [Source:MGI

00000040209

Symbol;Acc:MGI:2180715]

ENSMUSG
Gyg
protein_coding
glycogenin [Source:MGI Symbol;Acc:MGI:1351614]

00000019528

ENSMUSG
Fndc3b
protein_coding
fibronectin type III domain containing 3B [Source:MGI

00000039286

Symbol;Acc:MGI:1919257]

ENSMUSG
Tnik
protein_coding
TRAF2 and NCK interacting kinase [Source:MGI

00000027692

Symbol;Acc:MGI:1916264]

ENSMUSG
Mecom
protein_coding
MDS1 and EVI1 complex locus [Source:MGI

00000027684

Symbol;Acc:MGI:95457]

ENSMUSG
Zfp639
protein_coding
zinc finger protein 639 [Source:MGI

00000027667

Symbol;Acc:MGI:1915028]

ENSMUSG
Gnb4
protein_coding
guanine nucleotide binding protein (G protein), beta 4

00000027669

[Source:MGI Symbol;Acc:MGI:104581]

ENSMUSG
Atp11b
protein_coding
ATPase, class VI, type 11B [Source:MGI

00000037400

Symbol;Acc:MGI:1923545]

ENSMUSG
Anxa5
protein_coding
annexin A5 [Source:MGI Symbol;Acc:MGI:106008]

00000027712

ENSMUSG
Ccna2
protein_coding
cyclin A2 [Source:MGI Symbol;Acc:MGI:108069]

00000027715

ENSMUSG
Pgrmc2
protein_coding
progesterone receptor membrane component 2

00000049940

[Source:MGI Symbol;Acc:MGI:1918054]

ENSMUSG
Elf2
protein_coding
E74-like factor 2 [Source:MGI

00000037174

Symbol;Acc:MGI:1916507]

ENSMUSG
Naa15
protein_coding
N(alpha)-acetyltransferase 15, NatA auxiliary subunit

00000063273

[Source:MGI Symbol;Acc:MGI:1922088]

ENSMUSG
Mgst2
protein_coding
microsomal glutathione S-transferase 2 [Source:MGI

00000074604

Symbol;Acc:MGI:2448481]

ENSMUSG
Maml3
protein_coding
mastermind like 3 (Drosophila) [Source:MGI

00000061143

Symbol;Acc:MGI:2389461]

ENSMUSG
Nbea
protein_coding
neurobeachin [Source:MGI Symbol;Acc:MGI:1347075]

00000027799

ENSMUSG
Mbnl1
protein_coding
muscleblind-like 1 (Drosophila) [Source:MGI

00000027763

Symbol;Acc:MGI:1928482]

ENSMUSG
Rap2b
protein_coding
RAP2B, member of RAS oncogene family [Source:MGI

00000036894

Symbol;Acc:MGI:1921262]

ENSMUSG
Smc4
protein_coding
structural maintenance of chromosomes 4

00000034349

[Source:MGI Symbol;Acc:MGI:1917349]

ENSMUSG
Kpna4
protein_coding
karyopherin (importin) alpha 4 [Source:MGI

00000027782

Symbol;Acc:MGI:1100848]

ENSMUSG
Accn5
protein_coding
amiloride-sensitive cation channel 5, intestinal

00000028008

[Source:MGI Symbol;Acc:MGI:1929259]

ENSMUSG
Gucy1a3
protein_coding
guanylate cyclase 1, soluble, alpha 3 [Source:MGI

00000033910

Symbol;Acc:MGI:1926562]

ENSMUSG
D930015E06Rik
protein_coding
RIKEN cDNA D930015E06 gene [Source:MGI

00000033767

Symbol;Acc:MGI:2443399]

ENSMUSG
Arfip1
protein_coding
ADP-ribosylation factor interacting protein 1

00000074513

[Source:MGI Symbol;Acc:MGI:1277120]

ENSMUSG
Rps3a
protein_coding
ribosomal protein S3A [Source:MGI

00000028081

Symbol;Acc:MGI:1202063]

ENSMUSG
Lrba
protein_coding
LPS-responsive beige-like anchor [Source:MGI

00000028080

Symbol;Acc:MGI:1933162]

ENSMUSG
Prcc
protein_coding
papillary renal cell carcinoma (translocation-

00000004895

associated) [Source:MGI Symbol;Acc:MGI:2137738]

ENSMUSG
Hdgf
protein_coding
hepatoma-derived growth factor [Source:MGI

00000004897

Symbol;Acc:MGI:1194494]

ENSMUSG
Iqgap3
protein_coding
IQ motif containing GTPase activating protein 3

00000028068

[Source:MGI Symbol;Acc:MGI:3028642]

ENSMUSG
Smg5
protein_coding
Smg-5 homolog, nonsense mediated mRNA decay

00000001415

factor (C. elegans) [Source:MGI

Symbol;Acc:MGI:2447364]

ENSMUSG
Ubqln4
protein_coding
ubiquilin 4 [Source:MGI Symbol;Acc:MGI:2150152]

00000008604

ENSMUSG
Gon4l
protein_coding
gon-4-like (C.elegans) [Source:MGI

00000054199

Symbol;Acc:MGI:1917579]

ENSMUSG
Fdps
protein_coding
farnesyl diphosphate synthetase [Source:MGI

00000059743

Symbol;Acc:MGI:104888]

ENSMUSG
Il6ra
protein_coding
interleukin 6 receptor, alpha [Source:MGI

00000027947

Symbol;Acc:MGI:105304]

ENSMUSG
Ubap21
protein_coding
ubiquitin associated protein 2-like [Source:MGI

00000042520

Symbol;Acc:MGI:1921633]

ENSMUSG
Pogz
protein_coding
pogo transposable element with ZNF domain

00000038902

[Source:MGI Symbol;Acc:MGI:2442117]

ENSMUSG
Pi4kb
protein_coding
phosphatidylinositol 4-kinase, catalytic, beta

00000038861

polypeptide [Source:MGI Symbol;Acc:MGI:1334433]

ENSMUSG
Zfp687
protein_coding
zinc finger protein 687 [Source:MGI

00000019338

Symbol;Acc:MGI:1925516]

ENSMUSG
Pip5k1a
protein_coding
phosphatidylinositol-4-phosphate 5-kinase, type 1

00000028126

alpha [Source:MGI Symbol;Acc:MGI:107929]

ENSMUSG
Lass2
protein_coding
LAG1 homolog, ceramide synthase 2 [Source:MGI

00000015714

Symbol;Acc:MGI:1924143]

ENSMUSG
Golph3l
protein_coding
golgi phosphoprotein 3-like [Source:MGI

00000046519

Symbol;Acc:MGI:1917129]

ENSMUSG
Prpf3
protein_coding
PRP3 pre-mRNA processing factor 3 homolog (yeast)

00000015748

[Source:MGI Symbol;Acc:MGI:1918017]

ENSMUSG
Anp32e
protein_coding
acidic (leucine-rich) nuclear phosphoprotein 32

00000015749

family, member E [Source:MGI

Symbol;Acc:MGI:1913721]

ENSMUSG
Otud7b
protein_coding
OTU domain containing 7B [Source:MGI

00000038495

Symbol;Acc:MGI:2654703]

ENSMUSG
Hist2h2ac
protein_coding
histone cluster 2, H2ac [Source:MGI

00000068855

Symbol;Acc:MGI:2448316]

ENSMUSG
Bcl9
protein_coding
B cell CLL/lymphoma 9 [Source:MGI

00000038256

Symbol;Acc:MGI:1924828]

ENSMUSG
Pde4dip
protein_coding
phosphodiesterase 4D interacting protein

00000038170

(myomegalin) [Source:MGI Symbol;Acc:MGI:1891434]

ENSMUSG
Notch2
protein_coding
Notch gene homolog 2 (Drosophila) [Source:MGI

00000027878

Symbol;Acc:MGI:97364]

ENSMUSG
Csde1
protein_coding
cold shock domain containing E1, RNA binding

00000068823

[Source:MGI Symbol;Acc:MGI:92356]

ENSMUSG
Trim33
protein_coding
tripartite motif-containing 33 [Source:MGI

00000033014

Symbol;Acc:MGI:2137357]

ENSMUSG
Gm10964
protein_coding
predicted gene 10964 [Source:MGI

00000078620

Symbol;Acc:MGI:3779175]

ENSMUSG
Hipk1
protein_coding
homeodomain interacting protein kinase 1

00000008730

[Source:MGI Symbol;Acc:MGI:1314873]

ENSMUSG
Magi3
protein_coding
membrane associated guanylate kinase, WW and PDZ

00000052539

domain containing 3 [Source:MGI

Symbol;Acc:MGI:1923484]

ENSMUSG
Slc16a1
protein_coding
solute carrier family 16 (monocarboxylic acid

00000032902

transporters), member 1 [Source:MGI

Symbol;Acc:MGI:106013]

ENSMUSG
Atp5f1
protein_coding
ATP synthase, H+ transporting, mitochondrial F0

00000000563

complex, subunit B1 [Source:MGI

Symbol;Acc:MGI:1100495]

ENSMUSG
Ubl4b
protein_coding
ubiquitin-like 4B [Source:MGI

00000055891

Symbol;Acc:MGI:1914841]

ENSMUSG
Gnai3
protein_coding
guanine nucleotide binding protein (G protein), alpha

00000000001

inhibiting 3 [Source:MGI Symbol;Acc:MGI:95773]

ENSMUSG
Fndc7
protein_coding
fibronectin type III domain containing 7 [Source:MGI

00000045326

Symbol;Acc:MGI:2443535]

ENSMUSG
4921515J06Rik
protein_coding
RIKEN cDNA 4921515J06 gene [Source:MGI

00000045662

Symbol;Acc:MGI:1913965]

ENSMUSG
Bcar3
protein_coding
breast cancer anti-estrogen resistance 3 [Source:MGI

00000028121

Symbol;Acc:MGI:1352501]

ENSMUSG
Fnbp1l
protein_coding
formin binding protein 1-like [Source:MGI

00000039735

Symbol;Acc:MGI:1925642]

ENSMUSG
Tifa
protein_coding
TRAF-interacting protein with forkhead-associated

00000046688

domain [Source:MGI Symbol;Acc:MGI:2182965]

ENSMUSG
Enpep
protein_coding
glutamyl aminopeptidase [Source:MGI

00000028024

Symbol;Acc:MGI:106645]

ENSMUSG
Sec24b
protein_coding
Sec24 related gene family, member B (S. cerevisiae)

00000001052

[Source:MGI Symbol;Acc:MGI:2139764]

ENSMUSG
Hadh
protein_coding
hydroxyacyl-Coenzyme A dehydrogenase [Source:MGI

00000027984

Symbol;Acc:MGI:96009]

ENSMUSG
Mttp
protein_coding
microsomal triglyceride transfer protein [Source:MGI

00000028158

Symbol;Acc:MGI:106926]

ENSMUSG
Rap1gds1
protein_coding
RAP1, GTP-GDP dissociation stimulator 1 [Source:MGI

00000028149

Symbol;Acc:MGI:2385189]

ENSMUSG
B930007M17Rik
protein_coding
RIKEN cDNA B930007M17 gene [Source:MGI

00000047940

Symbol;Acc:MGI:2685863]

ENSMUSG
Znhit6
protein_coding
zinc finger, HIT type 6 [Source:MGI

00000074182

Symbol;Acc:MGI:1916996]

ENSMUSG
Syde2
protein_coding
synapse defective 1, Rho GTPase, homolog 2 (C.

00000036863

elegans) [Source:MGI Symbol;Acc:MGI:3036264]

ENSMUSG
Dnase2b
protein_coding
deoxyribonuclease II beta [Source:MGI

00000028185

Symbol;Acc:MGI:1913283]

ENSMUSG
Prkacb
protein_coding
protein kinase, cAMP dependent, catalytic, beta

00000005034

[Source:MGI Symbol;Acc:MGI:97594]

ENSMUSG
Rabggtb
protein_coding
RAB geranylgeranyl transferase, b subunit

00000038975

[Source:MGI Symbol;Acc:MGI:99537]

ENSMUSG
Lyn
protein_coding
Yamaguchi sarcoma viral (v-yes-1) oncogene homolog

00000042228

[Source:MGI Symbol;Acc:MGI:96892]

ENSMUSG
Rps20
protein_coding
ribosomal protein S20 [Source:MGI

00000028234

Symbol;Acc:MGI:1914677]

ENSMUSG
Impad1
protein_coding
inositol monophosphatase domain containing 1

00000066324

[Source:MGI Symbol;Acc:MGI:1915720]

ENSMUSG
Sdcbp
protein_coding
syndecan binding protein [Source:MGI

00000028249

Symbol;Acc:MGI:1337026]

ENSMUSG
Pdp1
protein_coding
pyruvate dehyrogenase phosphatase catalytic subunit

00000049225

1 [Source:MGI Symbol;Acc:MGI:2685870]

ENSMUSG
Runx1t1
protein_coding
runt-related transcription factor 1; translocated to, 1

00000006586

(cyclin D-related) [Source:MGI

Symbol;Acc:MGI:104793]

ENSMUSG
Tmem64
protein_coding
transmembrane protein 64 [Source:MGI

00000043252

Symbol;Acc:MGI:2140359]

ENSMUSG
Cpne3
protein_coding
copine III [Source:MGI Symbol;Acc:MGI:1917818]

00000028228

ENSMUSG
Prdm13
protein_coding
PR domain containing 13 [Source:MGI

00000040478

Symbol;Acc:MGI:2448528]

ENSMUSG
Mdn1
protein_coding
midasin homolog (yeast) [Source:MGI

00000058006

Symbol;Acc:MGI:1926159]

ENSMUSG
Pnrc1
protein_coding
proline-rich nuclear receptor coactivator 1

00000040128

[Source:MGI Symbol;Acc:MGI:1917838]

ENSMUSG
Zfp292
protein_coding
zinc finger protein 292 [Source:MGI

00000039967

Symbol;Acc:MGI:1353423]

ENSMUSG
Lingo2
protein_coding
leucine rich repeat and lg domain containing 2

00000045083

[Source:MGI Symbol;Acc:MGI:2442298]

ENSMUSG
Topors
protein_coding
topoisomerase I binding, arginine/serine-rich

00000036822

[Source:MGI Symbol;Acc:MGI:2146189]

ENSMUSG
B4galt1
protein_coding
UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase,

00000028413

polypeptide 1 [Source:MGI Symbol;Acc:MGI:95705]

ENSMUSG
Bag1
protein_coding
BCL2-associated athanogene 1 [Source:MGI

00000028416

Symbol;Acc:MGI:108047]

ENSMUSG
Ubap2
protein_coding
ubiquitin-associated protein 2 [Source:MGI

00000028433

Symbol;Acc:MGI:1916176]

ENSMUSG
Dcaf12
protein_coding
DDB1 and CUL4 associated factor 12 [Source:MGI

00000028436

Symbol;Acc:MGI:1916220]

ENSMUSG
Il11ra1
protein_coding
interleukin 11 receptor, alpha chain 1 [Source:MGI

00000073889

Symbol;Acc:MGI:107426]

ENSMUSG
Sigmar1
protein_coding
sigma non-opioid intracellular receptor 1 [Source:MGI

00000036078

Symbol;Acc:MGI:1195268]

ENSMUSG
1700022111Rik
protein_coding
RIKEN cDNA 1700022111 gene [Source:MGI

00000028451

Symbol;Acc:MGI:1914567]

ENSMUSG
Vcp
protein_coding
valosin containing protein [Source:MGI

00000028452

Symbol;Acc:MGI:99919]

ENSMUSG
Ccdc107
protein_coding
coiled-coil domain containing 107 [Source:MGI

00000028461

Symbol;Acc:MGI:1913423]

ENSMUSG
Tln1
protein_coding
talin 1 [Source:MGI Symbol;Acc:MGI:1099832]

00000028465

ENSMUSG
Rnf38
protein_coding
ring finger protein 38 [Source:MGI

00000035696

Symbol;Acc:MGI:1920719]

ENSMUSG
Frmpd1
protein_coding
FERM and PDZ domain containing 1 [Source:MGI

00000035615

Symbol;Acc:MGI:2446274]

ENSMUSG
Mcart1
protein_coding
mitochondrial carrier triple repeat 1 [Source:MGI

00000045973

Symbol;Acc:MGI:2684984]

ENSMUSG
Anp32b
protein_coding
acidic (leucine-rich) nuclear phosphoprotein 32

00000028333

family, member B [Source:MGI

Symbol;Acc:MGI:1914878]

ENSMUSG
Coro2a
protein_coding
coronin, actin binding protein 2A [Source:MGI

00000028337

Symbol;Acc:MGI:1345966]

ENSMUSG
Gabbr2
protein_coding
gamma-aminobutyric acid (GABA) B receptor, 2

00000039809

[Source:MGI Symbol;Acc:MGI:2386030]

ENSMUSG
Sec61b
protein_coding
Sec61 beta subunit [Source:MGI

00000053317

Symbol;Acc:MGI:1913462]

ENSMUSG
2810432L12Rik
protein_coding
RIKEN cDNA 2810432L12 gene [Source:MGI

00000039611

Symbol;Acc:MGI:1914313]

ENSMUSG
Rad23b
protein_coding
RAD23b homolog (S. cerevisiae) [Source:MGI

00000028426

Symbol;Acc:MGI:105128]

ENSMUSG
Epb4.1l4b
protein_coding
erythrocyte protein band 4.1-like 4b [Source:MGI

00000028434

Symbol;Acc:MGI:1859149]

ENSMUSG
Gm20459
protein_coding
predicted gene 20459 [Source:MGI

00000089945

Symbol;Acc:MGI:5141924]

ENSMUSG
Txn1
protein_coding
thioredoxin 1 [Source:MGI Symbol;Acc:MGI:98874]

00000028367

ENSMUSG
Txndc8
protein_coding
thioredoxin domain containing 8 [Source:MGI

00000038709

Symbol;Acc:MGI:1914652]

ENSMUSG
AI314180
protein_coding
expressed sequence AI314180 [Source:MGI

00000050812

Symbol;Acc:MGI:2140220]

ENSMUSG
AI481877
protein_coding
expressed sequence AI481877 [Source:MGI

00000038598

Symbol;Acc:MGI:2140313]

ENSMUSG
Susd1
protein_coding
sushi domain containing 1 [Source:MGI

00000038578

Symbol;Acc:MGI:3651543]

ENSMUSG
Rod1
protein_coding
ROD1 regulator of differentiation 1 (S. pombe)

00000028382

[Source:MGI Symbol;Acc:MGI:1923334]

ENSMUSG
E130308A19Rik
protein_coding
RIKEN cDNA E130308A19 gene [Source:MGI

00000045071

Symbol;Acc:MGI:2442164]

ENSMUSG
Alad
protein_coding
aminolevulinate, delta-, dehydratase [Source:MGI

00000028393

Symbol;Acc:MGI:96853]

ENSMUSG
Astn2
protein_coding
astrotactin 2 [Source:MGI Symbol;Acc:MGI:1889277]

00000028373

ENSMUSG
Ptprd
protein_coding
protein tyrosine phosphatase, receptor type, D

00000028399

[Source:MGI Symbol;Acc:MGI:97812]

ENSMUSG
Klhl9
protein_coding
kelch-like 9 (Drosophila) [Source:MGI

00000070923

Symbol;Acc:MGI:2180122]

ENSMUSG
Jun
protein_coding
Jun oncogene [Source:MGI Symbol;Acc:MGI:96646]

00000052684

ENSMUSG
Cyp2j12
protein_coding
cytochrome P450, family 2, subfamily j, polypeptide

00000081225

12 [Source:MGI Symbol;Acc:MGI:3717097]

ENSMUSG
Nfia
protein_coding
nuclear factor I/A [Source:MGI

00000028565

Symbol;Acc:MGI:108056]

ENSMUSG
Kank4
protein_coding
KN motif and ankyrin repeat domains 4 [Source:MGI

00000035407

Symbol;Acc:MGI:3043381]

ENSMUSG
Usp1
protein_coding
ubiquitin specific peptidase 1 [Source:MGI

00000028560

Symbol;Acc:MGI:2385198]

ENSMUSG
Jak1
protein_coding
Janus kinase 1 [Source:MGI Symbol;Acc:MGI:96628]

00000028530

ENSMUSG
Pde4b
protein_coding
phosphodiesterase 4B, cAMP specific [Source:MGI

00000028525

Symbol;Acc:MGI:99557]

ENSMUSG
Gm10963
protein_coding
predicted gene 10963 [Source:MGI

00000078617

Symbol;Acc:MGI:3779174]

ENSMUSG
Dab1
protein_coding
disabled homolog 1 (Drosophila) [Source:MGI

00000028519

Symbol;Acc:MGI:108554]

ENSMUSG
Usp24
protein_coding
ubiquitin specific peptidase 24 [Source:MGI

00000028514

Symbol;Acc:MGI:1919936]

ENSMUSG
Dhcr24
protein_coding
24-dehydrocholesterol reductase [Source:MGI

00000034926

Symbol;Acc:MGI:1922004]

ENSMUSG
Fam151a
protein_coding
family with sequence simliarity 151, member A

00000034871

[Source:MGI Symbol;Acc:MGI:2657115]

ENSMUSG
Acot11
protein_coding
acyl-CoA thioesterase 11 [Source:MGI

00000034853

Symbol;Acc:MGI:1913736]

ENSMUSG
Ssbp3
protein_coding
single-stranded DNA binding protein 3 [Source:MGI

00000061887

Symbol;Acc:MGI:1919725]

ENSMUSG
Dmrta2
protein_coding
doublesex and mab-3 related transcription factor like

00000047143

family A2 [Source:MGI Symbol;Acc:MGI:2653629]

ENSMUSG
Agbl4
protein_coding
ATP/GTP binding protein-like 4 [Source:MGI

00000061298

Symbol;Acc:MGI:1918244]

ENSMUSG
Skint5
protein_coding
selection and upkeep of intraepithelial T cells 5

00000078598

[Source:MGI Symbol;Acc:MGI:3650151]

ENSMUSG
Stil
protein_coding
Scl/Tal1 interrupting locus [Source:MGI

00000028718

Symbol;Acc:MGI:107477]

ENSMUSG
Tal1
protein_coding
T cell acute lymphocytic leukemia 1 [Source:MGI

00000028717

Symbol;Acc:MGI:98480]

ENSMUSG
Dmbx1
protein_coding
diencephalon/mesencephalon homeobox 1

00000028707

[Source:MGI Symbol;Acc:MGI:2153518]

ENSMUSG
Akr1a1
protein_coding
aldo-keto reductase family 1, member A1 (aldehyde

00000028692

reductase) [Source:MGI Symbol;Acc:MGI:1929955]

ENSMUSG
Rps8
protein_coding
ribosomal protein S8 [Source:MGI

00000047675

Symbol;Acc:MGI:98166]

ENSMUSG
Rnf220
protein_coding
ring finger protein 220 [Source:MGI

00000028677

Symbol;Acc:MGI:1913993]

ENSMUSG
Atp6v0b
protein_coding
ATPase, H+ transporting, lysosomal V0 subunit B

00000033379

[Source:MGI Symbol;Acc:MGI:1890510]

ENSMUSG
Kdm4a
protein_coding
lysine (K)-specific demethylase 4A [Source:MGI

00000033326

Symbol;Acc:MGI:2446210]

ENSMUSG
Elovl1
protein_coding
elongation of very long chain fatty acids (FEN1/Elo2,

00000006390

SUR4/Elo3, yeast)-like 1 [Source:MGI

Symbol;Acc:MGI:1858959]

ENSMUSG
Mpl
protein_coding
myeloproliferative leukemia virus oncogene

00000006389

[Source:MGI Symbol;Acc:MGI:97076]

ENSMUSG
Slc2a1
protein_coding
solute carrier family 2 (facilitated glucose

00000028645

transporter), member 1 [Source:MGI

Symbol;Acc:MGI:95755]

ENSMUSG
Ermap
protein_coding
erythroblast membrane-associated protein

00000028644

[Source:MGI Symbol;Acc:MGI:1349816]

ENSMUSG
AU022252
protein_coding
expressed sequence AU022252 [Source:MGI

00000078584

Symbol;Acc:MGI:2140466]

ENSMUSG
Ybx1
protein_coding
Y box protein 1 [Source:MGI Symbol;Acc:MGI:99146]

00000028639

ENSMUSG
Nfyc
protein_coding
nuclear transcription factor-Y gamma [Source:MGI

00000032897

Symbol;Acc:MGI:107901]

ENSMUSG
Zfp69
protein_coding
zinc finger protein 69 [Source:MGI

00000064141

Symbol;Acc:MGI:107794]

ENSMUSG
Smap2
protein_coding
stromal membrane-associated GTPase-activating

00000032870

protein 2 [Source:MGI Symbol;Acc:MGI:1917030]

ENSMUSG
Ppt1
protein_coding
palmitoyl-protein thioesterase 1 [Source:MGI

00000028657

Symbol;Acc:MGI:1298204]

ENSMUSG
Cap1
protein_coding
CAP, adenylate cyclase-associated protein 1 (yeast)

00000028656

[Source:MGI Symbol;Acc:MGI:88262]

ENSMUSG
Macf1
protein_coding
microtubule-actin crosslinking factor 1 [Source:MGI

00000028649

Symbol;Acc:MGI:108559]

ENSMUSG
Rragc
protein_coding
Ras-related GTP binding C [Source:MGI

00000028646

Symbol;Acc:MGI:1858751]

ENSMUSG
Inpp5b
protein_coding
inositol polyphosphate-5-phosphatase B [Source:MGI

00000028894

Symbol;Acc:MGI:103257]

ENSMUSG
1110065P20Rik
protein_coding
RIKEN cDNA 1110065P20 gene [Source:MGI

00000078570

Symbol;Acc:MGI:1916170]

ENSMUSG
Grik3
protein_coding
glutamate receptor, ionotropic, kainate 3

00000001985

[Source:MGI Symbol;Acc:MGI:95816]

ENSMUSG
Thrap3
protein_coding
thyroid hormone receptor associated protein 3

00000043962

[Source:MGI Symbol;Acc:MGI:2442637]

ENSMUSG
Mtap7d1
protein_coding
microtubule-associated protein 7 domain containing

00000028849

1 [Source:MGI Symbol;Acc:MGI:2384297]

ENSMUSG
Adprhl2
protein_coding
ADP-ribosylhydrolase like 2 [Source:MGI

00000042558

Symbol;Acc:MGI:2140364]

ENSMUSG
Psmb2
protein_coding
proteasome (prosome, macropain) subunit, beta type

00000028837

2 [Source:MGI Symbol;Acc:MGI:1347045]

ENSMUSG
Sfpq
protein_coding
splicing factor proline/glutamine rich (polypyrimidine

00000028820

tract binding protein associated) [Source:MGI

Symbol;Acc:MGI:1918764]

ENSMUSG
Csmd2
protein_coding
CUB and Sushi multiple domains 2 [Source:MGI

00000028804

Symbol;Acc:MGI:2386401]

ENSMUSG
Phc2
protein_coding
polyhomeotic-like 2 (Drosophila) [Source:MGI

00000028796

Symbol;Acc:MGI:1860454]

ENSMUSG
Ak2
protein_coding
adenylate kinase 2 [Source:MGI

00000028792

Symbol;Acc:MGI:87978]

ENSMUSG
Yars
protein_coding
tyrosyl-tRNA synthetase [Source:MGI

00000028811

Symbol;Acc:MGI:2147627]

ENSMUSG
Rbbp4
protein_coding
retinoblastoma binding protein 4 [Source:MGI

00000057236

Symbol;Acc:MGI:1194912]

ENSMUSG
Marcksl1
protein_coding
MARCKS-like 1 [Source:MGI Symbol;Acc:MGI:97143]

00000047945

ENSMUSG
Txlna
protein_coding
taxilin alpha [Source:MGI Symbol;Acc:MGI:105968]

00000053841

ENSMUSG
Khdrbs1
protein_coding
KH domain containing, RNA binding, signal

00000028790

transduction associated 1 [Source:MGI

Symbol;Acc:MGI:893579]

ENSMUSG
Ptp4a2
protein_coding
protein tyrosine phosphatase 4a2 [Source:MGI

00000028788

Symbol;Acc:MGI:1277117]

ENSMUSG
Snrnp40
protein_coding
small nuclear ribonucleoprotein 40 (U5) [Source:MGI

00000074088

Symbol;Acc:MGI:1913835]

ENSMUSG
Pum1
protein_coding
pumilio 1 (Drosophila) [Source:MGI

00000028580

Symbol;Acc:MGI:1931749]

ENSMUSG
Laptm5
protein_coding
lysosomal-associated protein transmembrane 5

00000028581

[Source:MGI Symbol;Acc:MGI:108046]

ENSMUSG
Srsf4
protein_coding
serine/arginine-rich splicing factor 4 [Source:MGI

00000028911

Symbol;Acc:MGI:1890577]

ENSMUSG
Epb4.1
protein_coding
erythrocyte protein band 4.1 [Source:MGI

00000028906

Symbol;Acc:MGI:95401]

ENSMUSG
Ythdf2
protein_coding
YTH domain family 2 [Source:MGI

00000040025

Symbol;Acc:MGI:2444233]

ENSMUSG
Rcc1
protein_coding
regulator of chromosome condensation 1

00000028896

[Source:MGI Symbol;Acc:MGI:1913989]

ENSMUSG
Eya3
protein_coding
eyes absent 3 homolog (Drosophila) [Source:MGI

00000028886

Symbol;Acc:MGI:109339]

ENSMUSG
Ppp1r8
protein_coding
protein phosphatase 1, regulatory (inhibitor) subunit

00000028882

8 [Source:MGI Symbol;Acc:MGI:2140494]

ENSMUSG
Ahdc1
protein_coding
AT hook, DNA binding motif, containing 1

00000037692

[Source:MGI Symbol;Acc:MGI:2444218]

ENSMUSG
Wasf2
protein_coding
WAS protein family, member 2 [Source:MGI

00000028868

Symbol;Acc:MGI:1098641]

ENSMUSG
Nudc
protein_coding
nuclear distribution gene C homolog (Aspergillus)

00000028851

[Source:MGI Symbol;Acc:MGI:106014]

ENSMUSG
Arid1a
protein_coding
AT rich interactive domain 1A (SWI-like) [Source:MGI

00000007880

Symbol;Acc:MGI:1935147]

ENSMUSG
Rps6ka1
protein_coding
ribosomal protein S6 kinase polypeptide 1

00000003644

[Source:MGI Symbol;Acc:MGI:104558]

ENSMUSG
2610002D18Rik
protein_coding
RIKEN cDNA 2610002D18 gene [Source:MGI

00000078521

Symbol;Acc:MGI:1917135]

ENSMUSG
D4Wsu53e
protein_coding
DNA segment, Chr 4, Wayne State University 53,

00000037266

expressed [Source:MGI Symbol;Acc:MGI:106498]

ENSMUSG
Srrm1
protein_coding
serine/arginine repetitive matrix 1 [Source:MGI

00000028809

Symbol;Acc:MGI:1858303]

ENSMUSG
Hnrnpr
protein_coding
heterogeneous nuclear ribonucleoprotein R

00000066037

[Source:MGI Symbol;Acc:MGI:1891692]

ENSMUSG
Htr1d
protein_coding
5-hydroxytryptamine (serotonin) receptor 1D

00000070687

[Source:MGI Symbol;Acc:MGI:96276]

ENSMUSG
Kdm1a
protein_coding
lysine (K)-specific demethylase 1A [Source:MGI

00000036940

Symbol;Acc:MGI:1196256]

ENSMUSG
Ece1
protein_coding
endothelin converting enzyme 1 [Source:MGI

00000057530

Symbol;Acc:MGI:1101357]

ENSMUSG
Ubr4
protein_coding
ubiquitin protein ligase E3 component n-recognin 4

00000066036

[Source:MGI Symbol;Acc:MGI:1916366]

ENSMUSG
Iffo2
protein_coding
intermediate filament family orphan 2 [Source:MGI

00000041025

Symbol;Acc:MGI:2140675]

ENSMUSG
Igsf21
protein_coding
immunoglobin superfamily, member 21 [Source:MGI

00000040972

Symbol;Acc:MGI:2681842]

ENSMUSG
Rcc2
protein_coding
regulator of chromosome condensation 2

00000040945

[Source:MGI Symbol;Acc:MGI:1919784]

ENSMUSG
Atp13a2
protein_coding
ATPase type 13A2 [Source:MGI

00000036622

Symbol;Acc:MGI:1922022]

ENSMUSG
Spen
protein_coding
SPEN homolog, transcriptional regulator (Drosophila)

00000040761

[Source:MGI Symbol;Acc:MGI:1891706]

ENSMUSG
Ddi2
protein_coding
DNA-damage inducible protein 2 [Source:MGI

00000078515

Symbol;Acc:MGI:1917244]

ENSMUSG
2510039O18Rik
protein_coding
RIKEN cDNA 2510039O18 gene [Source:MGI

00000044496

Symbol;Acc:MGI:1924284]

ENSMUSG
Mtor
protein_coding
mechanistic target of rapamycin (serine/threonine

00000028991

kinase) [Source:MGI Symbol;Acc:MGI:1928394]

ENSMUSG
Tardbp
protein_coding
TAR DNA binding protein [Source:MGI

00000041459

Symbol;Acc:MGI:2387629]

ENSMUSG
Casz1
protein_coding
castor homolog 1, zinc finger (Drosophila)

00000028977

[Source:MGI Symbol;Acc:MGI:1196251]

ENSMUSG
Pgd
protein_coding
phosphogluconate dehydrogenase [Source:MGI

00000028961

Symbol;Acc:MGI:97553]

ENSMUSG
Eno1
protein_coding
enolase 1, alpha non-neuron [Source:MGI

00000063524

Symbol;Acc:MGI:95393]

ENSMUSG
Rere
protein_coding
arginine glutamic acid dipeptide (RE) repeats

00000039852

[Source:MGI Symbol;Acc:MGI:2683486]

ENSMUSG
Park7
protein_coding
Parkinson disease (autosomal recessive, early onset)

00000028964

7 [Source:MGI Symbol;Acc:MGI:2135637]

ENSMUSG
Camta1
protein_coding
calmodulin binding transcription activator 1

00000014592

[Source:MGI Symbol;Acc:MGI:2140230]

ENSMUSG
Nol9
protein_coding
nucleolar protein 9 [Source:MGI

00000028948

Symbol;Acc:MGI:1921285]

ENSMUSG
Acot7
protein_coding
acyl-CoA thioesterase 7 [Source:MGI

00000028937

Symbol;Acc:MGI:1917275]

ENSMUSG
Rpl22
protein_coding
ribosomal protein L22 [Source:MGI

00000028936

Symbol;Acc:MGI:99262]

ENSMUSG
Kcnab2
protein_coding
potassium voltage-gated channel, shaker-related

00000028931

subfamily, beta member 2 [Source:MGI

Symbol;Acc:MGI:109239]

ENSMUSG
Nphp4
protein_coding
nephronophthisis 4 (juvenile) homolog (human)

00000039577

[Source:MGI Symbol;Acc:MGI:2384210]

ENSMUSG
Megf6
protein_coding
multiple EGF-like-domains 6 [Source:MGI

00000057751

Symbol;Acc:MGI:1919351]

ENSMUSG
Prdm16
protein_coding
PR domain containing 16 [Source:MGI

00000039410

Symbol;Acc:MGI:1917923]

ENSMUSG
Ski
protein_coding
ski sarcoma viral oncogene homolog (avian)

00000029050

[Source:MGI Symbol;Acc:MGI:98310]

ENSMUSG
Slc35e2
protein_coding
solute carrier family 35, member E2 [Source:MGI

00000042202

Symbol;Acc:MGI:2444240]

ENSMUSG
Cdk6
protein_coding
cyclin-dependent kinase 6 [Source:MGI

00000040274

Symbol;Acc:MGI:1277162]

ENSMUSG
Ankib1
protein_coding
ankyrin repeat and IBR domain containing 1

00000040351

[Source:MGI Symbol;Acc:MGI:1918047]

ENSMUSG
Cyp51
protein_coding
cytochrome P450, family 51 [Source:MGI

00000001467

Symbol;Acc:MGI:106040]

ENSMUSG
Cdk14
protein_coding
cyclin-dependent kinase 14 [Source:MGI

00000028926

Symbol;Acc:MGI:894318]

ENSMUSG
Dbf4
protein_coding
DBF4 homolog (S. cerevisiae) [Source:MGI

00000002297

Symbol;Acc:MGI:1351328]

ENSMUSG
Cacna2d1
protein_coding
calcium channel, voltage-dependent, alpha2/delta

00000040118

subunit 1 [Source:MGI Symbol;Acc:MGI:88295]

ENSMUSG
Sema3c
protein_coding
sema domain, immunoglobulin domain (Ig), short

00000028780

basic domain, secreted, (semaphorin) 3C [Source:MGI

Symbol;Acc:MGI:107557]

ENSMUSG
Magi2
protein_coding
membrane associated guanylate kinase, WW and PDZ

00000040003

domain containing 2 [Source:MGI

Symbol;Acc:MGI:1354953]

ENSMUSG
Rsbn1l
protein_coding
round spermatid basic protein 1-like [Source:MGI

00000039968

Symbol;Acc:MGI:3036237]

ENSMUSG
Ptpn12
protein_coding
protein tyrosine phosphatase, non-receptor type 12

00000028771

[Source:MGI Symbol;Acc:MGI:104673]

ENSMUSG
Psmc2
protein_coding
proteasome (prosome, macropain) 26S subunit,

00000028932

ATPase 2 [Source:MGI Symbol;Acc:MGI:109555]

ENSMUSG
Mll5
protein_coding
myeloid/lymphoid or mixed-lineage leukemia 5

00000029004

[Source:MGI Symbol;Acc:MGI:1924825]

ENSMUSG
Abcb8
protein_coding
ATP-binding cassette, sub-family B (MDR/TAP),

00000028973

member 8 [Source:MGI Symbol;Acc:MGI:1351667]

ENSMUSG
Cdk5
protein_coding
cyclin-dependent kinase 5 [Source:MGI

00000028969

Symbol;Acc:MGI:101765]

ENSMUSG
Chpf2
protein_coding
chondroitin polymerizing factor 2 [Source:MGI

00000038181

Symbol;Acc:MGI:1917522]

ENSMUSG
Smarcd3
protein_coding
SWI/SNF related, matrix associated, actin dependent

00000028949

regulator of chromatin, subfamily d, member 3

[Source:MGI Symbol;Acc:MGI:1914243]

ENSMUSG
Tyms
protein_coding
thymidylate synthase [Source:MGI

00000025747

Symbol;Acc:MGI:98878]

ENSMUSG
Emilin1
protein_coding
elastin microfibril interfacer 1 [Source:MGI

00000029163

Symbol;Acc:MGI:1926189]

ENSMUSG
Preb
protein_coding
prolactin regulatory element binding [Source:MGI

00000045302

Symbol;Acc:MGI:1355326]

ENSMUSG
Cad
protein_coding
carbamoyl-phosphate synthetase 2, aspartate

00000013629

transcarbamylase, and dihydroorotase [Source:MGI

Symbol;Acc:MGI:1916969]

ENSMUSG
Ppm1g
protein_coding
protein phosphatase 1G (formerly 2C), magnesium-

00000029147

dependent, gamma isoform [Source:MGI

Symbol;Acc:MGI:106065]

ENSMUSG
Gckr
protein_coding
glucokinase regulatory protein [Source:MGI

00000059434

Symbol;Acc:MGI:1096345]

ENSMUSG
RP23-118L1.1
protein_coding
MCG2531, isoform CRA_h; Uncharacterized protein

00000093574

[Source:UniProtKB/TrEMBL;Acc:Q3TJ76]

ENSMUSG
C330019G07Rik
protein_coding
RIKEN cDNA C330019G07 gene [Source:MGI

00000054280

Symbol;Acc:MGI:2443658]

ENSMUSG
Ctbp1
protein_coding
C-terminal binding protein 1 [Source:MGI

00000037373

Symbol;Acc:MGI:1201685]

ENSMUSG
Maea
protein_coding
macrophage erythroblast attacher [Source:MGI

00000079562

Symbol;Acc:MGI:1891748]

ENSMUSG
Rnf4
protein_coding
ring finger protein 4 [Source:MGI

00000029110

Symbol;Acc:MGI:1201691]

ENSMUSG
Fam193a
protein_coding
family with sequence similarity 193, member A

00000037210

[Source:MGI Symbol;Acc:MGI:2447768]

ENSMUSG
Htt
protein_coding
huntingtin [Source:MGI Symbol;Acc:MGI:96067]

00000029104

ENSMUSG
Adra2c
protein_coding
adrenergic receptor, alpha 2c [Source:MGI

00000045318

Symbol;Acc:MGI:87936]

ENSMUSG
Afap1
protein_coding
actin filament associated protein 1 [Source:MGI

00000029094

Symbol;Acc:MGI:1917542]

ENSMUSG
Sorcs2
protein_coding
sortilin-related VPS10 domain containing receptor 2

00000029093

[Source:MGI Symbol;Acc:MGI:1932289]

ENSMUSG
Psapl1
protein_coding
prosaposin-like 1 [Source:MGI

00000043430

Symbol;Acc:MGI:1924193]

ENSMUSG
D5Ertd579e
protein_coding
DNA segment, Chr 5, ERATO Doi 579, expressed

00000029190

[Source:MGI Symbol;Acc:MGI:1261849]

ENSMUSG
Evc
protein_coding
Ellis van Creveld gene homolog (human) [Source:MGI

00000029122

Symbol;Acc:MGI:1890596]

ENSMUSG
Wdr1
protein_coding
WD repeat domain 1 [Source:MGI

00000005103

Symbol;Acc:MGI:1337100]

ENSMUSG
Kcnip4
protein_coding
Kv channel interacting protein 4 [Source:MGI

00000029088

Symbol;Acc:MGI:1933131]

ENSMUSG
Pcdh7
protein_coding
protocadherin 7 [Source:MGI

00000029108

Symbol;Acc:MGI:1860487]

ENSMUSG
N4bp2
protein_coding
NEDD4 binding protein 2 [Source:MGI

00000037795

Symbol;Acc:MGI:2684414]

ENSMUSG
Nsun7
protein_coding
NOL1/NOP2/Sun domain family, member 7

00000029206

[Source:MGI Symbol;Acc:MGI:1918168]

ENSMUSG
Bend4
protein_coding
BEN domain containing 4 [Source:MGI

00000092060

Symbol;Acc:MGI:3648414]

ENSMUSG
Gabrb1
protein_coding
gamma-aminobutyric acid (GABA) A receptor, subunit

00000029212

beta 1 [Source:MGI Symbol;Acc:MGI:95619]

ENSMUSG
Fryl
protein_coding
furry homolog-like (Drosophila) [Source:MGI

00000070733

Symbol;Acc:MGI:1919563]

ENSMUSG
Fip1|1
protein_coding
FIP1 like 1 (S. cerevisiae) [Source:MGI

00000029227

Symbol;Acc:MGI:1914149]

ENSMUSG
Kit
protein_coding
kit oncogene [Source:MGI Symbol;Acc:MGI:96677]

00000005672

ENSMUSG
C530008M17Rik
protein_coding
RIKEN cDNA C530008M17 gene [Source:MGI

00000036377

Symbol;Acc:MGI:2444817]

ENSMUSG
Rest
protein_coding
RE1-silencing transcription factor [Source:MGI

00000029249

Symbol;Acc:MGI:104897]

ENSMUSG
Polr2b
protein_coding
polymerase (RNA) II (DNA directed) polypeptide B

00000029250

[Source:MGI Symbol;Acc:MGI:2388280]

ENSMUSG
Ankrd17
protein_coding
ankyrin repeat domain 17 [Source:MGI

00000055204

Symbol;Acc:MGI:1932101]

ENSMUSG
Pf4
protein_coding
platelet factor 4 [Source:MGI

00000029373

Symbol;Acc:MGI:1888711]

ENSMUSG
G3bp2
protein_coding
GTPase activating protein (SH3 domain) binding

00000029405

protein 2 [Source:MGI Symbol;Acc:MGI:2442040]

ENSMUSG
Bmp2k
protein_coding
BMP2 inducible kinase [Source:MGI

00000034663

Symbol;Acc:MGI:2155456]

ENSMUSG
Paqr3
protein_coding
progestin and adipoQ receptor family member III

00000055725

[Source:MGI Symbol;Acc:MGI:2679683]

ENSMUSG
1700007G11Rik
protein_coding
RIKEN cDNA 1700007G11 gene [Source:MGI

00000057816

Symbol;Acc:MGI:1916571]

ENSMUSG
Rasgef1b
protein_coding
RasGEF domain family, member 1B [Source:MGI

00000029333

Symbol;Acc:MGI:2443755]

ENSMUSG
Hnrnpd
protein_coding
heterogeneous nuclear ribonucleoprotein D

00000000568

[Source:MGI Symbol;Acc:MGI:101947]

ENSMUSG
Hnrpdl
protein_coding
heterogeneous nuclear ribonucleoprotein D-like

00000029328

[Source:MGI Symbol;Acc:MGI:1355299]

ENSMUSG
Sec31a
protein_coding
Sec31 homolog A (S. cerevisiae) [Source:MGI

00000035325

Symbol;Acc:MGI:1916412]

ENSMUSG
Hpse
protein_coding
heparanase [Source:MGI Symbol;Acc:MGI:1343124]

00000035273

ENSMUSG
Wdfy3
protein_coding
WD repeat and FYVE domain containing 3

00000043940

[Source:MGI Symbol;Acc:MGI:1096875]

ENSMUSG
Aff1
protein_coding
AF4/FMR2 family, member 1 [Source:MGI

00000029313

Symbol;Acc:MGI:1100819]

ENSMUSG
Lrrc8b
protein_coding
leucine rich repeat containing 8 family, member B

00000070639

[Source:MGI Symbol;Acc:MGI:2141353]

ENSMUSG
Lrrc8d
protein_coding
leucine rich repeat containing 8D [Source:MGI

00000046079

Symbol;Acc:MGI:1922368]

ENSMUSG
Zfp644
protein_coding
zinc finger protein 644 [Source:MGI

00000049606

Symbol;Acc:MGI:1277212]

ENSMUSG
Evi5
protein_coding
ecotropic viral integration site 5 [Source:MGI

00000011831

Symbol;Acc:MGI:104736]

ENSMUSG
Rpl5
protein_coding
ribosomal protein L5 [Source:MGI

00000058558

Symbol;Acc:MGI:102854]

ENSMUSG
Mtf2
protein_coding
metal response element binding transcription factor 2

00000029267

[Source:MGI Symbol;Acc:MGI:105050]

ENSMUSG
Gak
protein_coding
cyclin G associated kinase [Source:MGI

00000062234

Symbol;Acc:MGI:2442153]

ENSMUSG
Fbrsl1
protein_coding
fibrosin-like 1 [Source:MGI Symbol;Acc:MGI:1920907]

00000043323

ENSMUSG
Pus1
protein_coding
pseudouridine synthase 1 [Source:MGI

00000029507

Symbol;Acc:MGI:1929237]

ENSMUSG
Tfip11
protein_coding
tuftelin interacting protein 11 [Source:MGI

00000029345

Symbol;Acc:MGI:1930075]

ENSMUSG
Sez6l
protein_coding
seizure related 6 homolog like [Source:MGI

00000058153

Symbol;Acc:MGI:1935121]

ENSMUSG
Selplg
protein_coding
selectin, platelet (p-selectin) ligand [Source:MGI

00000048163

Symbol;Acc:MGI:106689]

ENSMUSG
Coro1c
protein_coding
coronin, actin binding protein 1C [Source:MGI

00000004530

Symbol;Acc:MGI:1345964]

ENSMUSG
Acacb
protein_coding
acetyl-Coenzyme A carboxylase beta [Source:MGI

00000042010

Symbol;Acc:MGI:2140940]

ENSMUSG
Myo1h
protein_coding
myosin 1H [Source:MGI Symbol;Acc:MGI:1914674]

00000066952

ENSMUSG
Kctd10
protein_coding
potassium channel tetramerisation domain

00000001098

containing 10 [Source:MGI Symbol;Acc:MGI:2141207]

ENSMUSG
Ube3b
protein_coding
ubiquitin protein ligase E3B [Source:MGI

00000029577

Symbol;Acc:MGI:1891295]

ENSMUSG
Git2
protein_coding
G protein-coupled receptor kinase-interactor 2

00000041890

[Source:MGI Symbol;Acc:MGI:1347053]

ENSMUSG
Ankrd13a
protein_coding
ankyrin repeat domain 13a [Source:MGI

00000041870

Symbol;Acc:MGI:1915670]

ENSMUSG
Sppl3
protein_coding
signal peptide peptidase 3 [Source:MGI

00000029550

Symbol;Acc:MGI:1891433]

ENSMUSG
Gm10401
protein_coding
predicted gene 10401 [Source:MGI

00000072693

Symbol;Acc:MGI:3704254]

ENSMUSG
Mlec
protein_coding
malectin [Source:MGI Symbol;Acc:MGI:1924015]

00000048578

ENSMUSG
Rnf10
protein_coding
ring finger protein 10 [Source:MGI

00000041740

Symbol;Acc:MGI:1859162]

ENSMUSG
Triap1
protein_coding
TP53 regulated inhibitor of apoptosis 1 [Source:MGI

00000029535

Symbol;Acc:MGI:1916326]

ENSMUSG
Cox6a1
protein_coding
cytochrome c oxidase, subunit VI a, polypeptide 1

00000041697

[Source:MGI Symbol;Acc:MGI:103099]

ENSMUSG
Pxn
protein_coding
paxillin [Source:MGI Symbol;Acc:MGI:108295]

00000029528

ENSMUSG
Rab35
protein_coding
RAB35, member RAS oncogene family [Source:MGI

00000029518

Symbol;Acc:MGI:1924657]

ENSMUSG
Cit
protein_coding
citron [Source:MGI Symbol;Acc:MGI:105313]

00000029516

ENSMUSG
Rfc5
protein_coding
replication factor C (activator 1) 5 [Source:MGI

00000029363

Symbol;Acc:MGI:1919401]

ENSMUSG
Nos1
protein_coding
nitric oxide synthase 1, neuronal [Source:MGI

00000029361

Symbol;Acc:MGI:97360]

ENSMUSG
Med13l
protein_coding
mediator complex subunit 13-like [Source:MGI

00000018076

Symbol;Acc:MGI:2670178]

ENSMUSG
Ptpn11
protein_coding
protein tyrosine phosphatase, non-receptor type 11

00000043733

[Source:MGI Symbol;Acc:MGI:99511]

ENSMUSG
Rpl6
protein_coding
ribosomal protein L6 [Source:MGI

00000029614

Symbol;Acc:MGI:108057]

ENSMUSG
Gm15800
protein_coding
predicted gene 15800 [Source:MGI

00000042744

Symbol;Acc:MGI:3647820]

ENSMUSG
Erp29
protein_coding
endoplasmic reticulum protein 29 [Source:MGI

00000029616

Symbol;Acc:MGI:1914647]

ENSMUSG
Aldh2
protein_coding
aldehyde dehydrogenase 2, mitochondrial

00000029455

[Source:MGI Symbol;Acc:MGI:99600]

ENSMUSG
Sh2b3
protein_coding
SH2B adaptor protein 3 [Source:MGI

00000042594

Symbol;Acc:MGI:893598]

ENSMUSG
Cux2
protein_coding
cut-like homeobox 2 [Source:MGI

00000042589

Symbol;Acc:MGI:107321]

ENSMUSG
Hvcn1
protein_coding
hydrogen voltage-gated channel 1 [Source:MGI

00000064267

Symbol;Acc:MGI:1921346]

ENSMUSG
Pptc7
protein_coding
PTC7 protein phosphatase homolog (S. cerevisiae)

00000038582

[Source:MGI Symbol;Acc:MGI:2444593]

ENSMUSG
Arpc3
protein_coding
actin related protein 2/3 complex, subunit 3

00000029465

[Source:MGI Symbol;Acc:MGI:1928375]

ENSMUSG
Atp2a2
protein_coding
ATPase, Ca++ transporting, cardiac muscle, slow

00000029467

twitch 2 [Source:MGI Symbol;Acc:MGI:88110]

ENSMUSG
Kdm2b
protein_coding
lysine (K)-specific demethylase 2B [Source:MGI

00000029475

Symbol;Acc:MGI:1354737]

ENSMUSG
Orai1
protein_coding
ORAI calcium release-activated calcium modulator 1

00000049686

[Source:MGI Symbol;Acc:MGI:1925542]

ENSMUSG
Setd1b
protein_coding
SET domain containing 1B [Source:MGI

00000038384

Symbol;Acc:MGI:2652820]

ENSMUSG
Bcl7a
protein_coding
B cell CLL/lymphoma 7A [Source:MGI

00000029438

Symbol;Acc:MGI:1924295]

ENSMUSG
Mlxip
protein_coding
MLX interacting protein [Source:MGI

00000038342

Symbol;Acc:MGI:2141183]

ENSMUSG
Denr
protein_coding
density-regulated protein [Source:MGI

00000023106

Symbol;Acc:MGI:1915434]

ENSMUSG
Cdk2ap1
protein_coding
CDK2 (cyclin-dependent kinase 2)-associated protein

00000029394

1 [Source:MGI Symbol;Acc:MGI:1202069]

ENSMUSG
Sbno1
protein_coding
sno, strawberry notch homolog 1 (Drosophila)

00000038095

[Source:MGI Symbol;Acc:MGI:2384298]

ENSMUSG
Atp6v0a2
protein_coding
ATPase, H+ transporting, lysosomal V0 subunit A2

00000038023

[Source:MGI Symbol;Acc:MGI:104855]

ENSMUSG
Zfp664
protein_coding
zinc finger protein 664 [Source:MGI

00000079215

Symbol;Acc:MGI:2442505]

ENSMUSG
Zfp664
protein_coding
zinc finger protein 664 [Source:MGI

00000051911

Symbol;Acc:MGI:2442505]

ENSMUSG
Ncor2
protein_coding
nuclear receptor co-repressor 2 [Source:MGI

00000029478

Symbol;Acc:MGI:1337080]

ENSMUSG
Bri3bp
protein_coding
Bri3 binding protein [Source:MGI

00000037905

Symbol;Acc:MGI:1924059]

ENSMUSG
Aacs
protein_coding
acetoacetyl-CoA synthetase [Source:MGI

00000029482

Symbol;Acc:MGI:1926144]

ENSMUSG
Tmem132c
protein_coding
transmembrane protein 132C [Source:MGI

00000034324

Symbol;Acc:MGI:2443061]

ENSMUSG
Tmem132d
protein_coding
transmembrane protein 132D [Source:MGI

00000034310

Symbol;Acc:MGI:3044963]

ENSMUSG
Rimbp2
protein_coding
RIMS binding protein 2 [Source:MGI

00000029420

Symbol;Acc:MGI:2443235]

ENSMUSG
Sfswap
protein_coding
splicing factor, suppressor of white-apricot homolog

00000029439

(Drosophila) [Source:MGI Symbol;Acc:MGI:101760]

ENSMUSG
Cct6a
protein_coding
chaperonin containing Tcp1, subunit 6a (zeta)

00000029447

[Source:MGI Symbol;Acc:MGI:107943]

ENSMUSG
Phkg1
protein_coding
phosphorylase kinase gamma 1 [Source:MGI

00000025537

Symbol;Acc:MGI:97579]

ENSMUSG
Rabgef1
protein_coding
RAB guanine nucleotide exchange factor (GEF) 1

00000025340

[Source:MGI Symbol;Acc:MGI:1929459]

ENSMUSG
Wbscr17
protein_coding
Williams-Beuren syndrome chromosome region 17

00000034040

homolog (human) [Source:MGI

Symbol;Acc:MGI:2137594]

ENSMUSG
Auts2
protein_coding
autism susceptibility candidate 2 [Source:MGI

00000029673

Symbol;Acc:MGI:1919847]

ENSMUSG
Gtf2i
protein_coding
general transcription factor II I [Source:MGI

00000060261

Symbol;Acc:MGI:1202722]

ENSMUSG
Clip2
protein_coding
CAP-GLY domain containing linker protein 2

00000063146

[Source:MGI Symbol;Acc:MGI:1313136]

ENSMUSG
Eif4h
protein_coding
eukaryotic translation initiation factor 4H

00000040731

[Source:MGI Symbol;Acc:MGI:1341822]

ENSMUSG
Baz1b
protein_coding
bromodomain adjacent to zinc finger domain, 1B

00000002748

[Source:MGI Symbol;Acc:MGI:1353499]

ENSMUSG
Pom121
protein_coding
nuclear pore membrane protein 121 [Source:MGI

00000053293

Symbol;Acc:MGI:2137624]

ENSMUSG
Mdh2
protein_coding
malate dehydrogenase 2, NAD (mitochondrial)

00000019179

[Source:MGI Symbol;Acc:MGI:97050]

ENSMUSG
Ywhag
protein_coding
tyrosine 3-monooxygenase/tryptophan 5-

00000051391

monooxygenase activation protein, gamma

polypeptide [Source:MGI Symbol;Acc:MGI:108109]

ENSMUSG
Cux1
protein_coding
cut-like homeobox 1 [Source:MGI

00000029705

Symbol;Acc:MGI:88568]

ENSMUSG
Serpine1
protein_coding
serine (or cysteine) peptidase inhibitor, clade E,

00000037411

member 1 [Source:MGI Symbol;Acc:MGI:97608]

ENSMUSG
Trim56
protein_coding
tripartite motif-containing 56 [Source:MGI

00000043279

Symbol;Acc:MGI:2685298]

ENSMUSG
Srrt
protein_coding
serrate RNA effector molecule homolog (Arabidopsis)

00000037364

[Source:MGI Symbol;Acc:MGI:1933527]

ENSMUSG
Epo
protein_coding
erythropoietin [Source:MGI Symbol;Acc:MGI:95407]

00000029711

ENSMUSG
Tsc22d4
protein_coding
TSC22 domain family, member 4 [Source:MGI

00000029723

Symbol;Acc:MGI:1926079]

ENSMUSG
Mepce
protein_coding
methylphosphate capping enzyme [Source:MGI

00000029726

Symbol;Acc:MGI:106477]

ENSMUSG
Mcm7
protein_coding
minichromosome maintenance deficient 7 (S.

00000029730

cerevisiae) [Source:MGI Symbol;Acc:MGI:1298398]

ENSMUSG
Get4
protein_coding
golgi to ER traffic protein 4 homolog (S. cerevisiae)

00000025858

[Source:MGI Symbol;Acc:MGI:1914854]

ENSMUSG
Ints1
protein_coding
integrator complex subunit 1 [Source:MGI

00000029547

Symbol;Acc:MGI:1915760]

ENSMUSG
Mad1|1
protein_coding
MAD1 mitotic arrest deficient 1-like 1 [Source:MGI

00000029554

Symbol;Acc:MGI:1341857]

ENSMUSG
Eif3b
protein_coding
eukaryotic translation initiation factor 3, subunit B

00000056076

[Source:MGI Symbol;Acc:MGI:106478]

ENSMUSG
Ttyh3
protein_coding
tweety homolog 3 (Drosophila) [Source:MGI

00000036565

Symbol;Acc:MGI:1925589]

ENSMUSG
Amz1
protein_coding
archaelysin family metallopeptidase 1 [Source:MGI

00000050022

Symbol;Acc:MGI:2442258]

ENSMUSG
Gna12
protein_coding
guanine nucleotide binding protein, alpha 12

00000000149

[Source:MGI Symbol;Acc:MGI:95767]

ENSMUSG
Sdk1
protein_coding
sidekick homolog 1 (chicken) [Source:MGI

00000039683

Symbol;Acc:MGI:2444413]

ENSMUSG
Foxk1
protein_coding
forkhead box K1 [Source:MGI

00000056493

Symbol;Acc:MGI:1347488]

ENSMUSG
Actb
protein_coding
actin, beta [Source:MGI Symbol;Acc:MGI:87904]

00000029580

ENSMUSG
E130309D02Rik
protein_coding
RIKEN cDNA E130309D02 gene [Source:MGI

00000039244

Symbol;Acc:MGI:2442621]

ENSMUSG
Rac1
protein_coding
RAS-related C3 botulinum substrate 1 [Source:MGI

00000001847

Symbol;Acc:MGI:97845]

ENSMUSG
Usp42
protein_coding
ubiquitin specific peptidase 42 [Source:MGI

00000051306

Symbol;Acc:MGI:1924050]

ENSMUSG
Trrap
protein_coding
transformation/transcription domain-associated

00000045482

protein [Source:MGI Symbol;Acc:MGI:2153272]

ENSMUSG
Arpc1b
protein_coding
actin related protein 2/3 complex, subunit 1B

00000029622

[Source:MGI Symbol;Acc:MGI:1343142]

ENSMUSG
Pdap1
protein_coding
PDGFA associated protein 1 [Source:MGI

00000029623

Symbol;Acc:MGI:2448536]

ENSMUSG
Mtif3
protein_coding
mitochondrial translational initiation factor 3

00000016510

[Source:MGI Symbol;Acc:MGI:1923616]

ENSMUSG
Pan3
protein_coding
PAN3 polyA specific ribonuclease subunit homolog (S.

00000029647

cerevisiae) [Source:MGI Symbol;Acc:MGI:1919837]

ENSMUSG
Mtus2
protein_coding
microtubule associated tumor suppressor candidate 2

00000029651

[Source:MGI Symbol;Acc:MGI:1915388]

ENSMUSG
Slc7a1
protein_coding
solute carrier family 7 (cationic amino acid

00000041313

transporter, y+ system), member 1 [Source:MGI

Symbol;Acc:MGI:88117]

ENSMUSG
Wdr95
protein_coding
WD40 repeat domain 95 [Source:MGI

00000029658

Symbol;Acc:MGI:1923042]

ENSMUSG
Hsph1
protein_coding
heat shock 105 kDa/110k Da protein 1 [Source:MGI

00000029657

Symbol;Acc:MGI:105053]

ENSMUSG
Pds5b
protein_coding
PDS5, regulator of cohesion maintenance, homolog B

00000034021

(S. cerevisiae) [Source:MGI Symbol;Acc:MGI:2140945]

ENSMUSG
Ccdc132
protein_coding
coiled-coil domain containing 132 [Source:MGI

00000001376

Symbol;Acc:MGI:1920538]

ENSMUSG
Ppp1r9a
protein_coding
protein phosphatase 1, regulatory (inhibitor) subunit

00000032827

9A [Source:MGI Symbol;Acc:MGI:2442401]

ENSMUSG
Kcnd2
protein_coding
potassium voltage-gated channel, Shal-related family,

00000060882

member 2 [Source:MGI Symbol;Acc:MGI:102663]

ENSMUSG
A430107O13Rik
protein_coding
RIKEN cDNA A430107O13 gene [Source:MGI

00000062980

Symbol;Acc:MGI:2444814]

ENSMUSG
Cadps2
protein_coding
Ca2+-dependent activator protein for secretion 2

00000017978

[Source:MGI Symbol;Acc:MGI:2443963]

ENSMUSG
Gcc1
protein_coding
golgi coiled coil 1 [Source:MGI

00000029708

Symbol;Acc:MGI:1921625]

ENSMUSG
Snd1
protein_coding
staphylococcal nuclease and tudor domain containing

00000001424

1 [Source:MGI Symbol;Acc:MGI:1929266]

ENSMUSG
Lrrc4
protein_coding
leucine rich repeat containing 4 [Source:MGI

00000049939

Symbol;Acc:MGI:2182081]

ENSMUSG
Opn1sw
protein_coding
opsin 1 (cone pigments), short-wave-sensitive (color

00000058831

blindness, tritan) [Source:MGI

Symbol;Acc:MGI:99438]

ENSMUSG
Klf14
protein_coding
Kruppel-like factor 14 [Source:MGI

00000073209

Symbol;Acc:MGI:3577024]

ENSMUSG
Plxna4
protein_coding
plexin A4 [Source:MGI Symbol;Acc:MGI:2179061]

00000029765

ENSMUSG
Slc35b4
protein_coding
solute carrier family 35, member B4 [Source:MGI

00000018999

Symbol;Acc:MGI:1931249]

ENSMUSG
Nup205
protein_coding
nucleoporin 205 [Source:MGI

00000038759

Symbol;Acc:MGI:2141625]

ENSMUSG
Mtpn
protein_coding
myotrophin [Source:MGI Symbol;Acc:MGI:99445]

00000029840

ENSMUSG
Trim24
protein_coding
tripartite motif-containing 24 [Source:MGI

00000029833

Symbol;Acc:MGI:109275]

ENSMUSG
Zc3hav1l
protein_coding
zinc finger CCCH-type, antiviral 1-like [Source:MGI

00000047749

Symbol;Acc:MGI:2443387]

ENSMUSG
Zc3hav1
protein_coding
zinc finger CCCH type, antiviral 1 [Source:MGI

00000029826

Symbol;Acc:MGI:1926031]

ENSMUSG
Hipk2
protein_coding
homeodomain interacting protein kinase 2

00000061436

[Source:MGI Symbol;Acc:MGI:1314872]

ENSMUSG
Jhdm1d
protein_coding
jumonji C domain-containing histone demethylase 1

00000042599

homolog D (S. cerevisiae) [Source:MGI

Symbol;Acc:MGI:2443388]

ENSMUSG
Casp2
protein_coding
caspase 2 [Source:MGI Symbol;Acc:MGI:97295]

00000029863

ENSMUSG
Zyx
protein_coding
zyxin [Source:MGI Symbol;Acc:MGI:103072]

00000029860

ENSMUSG
Cntnap2
protein_coding
contactin associated protein-like 2 [Source:MGI

00000039419

Symbol;Acc:MGI:1914047]

ENSMUSG
Zfp282
protein_coding
zinc finger protein 282 [Source:MGI

00000025821

Symbol;Acc:MGI:2141413]

ENSMUSG
Zfp777
protein_coding
zinc finger protein 777 [Source:MGI

00000071477

Symbol;Acc:MGI:1919556]

ENSMUSG
Zfp746
protein_coding
zinc finger protein 746 [Source:MGI

00000057691

Symbol;Acc:MGI:1916478]

ENSMUSG
Tmem176b
protein_coding
transmembrane protein 176B [Source:MGI

00000029810

Symbol;Acc:MGI:1916348]

ENSMUSG
Hnrnpa2b1
protein_coding
heterogeneous nuclear ribonucleoprotein A2/B1

00000004980

[Source:MGI Symbol;Acc:MGI:104819]

ENSMUSG
Skap2
protein_coding
src family associated phosphoprotein 2 [Source:MGI

00000059182

Symbol;Acc:MGI:1889206]

ENSMUSG
Hoxa1
protein_coding
homeobox A1 [Source:MGI Symbol;Acc:MGI:96170]

00000029844

ENSMUSG
RP23-103L13.8
protein_coding

00000093412

ENSMUSG
Hoxa3
protein_coding
homeobox A3 [Source:MGI Symbol;Acc:MGI:96175]

00000079560

ENSMUSG
Hoxa3
protein_coding
homeobox A3 [Source:MGI Symbol;Acc:MGI:96175]

00000059723

ENSMUSG
Tax1bp1
protein_coding
Tax1 (human T cell leukemia virus type I) binding

00000004535

protein 1 [Source:MGI Symbol;Acc:MGI:1289308]

ENSMUSG
Jazf1
protein_coding
JAZF zinc finger 1 [Source:MGI

00000063568

Symbol;Acc:MGI:2141450]

ENSMUSG
Ppp1r17
protein_coding
protein phosphatase 1, regulatory subunit 17

00000002930

[Source:MGI Symbol;Acc:MGI:1333876]

ENSMUSG
Fam190a
protein_coding
family with sequence similarity 190, member A

00000039578

[Source:MGI Symbol;Acc:MGI:3045354]

ENSMUSG
Grid2
protein_coding
glutamate receptor, ionotropic, delta 2 [Source:MGI

00000071424

Symbol;Acc:MGI:95813]

ENSMUSG
A930038C07Rik
protein_coding
RIKEN cDNA A930038C07 gene [Source:MGI

00000049001

Symbol;Acc:MGI:1915419]

ENSMUSG
Serbp1
protein_coding
serpine1 mRNA binding protein 1 [Source:MGI

00000036371

Symbol;Acc:MGI:1914120]

ENSMUSG
Rmnd5a
protein_coding
required for meiotic nuclear division 5 homolog A (S.

00000002222

cerevisiae) [Source:MGI Symbol;Acc:MGI:1915727]

ENSMUSG
Ptcd3
protein_coding
pentatricopeptide repeat domain 3 [Source:MGI

00000063884

Symbol;Acc:MGI:1917206]

ENSMUSG
Atoh8
protein_coding
atonal homolog 8 (Drosophila) [Source:MGI

00000037621

Symbol;Acc:MGI:1918343]

ENSMUSG
Usp39
protein_coding
ubiquitin specific peptidase 39 [Source:MGI

00000056305

Symbol;Acc:MGI:107622]

ENSMUSG
Mat2a
protein_coding
methionine adenosyltransferase II, alpha [Source:MGI

00000053907

Symbol;Acc:MGI:2443731]

ENSMUSG
Tcf7l1
protein_coding
transcription factor 7 like 1 (T cell specific, HMG box)

00000055799

[Source:MGI Symbol;Acc:MGI:1202876]

ENSMUSG
Lrrtm4
protein_coding
leucine rich repeat transmembrane neuronal 4

00000052581

[Source:MGI Symbol;Acc:MGI:2389180]

ENSMUSG
Mogs
protein_coding
mannosyl-oligosaccharide glucosidase [Source:MGI

00000030036

Symbol;Acc:MGI:1929872]

ENSMUSG
Tet3
protein_coding
tet methylcytosine dioxygenase 3 [Source:MGI

00000034832

Symbol;Acc:MGI:2446229]

ENSMUSG
Cyp26b1
protein_coding
cytochrome P450, family 26, subfamily b, polypeptide

00000063415

1 [Source:MGI Symbol;Acc:MGI:2176159]

ENSMUSG
Exoc6b
protein_coding
exocyst complex component 6B [Source:MGI

00000033769

Symbol;Acc:MGI:1923164]

ENSMUSG
Cct7
protein_coding
chaperonin containing Tcp1, subunit 7 (eta)

00000030007

[Source:MGI Symbol;Acc:MGI:107184]

ENSMUSG
Pcbp1
protein_coding
poly(rC) binding protein 1 [Source:MGI

00000051695

Symbol;Acc:MGI:1345635]

ENSMUSG
Gfpt1
protein_coding
glutamine fructose-6-phosphate transaminase 1

00000029992

[Source:MGI Symbol;Acc:MGI:95698]

ENSMUSG
Arhgap25
protein_coding
Rho GTPase activating protein 25 [Source:MGI

00000030047

Symbol;Acc:MGI:2443687]

ENSMUSG
Rab43
protein_coding
RAB43, member RAS oncogene family [Source:MGI

00000030055

Symbol;Acc:MGI:1917084]

ENSMUSG
Cnbp
protein_coding
cellular nucleic acid binding protein [Source:MGI

00000030057

Symbol;Acc:MGI:88431]

ENSMUSG
Gm5577
protein_coding
predicted gene 5577 [Source:MGI

00000084950

Symbol;Acc:MGI:3648213]

ENSMUSG
Rab7
protein_coding
RAB7, member RAS oncogene family [Source:MGI

00000079477

Symbol;Acc:MGI:105068]

ENSMUSG
Gata2
protein_coding
GATA binding protein 2 [Source:MGI

00000015053

Symbol;Acc:MGI:95662]

ENSMUSG
Eefsec
protein_coding
eukaryotic elongation factor, selenocysteine-tRNA-

00000033216

specific [Source:MGI Symbol;Acc:MGI:2137092]

ENSMUSG
Sec61a1
protein_coding
Sec61 alpha 1 subunit (S. cerevisiae) [Source:MGI

00000030082

Symbol;Acc:MGI:1858417]

ENSMUSG
Mcm2
protein_coding
minichromosome maintenance deficient 2 mitotin (S.

00000002870

cerevisiae) [Source:MGI Symbol;Acc:MGI:105380]

ENSMUSG
Chchd6
protein_coding
coiled-coil-helix-coiled-coil-helix domain containing 6

00000030086

[Source:MGI Symbol;Acc:MGI:1913348]

ENSMUSG
Zxdc
protein_coding
ZXD family zinc finger C [Source:MGI

00000034430

Symbol;Acc:MGI:1933108]

ENSMUSG
Nup210
protein_coding
nucleoporin 210 [Source:MGI

00000030091

Symbol;Acc:MGI:1859555]

ENSMUSG
Slc6a6
protein_coding
solute carrier family 6 (neurotransmitter transporter,

00000030096

taurine), member 6 [Source:MGI

Symbol;Acc:MGI:98488]

ENSMUSG
Magi1
protein_coding
membrane associated guanylate kinase, WW and PDZ

00000045095

domain containing 1 [Source:MGI

Symbol;Acc:MGI:1203522]

ENSMUSG
Gm765
protein_coding
predicted gene 765 [Source:MGI

00000090667

Symbol;Acc:MGI:2685611]

ENSMUSG
Foxp1
protein_coding
forkhead box P1 [Source:MGI

00000030067

Symbol;Acc:MGI:1914004]

ENSMUSG
Srgap3
protein_coding
SLIT-ROBO Rho GTPase activating protein 3

00000030257

[Source:MGI Symbol;Acc:MGI:2152938]

ENSMUSG
Setd5
protein_coding
SET domain containing 5 [Source:MGI

00000034269

Symbol;Acc:MGI:1920145]

ENSMUSG
Arpc4
protein_coding
actin related protein 2/3 complex, subunit 4

00000079426

[Source:MGI Symbol;Acc:MGI:1915339]

ENSMUSG
Ttll3
protein_coding
tubulin tyrosine ligase-like family, member 3

00000030276

[Source:MGI Symbol;Acc:MGI:2141418]

ENSMUSG
Jagn1
protein_coding
jagunal homolog 1 (Drosophila) [Source:MGI

00000051256

Symbol;Acc:MGI:1915017]

ENSMUSG
Atg7
protein_coding
autophagy-related 7 (yeast) [Source:MGI

00000030314

Symbol;Acc:MGI:1921494]

ENSMUSG
Raf1
protein_coding
v-raf-leukemia viral oncogene 1 [Source:MGI

00000000441

Symbol;Acc:MGI:97847]

ENSMUSG
Rpl32
protein_coding
ribosomal protein L32 [Source:MGI

00000057841

Symbol;Acc:MGI:98038]

ENSMUSG
Plxnd1
protein_coding
plexin D1 [Source:MGI Symbol;Acc:MGI:2154244]

00000030123

ENSMUSG
Tmcc1
protein_coding
transmembrane and coiled coil domains 1

00000030126

[Source:MGI Symbol;Acc:MGI:2442368]

ENSMUSG
Hnrnpf
protein_coding
heterogeneous nuclear ribonucleoprotein F

00000042079

[Source:MGI Symbol;Acc:MGI:2138741]

ENSMUSG
Ret
protein_coding
ret proto-oncogene [Source:MGI

00000030110

Symbol;Acc:MGI:97902]

ENSMUSG
Adipor2
protein_coding
adiponectin receptor 2 [Source:MGI

00000030168

Symbol;Acc:MGI:93830]

ENSMUSG
Wnt5b
protein_coding
wingless-related MMTV integration site 5B

00000030170

[Source:MGI Symbol;Acc:MGI:98959]

ENSMUSG
Fbxl14
protein_coding
F-box and leucine-rich repeat protein 14 [Source:MGI

00000030019

Symbol;Acc:MGI:2141676]

ENSMUSG
Wnk1
protein_coding
WNK lysine deficient protein kinase 1 [Source:MGI

00000045962

Symbol;Acc:MGI:2442092]

ENSMUSG
Kdm5a
protein_coding
lysine (K)-specific demethylase 5A [Source:MGI

00000030180

Symbol;Acc:MGI:2136980]

ENSMUSG
Il17ra
protein_coding
interleukin 17 receptor A [Source:MGI

00000002897

Symbol;Acc:MGI:107399]

ENSMUSG
Mical3
protein_coding
microtubule associated monoxygenase, calponin and

00000051586

LIM domain containing 3 [Source:MGI

Symbol;Acc:MGI:2442733]

ENSMUSG
Phc1
protein_coding
polyhomeotic-like 1 (Drosophila) [Source:MGI

00000040669

Symbol;Acc:MGI:103248]

ENSMUSG
Phb2
protein_coding
prohibitin 2 [Source:MGI Symbol;Acc:MGI:102520]

00000004264

ENSMUSG
Ptpn6
protein_coding
protein tyrosine phosphatase, non-receptor type 6

00000004266

[Source:MGI Symbol;Acc:MGI:96055]

ENSMUSG
Atn1
protein_coding
atrophin 1 [Source:MGI Symbol;Acc:MGI:104725]

00000004263

ENSMUSG
Chd4
protein_coding
chromodomain helicase DNA binding protein 4

00000063870

[Source:MGI Symbol;Acc:MGI:1344380]

ENSMUSG
Ncapd2
protein_coding
non-SMC condensin I complex, subunit D2

00000038252

[Source:MGI Symbol;Acc:MGI:1915548]

ENSMUSG
Ltbr
protein_coding
lymphotoxin B receptor [Source:MGI

00000030339

Symbol;Acc:MGI:104875]

ENSMUSG
Cd9
protein_coding
CD9 antigen [Source:MGI Symbol;Acc:MGI:88348]

00000030342

ENSMUSG
Ano2
protein_coding
anoctamin 2 [Source:MGI Symbol;Acc:MGI:2387214]

00000038115

ENSMUSG
Ccnd2
protein_coding
cyclin D2 [Source:MGI Symbol;Acc:MGI:88314]

00000000184

ENSMUSG
Foxm1
protein_coding
forkhead box M1 [Source:MGI

00000001517

Symbol;Acc:MGI:1347487]

ENSMUSG
Fkbp4
protein_coding
FK506 binding protein 4 [Source:MGI

00000030357

Symbol;Acc:MGI:95543]

ENSMUSG
Gabarapl1
protein_coding
gamma-aminobutyric acid (GABA) A receptor-

00000030161

associated protein-like 1 [Source:MGI

Symbol;Acc:MGI:1914980]

ENSMUSG
Csda
protein_coding
cold shock domain protein A [Source:MGI

00000030189

Symbol;Acc:MGI:2137670]

ENSMUSG
Etv6
protein_coding
ets variant gene 6 (TEL oncogene) [Source:MGI

00000030199

Symbol;Acc:MGI:109336]

ENSMUSG
Lrp6
protein_coding
low density lipoprotein receptor-related protein 6

00000030201

[Source:MGI Symbol;Acc:MGI:1298218]

ENSMUSG
Dusp 16
protein_coding
dual specificity phosphatase 16 [Source:MGI

00000030203

Symbol;Acc:MGI:1917936]

ENSMUSG
Ddx47
protein_coding
DEAD (Asp-Glu-Ala-Asp) box polypeptide 47

00000030204

[Source:MGI Symbol;Acc:MGI:1915005]

ENSMUSG
Grin2b
protein_coding
glutamate receptor, ionotropic, NMDA2B (epsilon 2)

00000030209

[Source:MGI Symbol;Acc:MGI:95821]

ENSMUSG
Arhgdib
protein_coding
Rho, GDP dissociation inhibitor (GDI) beta

00000030220

[Source:MGI Symbol;Acc:MGI:101940]

ENSMUSG
Strap
protein_coding
serine/threonine kinase receptor associated protein

00000030224

[Source:MGI Symbol;Acc:MGI:1329037]

ENSMUSG
Pde3a
protein_coding
phosphodiesterase 3A, cGMP inhibited [Source:MGI

00000041741

Symbol;Acc:MGI:1860764]

ENSMUSG
5730419I09Rik
protein_coding
RIKEN cDNA 5730419I09 gene [Source:MGI

00000030279

Symbol;Acc:MGI:1921991]

ENSMUSG
Etnk1
protein_coding
ethanolamine kinase 1 [Source:MGI

00000030275

Symbol;Acc:MGI:1922570]

ENSMUSG
Sox5
protein_coding
SRY-box containing gene 5 [Source:MGI

00000041540

Symbol;Acc:MGI:98367]

ENSMUSG
Tmtc1
protein_coding
transmembrane and tetratricopeptide repeat

00000030306

containing 1 [Source:MGI Symbol;Acc:MGI:3039590]

ENSMUSG
Ipo8
protein_coding
importin 8 [Source:MGI Symbol;Acc:MGI:2444611]

00000040029

ENSMUSG
Rps9
protein_coding
ribosomal protein S9 [Source:MGI

00000006333

Symbol;Acc:MGI:1924096]

ENSMUSG
Ppp1r12c
protein_coding
protein phosphatase 1, regulatory (inhibitor) subunit

00000019254

12C [Source:MGI Symbol;Acc:MGI:1924258]

ENSMUSG
Hspbp1
protein_coding
HSPA (heat shock 70 kDa) binding protein, cytoplasmic

00000063802

cochaperone 1 [Source:MGI Symbol;Acc:MGI:1913495]

ENSMUSG
U2af2
protein_coding
U2 small nuclear ribonucleoprotein auxiliary factor

00000030435

(U2AF) 2 [Source:MGI Symbol;Acc:MGI:98886]

ENSMUSG
Epn1
protein_coding
epsin 1 [Source:MGI Symbol;Acc:MGI:1333763]

00000035203

ENSMUSG
Rps5
protein_coding
ribosomal protein S5 [Source:MGI

00000012848

Symbol;Acc:MGI:1097682]

ENSMUSG
Zbtb45
protein_coding
zinc finger and BTB domain containing 45

00000049600

[Source:MGI Symbol;Acc:MGI:2685003]

ENSMUSG
Trim28
protein_coding
tripartite motif-containing 28 [Source:MGI

00000005566

Symbol;Acc:MGI:109274]

ENSMUSG
Gltscr2
protein_coding
glioma tumor suppressor candidate region gene 2

00000041560

[Source:MGI Symbol;Acc:MGI:2154441]

ENSMUSG
Napa
protein_coding
N-ethylmaleimide sensitive fusion protein attachment

00000006024

protein alpha [Source:MGI Symbol;Acc:MGI:104563]

ENSMUSG
Sae1
protein_coding
SUMO1 activating enzyme subunit 1 [Source:MGI

00000052833

Symbol;Acc:MGI:1929264]

ENSMUSG
Zc3h4
protein_coding
zinc finger CCCH-type containing 4 [Source:MGI

00000059273

Symbol;Acc:MGI:2682314]

ENSMUSG
Tmem160
protein_coding
transmembrane protein 160 [Source:MGI

00000019158

Symbol;Acc:MGI:1916344]

ENSMUSG
Grlf1
protein_coding
glucocorticoid receptor DNA binding factor 1

00000058230

[Source:MGI Symbol;Acc:MGI:1929494]

ENSMUSG
Fkrp
protein_coding
fukutin related protein [Source:MGI

00000048920

Symbol;Acc:MGI:2447586]

ENSMUSG
Calm3
protein_coding
calmodulin 3 [Source:MGI Symbol;Acc:MGI:103249]

00000019370

ENSMUSG
Irf2bp1
protein_coding
interferon regulatory factor 2 binding protein 1

00000044030

[Source:MGI Symbol;Acc:MGI:2442159]

ENSMUSG
Sympk
protein_coding
symplekin [Source:MGI Symbol;Acc:MGI:1915438]

00000023118

ENSMUSG
Eml2
protein_coding
echinoderm microtubule associated protein like 2

00000040811

[Source:MGI Symbol;Acc:MGI:1919455]

ENSMUSG
Vasp
protein_coding
vasodilator-stimulated phosphoprotein [Source:MGI

00000030403

Symbol;Acc:MGI:109268]

ENSMUSG
Ppp1r13I
protein_coding
protein phosphatase 1, regulatory (inhibitor) subunit

00000040734

13 like [Source:MGI Symbol;Acc:MGI:3525053]

ENSMUSG
Gemin7
protein_coding
gem (nuclear organelle) associated protein 7

00000044709

[Source:MGI Symbol;Acc:MGI:1916981]

ENSMUSG
Tomm40
protein_coding
translocase of outer mitochondrial membrane 40

00000002984

homolog (yeast) [Source:MGI

Symbol;Acc:MGI:1858259]

ENSMUSG
Arhgef1
protein_coding
Rho guanine nucleotide exchange factor (GEF) 1

00000040940

[Source:MGI Symbol;Acc:MGI:1353510]

ENSMUSG
Grik5
protein_coding
glutamate receptor, ionotropic, kainate 5 (gamma 2)

00000003378

[Source:MGI Symbol;Acc:MGI:95818]

ENSMUSG
Zfp574
protein_coding
zinc finger protein 574 [Source:MGI

00000045252

Symbol;Acc:MGI:2442951]

ENSMUSG
Gsk3a
protein_coding
glycogen synthase kinase 3 alpha [Source:MGI

00000057177

Symbol;Acc:MGI:2152453]

ENSMUSG
Erf
protein_coding
Ets2 repressor factor [Source:MGI

00000040857

Symbol;Acc:MGI:109637]

ENSMUSG
Cic
protein_coding
capicua homolog (Drosophila) [Source:MGI

00000005442

Symbol;Acc:MGI:1918972]

ENSMUSG
Exosc5
protein_coding
exosome component 5 [Source:MGI

00000061286

Symbol;Acc:MGI:107889]

ENSMUSG
Tgfb1
protein_coding
transforming growth factor, beta 1 [Source:MGI

00000002603

Symbol;Acc:MGI:98725]

ENSMUSG
Ccdc97
protein_coding
coiled-coil domain containing 97 [Source:MGI

00000002608

Symbol;Acc:MGI:1196455]

ENSMUSG
Hnrnpul1
protein_coding
heterogeneous nuclear ribonucleoprotein U-like 1

00000040725

[Source:MGI Symbol;Acc:MGI:2443517]

ENSMUSG
Egln2
protein_coding
EGL nine homolog 2 (C. elegans) [Source:MGI

00000058709

Symbol;Acc:MGI:1932287]

ENSMUSG
Spnb4
protein_coding
spectrin beta 4 [Source:MGI

00000011751

Symbol;Acc:MGI:1890574]

ENSMUSG
1700049G17Rik
protein_coding
RIKEN cDNA 1700049G17 gene [Source:MGI

00000070709

Symbol;Acc:MGI:1920680]

ENSMUSG
Supt5h
protein_coding
suppressor of Ty 5 homolog (S. cerevisiae)

00000003435

[Source:MGI Symbol;Acc:MGI:1202400]

ENSMUSG
Rps16
protein_coding
ribosomal protein S16 [Source:MGI

00000037563

Symbol;Acc:MGI:98118]

ENSMUSG
Lrfn1
protein_coding
leucine rich repeat and fibronectin type III domain

00000030600

containing 1 [Source:MGI Symbol;Acc:MGI:2136810]

ENSMUSG
Hnrnpl
protein_coding
heterogeneous nuclear ribonucleoprotein L

00000015165

[Source:MGI Symbol;Acc:MGI:104816]

ENSMUSG
Capn12
protein_coding
calpain 12 [Source:MGI Symbol;Acc:MGI:1891369]

00000054083

ENSMUSG
Psmd8
protein_coding
proteasome (prosome, macropain) 26S subunit, non-

00000030591

ATPase, 8 [Source:MGI Symbol;Acc:MGI:1888669]

ENSMUSG
Ppp1r14a
protein_coding
protein phosphatase 1, regulatory (inhibitor) subunit

00000037166

14A [Source:MGI Symbol;Acc:MGI:1931139]

ENSMUSG
Sipa1l3
protein_coding
signal-induced proliferation-associated 1 like 3

00000030583

[Source:MGI Symbol;Acc:MGI:1921456]

ENSMUSG
Capns1
protein_coding
calpain, small subunit 1 [Source:MGI

00000001794

Symbol;Acc:MGI:88266]

ENSMUSG
Sdhaf1
protein_coding
succinate dehydrogenase complex assembly factor 1

00000074211

[Source:MGI Symbol;Acc:MGI:1915582]

ENSMUSG
E130208F15Rik
protein_coding
RIKEN cDNA E130208F15 gene [Source:MGI

00000074210

Symbol;Acc:MGI:3767226]

ENSMUSG
Rbm42
protein_coding
RNA binding motif protein 42 [Source:MGI

00000036733

Symbol;Acc:MGI:1915285]

ENSMUSG
Haus5
protein_coding
HAUS augmin-like complex, subunit 5 [Source:MGI

00000078762

Symbol;Acc:MGI:1919159]

ENSMUSG
Usf2
protein_coding
upstream transcription factor 2 [Source:MGI

00000058239

Symbol;Acc:MGI:99961]

ENSMUSG
Uba2
protein_coding
ubiquitin-like modifier activating enzyme 2

00000052997

[Source:MGI Symbol;Acc:MGI:1858313]

ENSMUSG
Gpi1
protein_coding
glucose phosphate isomerase 1 [Source:MGI

00000036427

Symbol;Acc:MGI:95797]

ENSMUSG
Lsm14a
protein_coding
LSM14 homolog A (SCD6, S. cerevisiae) [Source:MGI

00000066568

Symbol;Acc:MGI:1914320]

ENSMUSG
Chst8
protein_coding
carbohydrate (N-acetylgalactosamine 4-0)

00000060402

sulfotransferase 8 [Source:MGI

Symbol;Acc:MGI:1916197]

ENSMUSG
Cebpa
protein_coding
CCAAT/enhancer binding protein (C/EBP), alpha

00000034957

[Source:MGI Symbol;Acc:MGI:99480]

ENSMUSG
Ankrd27
protein_coding
ankyrin repeat domain 27 (VPS9 domain) [Source:MGI

00000034867

Symbol;Acc:MGI:2444103]

ENSMUSG
Zfp507
protein_coding
zinc finger protein 507 [Source:MGI

00000044452

Symbol;Acc:MGI:1916378]

ENSMUSG
2410002F23Rik
protein_coding
RIKEN cDNA 2410002F23 gene [Source:MGI

00000045411

Symbol;Acc:MGI:1914226]

ENSMUSG
Lrrc4b
protein_coding
leucine rich repeat containing 4B [Source:MGI

00000047085

Symbol;Acc:MGI:3027390]

ENSMUSG
2310044H10Rik
protein_coding
RIKEN cDNA 2310044H10 gene [Source:MGI

00000008140

Symbol;Acc:MGI:1916933]

ENSMUSG
Kcnc3
protein_coding
potassium voltage gated channel, Shaw-related

00000062785

subfamily, member 3 [Source:MGI

Symbol;Acc:MGI:96669]

ENSMUSG
Myh14
protein_coding
myosin, heavy polypeptide 14 [Source:MGI

00000030739

Symbol;Acc:MGI:1919210]

ENSMUSG
Zfp473
protein_coding
zinc finger protein 473 [Source:MGI

00000048012

Symbol;Acc:MGI:2442697]

ENSMUSG
Vrk3
protein_coding
vaccinia related kinase 3 [Source:MGI

00000002205

Symbol;Acc:MGI:2182465]

ENSMUSG
Nup62
protein_coding
nucleoporin 62 [Source:MGI

00000043858

Symbol;Acc:MGI:1351500]

ENSMUSG
Il4i1
protein_coding
interleukin 4 induced 1 [Source:MGI

00000074141

Symbol;Acc:MGI:109552]

ENSMUSG
Ptov1
protein_coding
prostate tumor over expressed gene 1 [Source:MGI

00000038502

Symbol;Acc:MGI:1933946]

ENSMUSG
Scaf1
protein_coding
SR-related CTD-associated factor 1 [Source:MGI

00000038406

Symbol;Acc:MGI:2141980]

ENSMUSG
Prr12
protein_coding
proline rich 12 [Source:MGI

00000046574

Symbol;Acc:MGI:2679002]

ENSMUSG
Rps11
protein_coding
ribosomal protein S11 [Source:MGI

00000003429

Symbol;Acc:MGI:1351329]

ENSMUSG
Trpm4
protein_coding
transient receptor potential cation channel, subfamily

00000038260

M, member 4 [Source:MGI Symbol;Acc:MGI:1915917]

ENSMUSG
Snrnp70
protein_coding
small nuclear ribonucleoprotein 70 (U1) [Source:MGI

00000063511

Symbol;Acc:MGI:98341]

ENSMUSG
Bax
protein_coding
BCL2-associated X protein [Source:MGI

00000003873

Symbol;Acc:MGI:99702]

ENSMUSG
Bcat2
protein_coding
branched chain aminotransferase 2, mitochondrial

00000030826

[Source:MGI Symbol;Acc:MGI:1276534]

ENSMUSG
Sphk2
protein_coding
sphingosine kinase 2 [Source:MGI

00000057342

Symbol;Acc:MGI:1861380]

ENSMUSG
Grwd1
protein_coding
glutamate-rich WD repeat containing 1 [Source:MGI

00000053801

Symbol;Acc:MGI:2141989]

ENSMUSG
Grin2d
protein_coding
glutamate receptor, ionotropic, NMDA2D (epsilon 4)

00000002771

[Source:MGI Symbol;Acc:MGI:95823]

ENSMUSG
Kdelr1
protein_coding
KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum

00000002778

protein retention receptor 1 [Source:MGI

Symbol;Acc:MGI:1915387]

ENSMUSG
Nomo1
protein_coding
nodal modulator 1 [Source:MGI

00000030835

Symbol;Acc:MGI:2385850]

ENSMUSG
Abcc8
protein_coding
ATP-binding cassette, sub-family C (CFTR/MRP),

00000040136

member 8 [Source:MGI Symbol;Acc:MGI:1352629]

ENSMUSG
Sergef
protein_coding
secretion regulating guanine nucleotide exchange

00000030839

factor [Source:MGI Symbol;Acc:MGI:1351630]

ENSMUSG
Ldha
protein_coding
lactate dehydrogenase A [Source:MGI

00000063229

Symbol;Acc:MGI:96759]

ENSMUSG
Nav2
protein_coding
neuron navigator 2 [Source:MGI

00000052512

Symbol;Acc:MGI:2183691]

ENSMUSG
Nell1
protein_coding
NEL-like 1 (chicken) [Source:MGI

00000055409

Symbol;Acc:MGI:2443902]

ENSMUSG
Herc2
protein_coding
hect (homologous to the E6-AP (UBE3A) carboxyl

00000030451

terminus) domain and RCC1 (CHC1)-like domain (RLD)

2 [Source:MGI Symbol;Acc:MGI:103234]

ENSMUSG
Snrpn
protein_coding
small nuclear ribonucleoprotein N [Source:MGI

00000000948

Symbol;Acc:MGI:98347]

ENSMUSG
Klf13
protein_coding
Kruppel-like factor 13 [Source:MGI

00000052040

Symbol;Acc:MGI:1354948]

ENSMUSG
Fam189a1
protein_coding
family with sequence similarity 189, member A1

00000030518

[Source:MGI Symbol;Acc:MGI:1917888]

ENSMUSG
Lrrk1
protein_coding
leucine-rich repeat kinase 1 [Source:MGI

00000015133

Symbol;Acc:MGI:2142227]

ENSMUSG
Igf1r
protein_coding
insulin-like growth factor I receptor [Source:MGI

00000005533

Symbol;Acc:MGI:96433]

ENSMUSG
Chd2
protein_coding
chromodomain helicase DNA binding protein 2

00000078671

[Source:MGI Symbol;Acc:MGI:2448567]

ENSMUSG
Slco3a1
protein_coding
solute carrier organic anion transporter family,

00000025790

member 3a1 [Source:MGI Symbol;Acc:MGI:1351867]

ENSMUSG
Akap13
protein_coding
A kinase (PRKA) anchor protein 13 [Source:MGI

00000066406

Symbol;Acc:MGI:2676556]

ENSMUSG
Mfge8
protein_coding
milk fat globule-EGF factor 8 protein [Source:MGI

00000030605

Symbol;Acc:MGI:102768]

ENSMUSG
Vps33b
protein_coding
vacuolar protein sorting 33B (yeast) [Source:MGI

00000030534

Symbol;Acc:MGI:2446237]

ENSMUSG
Man2a2
protein_coding
mannosidase 2, alpha 2 [Source:MGI

00000038886

Symbol;Acc:MGI:2150656]

ENSMUSG
Furin
protein_coding
furin (paired basic amino acid cleaving enzyme)

00000030530

[Source:MGI Symbol;Acc:MGI:97513]

ENSMUSG
Iqgap1
protein_coding
IQ motif containing GTPase activating protein 1

00000030536

[Source:MGI Symbol;Acc:MGI:1352757]

ENSMUSG
Zfp592
protein_coding
zinc finger protein 592 [Source:MGI

00000005621

Symbol;Acc:MGI:2443541]

ENSMUSG
Mex3b
protein_coding
mex3 homolog B (C. elegans) [Source:MGI

00000057706

Symbol;Acc:MGI:1918252]

ENSMUSG
Gm7964
protein_coding
predicted gene 7964 [Source:MGI

00000063902

Symbol;Acc:MGI:3646150]

ENSMUSG
Mesdc1
protein_coding
mesoderm development candidate 1 [Source:MGI

00000070462

Symbol;Acc:MGI:1891420]

ENSMUSG
Fam108c
protein_coding
family with sequence similarity 108, member C

00000038459

[Source:MGI Symbol;Acc:MGI:1917428]

ENSMUSG
Picalm
protein_coding
phosphatidylinositol binding clathrin assembly

00000039361

protein [Source:MGI Symbol;Acc:MGI:2385902]

ENSMUSG
Dlg2
protein_coding
discs, large homolog 2 (Drosophila) [Source:MGI

00000052572

Symbol;Acc:MGI:1344351]

ENSMUSG
Pcf11
protein_coding
cleavage and polyadenylation factor subunit homolog

00000041328

(S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1919579]

ENSMUSG
Gm9934
protein_coding
predicted gene 9934 [Source:MGI

00000054061

Symbol;Acc:MGI:3641747]

ENSMUSG
Odz4
protein_coding
odd Oz/ten-m homolog 4 (Drosophila) [Source:MGI

00000048078

Symbol;Acc:MGI:2447063]

ENSMUSG
Rsf1
protein_coding
remodeling and spacing factor 1 [Source:MGI

00000035623

Symbol;Acc:MGI:2682305]

ENSMUSG
Prkrir
protein_coding
protein-kinase, interferon-inducible double stranded

00000030753

RNA dependent inhibitor, repressor of (P58

repressor) [Source:MGI Symbol;Acc:MGI:1920231]

ENSMUSG
Rps3
protein_coding
ribosomal protein S3 [Source:MGI

00000030744

Symbol;Acc:MGI:1350917]

ENSMUSG
Arrb1
protein_coding
arrestin, beta 1 [Source:MGI Symbol;Acc:MGI:99473]

00000018909

ENSMUSG
Rnf169
protein_coding
ring finger protein 169 [Source:MGI

00000058761

Symbol;Acc:MGI:1920257]

ENSMUSG
C2cd3
protein_coding
C2 calcium-dependent domain containing 3

00000047248

[Source:MGI Symbol;Acc:MGI:2142166]

ENSMUSG
Fam168a
protein_coding
family with sequence similarity 168, member A

00000029461

[Source:MGI Symbol;Acc:MGI:2442372]

ENSMUSG
Arhgef17
protein_coding
Rho guanine nucleotide exchange factor (GEF) 17

00000032875

[Source:MGI Symbol;Acc:MGI:2673002]

ENSMUSG
Numa1
protein_coding
nuclear mitotic apparatus protein 1 [Source:MGI

00000066306

Symbol;Acc:MGI:2443665]

ENSMUSG
Rhog
protein_coding
ras homolog gene family, member G [Source:MGI

00000073982

Symbol;Acc:MGI:1928370]

ENSMUSG
Rrm1
protein_coding
ribonucleotide reductase M1 [Source:MGI

00000030978

Symbol;Acc:MGI:98180]

ENSMUSG
Dchs1
protein_coding
dachsous 1 (Drosophila) [Source:MGI

00000036862

Symbol;Acc:MGI:2685011]

ENSMUSG
Eif3f
protein_coding
eukaryotic translation initiation factor 3, subunit F

00000031029

[Source:MGI Symbol;Acc:MGI:1913335]

ENSMUSG
Rpl27a
protein_coding
ribosomal protein L27A [Source:MGI

00000046364

Symbol;Acc:MGI:1347076]

ENSMUSG
Dennd5a
protein_coding
DENN/MADD domain containing 5A [Source:MGI

00000035901

Symbol;Acc:MGI:1201681]

ENSMUSG
Ipo7
protein_coding
importin 7 [Source:MGI Symbol;Acc:MGI:2152414]

00000066232

ENSMUSG
Swap 70
protein_coding
SWA-70 protein [Source:MGI

00000031015

Symbol;Acc:MGI:1298390]

ENSMUSG
Eif4g2
protein_coding
eukaryotic translation initiation factor 4, gamma 2

00000005610

[Source:MGI Symbol;Acc:MGI:109207]

ENSMUSG
Usp47
protein_coding
ubiquitin specific peptidase 47 [Source:MGI

00000059263

Symbol;Acc:MGI:1922246]

ENSMUSG
Btbd10
protein_coding
BTB (POZ) domain containing 10 [Source:MGI

00000038187

Symbol;Acc:MGI:1916065]

ENSMUSG
Sox6
protein_coding
SRY-box containing gene 6 [Source:MGI

00000051910

Symbol;Acc:MGI:98368]

ENSMUSG
Plekha7
protein_coding
pleckstrin homology domain containing, family A

00000045659

member 7 [Source:MGI Symbol;Acc:MGI:2445094]

ENSMUSG
Gga2
protein_coding
golgi associated, gamma adaptin ear containing, ARF

00000030872

binding protein 2 [Source:MGI

Symbol;Acc:MGI:1921355]

ENSMUSG
Ubfd1
protein_coding
ubiquitin family domain containing 1 [Source:MGI

00000030870

Symbol;Acc:MGI:107301]

ENSMUSG
Prkcb
protein_coding
protein kinase C, beta [Source:MGI

00000052889

Symbol;Acc:MGI:97596]

ENSMUSG
Rbbp6
protein_coding
retinoblastoma binding protein 6 [Source:MGI

00000030779

Symbol;Acc:MGI:894835]

ENSMUSG
Il4ra
protein_coding
interleukin 4 receptor, alpha [Source:MGI

00000030748

Symbol;Acc:MGI:105367]

ENSMUSG
Gsg1l
protein_coding
GSG1-like [Source:MGI Symbol;Acc:MGI:2685483]

00000046182

ENSMUSG
Xpo6
protein_coding
exportin 6 [Source:MGI Symbol;Acc:MGI:2429950]

00000000131

ENSMUSG
Atxn2l
protein_coding
ataxin 2-like [Source:MGI Symbol;Acc:MGI:2446242]

00000032637

ENSMUSG
Eif3c
protein_coding
eukaryotic translation initiation factor 3, subunit C

00000030738

[Source:MGI Symbol;Acc:MGI:1926966]

ENSMUSG
Corola
protein_coding
coronin, actin binding protein 1A [Source:MGI

00000030707

Symbol;Acc:MGI:1345961]

ENSMUSG
Aldoa
protein_coding
aldolase A, fructose-bisphosphate [Source:MGI

00000030695

Symbol;Acc:MGI:87994]

ENSMUSG
Ino80e
protein_coding
INO80 complex subunit E [Source:MGI

00000030689

Symbol;Acc:MGI:2141881]

ENSMUSG
2900092E17Rik
protein_coding
RIKEN cDNA 2900092E17 gene [Source:MGI

00000030680

Symbol;Acc:MGI:1914528]

ENSMUSG
Maz
protein_coding
MYC-associated zinc finger protein (purine-binding

00000030678

transcription factor) [Source:MGI

Symbol;Acc:MGI:1338823]

ENSMUSG
AI467606
protein_coding
expressed sequence Al467606 [Source:MGI

00000045165

Symbol;Acc:MGI:2141979]

ENSMUSG
Spn
protein_coding
sialophorin [Source:MGI Symbol;Acc:MGI:98384]

00000051457

ENSMUSG
Cd2bp2
protein_coding
CD2 antigen (cytoplasmic tail) binding protein 2

00000042502

[Source:MGI Symbol;Acc:MGI:1917483]

ENSMUSG
Sephs2
protein_coding
selenophosphate synthetase 2 [Source:MGI

00000049091

Symbol;Acc:MGI:108388]

ENSMUSG
Srcap
protein_coding
Snf2-related CREBBP activator protein [Source:MGI

00000053877

Symbol;Acc:MGI:2444036]

ENSMUSG
Srcap
protein_coding
Snf2-related CREBBP activator protein [Source:MGI

00000090663

Symbol;Acc:MGI:2444036]

ENSMUSG
Fbxl19
protein_coding
F-box and leucine-rich repeat protein 19 [Source:MGI

00000030811

Symbol;Acc:MGI:3039600]

ENSMUSG
Setd1a
protein_coding
SET domain containing 1A [Source:MGI

00000042308

Symbol;Acc:MGI:2446244]

ENSMUSG
Hsd3b7
protein_coding
hydroxy-delta-5-steroid dehydrogenase, 3 beta- and

00000042289

steroid delta-isomerase 7 [Source:MGI

Symbol;Acc:MGI:2141879]

ENSMUSG
Zfp646
protein_coding
zinc finger protein 646 [Source:MGI

00000049739

Symbol;Acc:MGI:3665412]

ENSMUSG
Fus
protein_coding
fusion, derived from t(12;16) malignant liposarcoma

00000030795

(human) [Source:MGI Symbol;Acc:MGI:1353633]

ENSMUSG
Itgam
protein_coding
integrin alpha M [Source:MGI

00000030786

Symbol;Acc:MGI:96607]

ENSMUSG
Tial1
protein_coding
Tia1 cytotoxic granule-associated RNA binding

00000030846

protein-like 1 [Source:MGI Symbol;Acc:MGI:107913]

ENSMUSG
Fgfr2
protein_coding
fibroblast growth factor receptor 2 [Source:MGI

00000030849

Symbol;Acc:MGI:95523]

ENSMUSG
Bub3
protein_coding
budding uninhibited by benzimidazoles 3 homolog (S.

00000066979

cerevisiae) [Source:MGI Symbol;Acc:MGI:1343463]

ENSMUSG
Gpr26
protein_coding
G protein-coupled receptor 26 [Source:MGI

00000040125

Symbol;Acc:MGI:2441758]

ENSMUSG
Chst15
protein_coding
carbohydrate (N-acetylgalactosamine 4-sulfate 6-0)

00000030930

sulfotransferase 15 [Source:MGI

Symbol;Acc:MGI:1924840]

ENSMUSG
Lhpp
protein_coding
phospholysine phosphohistidine inorganic

00000030946

pyrophosphate phosphatase [Source:MGI

Symbol;Acc:MGI:1923679]

ENSMUSG
Fam53b
protein_coding
family with sequence similarity 53, member B

00000030956

[Source:MGI Symbol;Acc:MGI:1925188]

ENSMUSG
Ctbp2
protein_coding
C-terminal binding protein 2 [Source:MGI

00000030970

Symbol;Acc:MGI:1201686]

ENSMUSG
Uros
protein_coding
uroporphyrinogen III synthase [Source:MGI

00000030979

Symbol;Acc:MGI:98917]

ENSMUSG
Ptpre
protein_coding
protein tyrosine phosphatase, receptor type, E

00000041836

[Source:MGI Symbol;Acc:MGI:97813]

ENSMUSG
Mki67
protein_coding
antigen identified by monoclonal antibody Ki 67

00000031004

[Source:MGI Symbol;Acc:MGI:106035]

ENSMUSG
Ifitm1
protein_coding
interferon induced transmembrane protein 1

00000025491

[Source:MGI Symbol;Acc:MGI:1915963]

ENSMUSG
Sct
protein_coding
secretin [Source:MGI Symbol;Acc:MGI:99466]

00000038580

ENSMUSG
Rplp2
protein_coding
ribosomal protein, large P2 [Source:MGI

00000025508

Symbol;Acc:MGI:1914436]

ENSMUSG
Muc2
protein_coding
mucin 2 [Source:MGI Symbol;Acc:MGI:1339364]

00000025515

ENSMUSG
Ctsd
protein_coding
cathepsin D [Source:MGI Symbol;Acc:MGI:88562]

00000007891

ENSMUSG
Kcnq1
protein_coding
potassium voltage-gated channel, subfamily Q,

00000009545

member 1 [Source:MGI Symbol;Acc:MGI:108083]

ENSMUSG
Nap1|4
protein_coding
nucleosome assembly protein 1-like 4 [Source:MGI

00000059119

Symbol;Acc:MGI:1316687]

ENSMUSG
Pnpla6
protein_coding
patatin-like phospholipase domain containing 6

00000004565

[Source:MGI Symbol;Acc:MGI:1354723]

ENSMUSG
Stxbp2
protein_coding
syntaxin binding protein 2 [Source:MGI

00000004626

Symbol;Acc:MGI:107370]

ENSMUSG
Elavl1
protein_coding
ELAV (embryonic lethal, abnormal vision, Drosophila)-

00000040028

like 1 (Hu antigen R) [Source:MGI

Symbol;Acc:MGI:1100851]

ENSMUSG
Lass4
protein_coding
LAG1 homolog, ceramide synthase 4 [Source:MGI

00000008206

Symbol;Acc:MGI:1914510]

ENSMUSG
Gm1840
protein_coding
predicted gene 1840 [Source:MGI

00000043192

Symbol;Acc:MGI:3037698]

ENSMUSG
Arglu1
protein_coding
arginine and glutamate rich 1 [Source:MGI

00000040459

Symbol;Acc:MGI:2442985]

ENSMUSG
Ing1
protein_coding
inhibitor of growth family, member 1 [Source:MGI

00000045969

Symbol;Acc:MGI:1349481]

ENSMUSG
Arhgef7
protein_coding
Rho guanine nucleotide exchange factor (GEF7)

00000031511

[Source:MGI Symbol;Acc:MGI:1860493]

ENSMUSG
A230072I06Rik
protein_coding
RIKEN cDNA A230072I06 gene [Source:MGI

00000074473

Symbol;Acc:MGI:3588221]

ENSMUSG
Atp11a
protein_coding
ATPase, class VI, type 11A [Source:MGI

00000031441

Symbol;Acc:MGI:1354735]

ENSMUSG
Cul4a
protein_coding
cullin 4A [Source:MGI Symbol;Acc:MGI:1914487]

00000031446

ENSMUSG
Lamp1
protein_coding
lysosomal-associated membrane protein 1

00000031447

[Source:MGI Symbol;Acc:MGI:96745]

ENSMUSG
Dcun1d2
protein_coding
DCN1, defective in cullin neddylation 1, domain

00000038506

containing 2 (S. cerevisiae) [Source:MGI

Symbol;Acc:MGI:2142792]

ENSMUSG
Dlgap2
protein_coding
discs, large (Drosophila) homolog-associated protein

00000047495

2 [Source:MGI Symbol;Acc:MGI:2443181]

ENSMUSG
Thsd1
protein_coding
thrombospondin, type I, domain 1 [Source:MGI

00000031480

Symbol;Acc:MGI:1929096]

ENSMUSG
Myst3
protein_coding
MYST histone acetyltransferase (monocytic leukemia)

00000031540

3 [Source:MGI Symbol;Acc:MGI:2442415]

ENSMUSG
Ank1
protein_coding
ankyrin 1, erythroid [Source:MGI

00000031543

Symbol;Acc:MGI:88024]

ENSMUSG
Zmat4
protein_coding
zinc finger, matrin type 4 [Source:MGI

00000037492

Symbol;Acc:MGI:2443497]

ENSMUSG
Plekha2
protein_coding
pleckstrin homology domain-containing, family A

00000031557

(phosphoinositide binding specific) member 2

[Source:MGI Symbol;Acc:MGI:1928144]

ENSMUSG
Tacc1
protein_coding
transforming, acidic coiled-coil containing protein 1

00000065954

[Source:MGI Symbol;Acc:MGI:2443510]

ENSMUSG
Whsc1|1
protein_coding
Wolf-Hirschhorn syndrome candidate 1-like 1

00000054823

(human) [Source:MGI Symbol;Acc:MGI:2142581]

ENSMUSG
Eif4ebp1
protein_coding
eukaryotic translation initiation factor 4E binding

00000031490

protein 1 [Source:MGI Symbol;Acc:MGI:103267]

ENSMUSG
Tti2
protein_coding
TELO2 interacting protein 2 [Source:MGI

00000031577

Symbol;Acc:MGI:2384576]

ENSMUSG
Rbpms
protein_coding
RNA binding protein gene with multiple splicing

00000031586

[Source:MGI Symbol;Acc:MGI:1334446]

ENSMUSG
Dctn6
protein_coding
dynactin 6 [Source:MGI Symbol;Acc:MGI:1343154]

00000031516

ENSMUSG
Dusp4
protein_coding
dual specificity phosphatase 4 [Source:MGI

00000031530

Symbol;Acc:MGI:2442191]

ENSMUSG
Tnks
protein_coding
tankyrase, TRF1-interacting ankyrin-related ADP-

00000031529

ribose polymerase [Source:MGI

Symbol;Acc:MGI:1341087]

ENSMUSG
Dlc1
protein_coding
deleted in liver cancer 1 [Source:MGI

00000031523

Symbol;Acc:MGI:1354949]

ENSMUSG
Mtmr7
protein_coding
myotubularin related protein 7 [Source:MGI

00000039431

Symbol;Acc:MGI:1891693]

ENSMUSG
Gm6180
protein_coding
predicted pseudogene 6180 [Source:MGI

00000053038

Symbol;Acc:MGI:3643972]

ENSMUSG
Snx25
protein_coding
sorting nexin 25 [Source:MGI

00000038291

Symbol;Acc:MGI:2142610]

ENSMUSG
Slc25a4
protein_coding
solute carrier family 25 (mitochondrial carrier,

00000031633

adenine nucleotide translocator), member 4

[Source:MGI Symbol;Acc:MGI:1353495]

ENSMUSG
Galnt7
protein_coding
UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-

00000031608

acetylgalactosaminyltransferase 7 [Source:MGI

Symbol;Acc:MGI:1349449]

ENSMUSG
Sh3rf1
protein_coding
SH3 domain containing ring finger 1 [Source:MGI

00000031642

Symbol;Acc:MGI:1913066]

ENSMUSG
Sc4mol
protein_coding
sterol-C4-methyl oxidase-like [Source:MGI

00000031604

Symbol;Acc:MGI:1913484]

ENSMUSG
Csgalnact1
protein_coding
chondroitin sulfate N-acetylgalactosaminyltransferase

00000036356

1 [Source:MGI Symbol;Acc:MGI:2442354]

ENSMUSG
Atp6v1b2
protein_coding
ATPase, H+ transporting, lysosomal V1 subunit B2

00000006273

[Source:MGI Symbol;Acc:MGI:109618]

ENSMUSG
Gatad2a
protein_coding
GATA zinc finger domain containing 2A [Source:MGI

00000036180

Symbol;Acc:MGI:2384585]

ENSMUSG
Homer3
protein_coding
homer homolog 3 (Drosophila) [Source:MGI

00000003573

Symbol;Acc:MGI:1347359]

ENSMUSG
Ddx49
protein_coding
DEAD (Asp-Glu-Ala-Asp) box polypeptide 49

00000057788

[Source:MGI Symbol;Acc:MGI:2136689]

ENSMUSG
Upf1
protein_coding
UPF1 regulator of nonsense transcripts homolog

00000058301

(yeast) [Source:MGI Symbol;Acc:MGI:107995]

ENSMUSG
EII
protein_coding
elongation factor RNA polymerase II [Source:MGI

00000070002

Symbol;Acc:MGI:109377]

ENSMUSG
Isyna1
protein_coding
myo-inositol 1-phosphate synthase A1 [Source:MGI

00000019139

Symbol;Acc:MGI:1919030]

ENSMUSG
Jund
protein_coding
Jun proto-oncogene related gene d [Source:MGI

00000071076

Symbol;Acc:MGI:96648]

ENSMUSG
Gm11175
protein_coding
predicted gene 11175 [Source:MGI

00000080058

Symbol;Acc:MGI:3809055]

ENSMUSG
Mast3
protein_coding
microtubule associated serine/threonine kinase 3

00000031833

[Source:MGI Symbol;Acc:MGI:2683541]

ENSMUSG
Mtap1s
protein_coding
microtubule-associated protein 1S [Source:MGI

00000019261

Symbol;Acc:MGI:2443304]

ENSMUSG
Myo9b
protein_coding
myosin IXb [Source:MGI Symbol;Acc:MGI:106624]

00000004677

ENSMUSG
Jak3
protein_coding
Janus kinase 3 [Source:MGI Symbol;Acc:MGI:99928]

00000031805

ENSMUSG
Rab8a
protein_coding
RAB8A, member RAS oncogene family [Source:MGI

00000003037

Symbol;Acc:MGI:96960]

ENSMUSG
Fam32a
protein_coding
family with sequence similarity 32, member A

00000003039

[Source:MGI Symbol;Acc:MGI:1915172]

ENSMUSG
Slc35e1
protein_coding
solute carrier family 35, member E1 [Source:MGI

00000019731

Symbol;Acc:MGI:2142403]

ENSMUSG
Sin3b
protein_coding
transcriptional regulator, SIN3B (yeast) [Source:MGI

00000031622

Symbol;Acc:MGI:107158]

ENSMUSG
Large
protein_coding
like-glycosyltransferase [Source:MGI

00000004383

Symbol;Acc:MGI:1342270]

ENSMUSG
Mcm5
protein_coding
minichromosome maintenance deficient 5, cell

00000005410

division cycle 46 (S. cerevisiae) [Source:MGI

Symbol;Acc:MGI:103197]

ENSMUSG
Slc10a7
protein_coding
solute carrier family 10 (sodium/bile acid

00000031684

cotransporter family), member 7 [Source:MGI

Symbol;Acc:MGI:1924025]

ENSMUSG
Smad1
protein_coding
MAD homolog 1 (Drosophila) [Source:MGI

00000031681

Symbol;Acc:MGI:109452]

ENSMUSG
Abce1
protein_coding
ATP-binding cassette, sub-family E (OABP), member 1

00000058355

[Source:MGI Symbol;Acc:MGI:1195458]

ENSMUSG
Smarca5
protein_coding
SWI/SNF related, matrix associated, actin dependent

00000031715

regulator of chromatin, subfamily a, member 5

[Source:MGI Symbol;Acc:MGI:1935129]

ENSMUSG
Usp38
protein_coding
ubiquitin specific peptidase 38 [Source:MGI

00000038250

Symbol;Acc:MGI:1922091]

ENSMUSG
Inpp4b
protein_coding
inositol polyphosphate-4-phosphatase, type II

00000037940

[Source:MGI Symbol;Acc:MGI:2158925]

ENSMUSG
Tecr
protein_coding
trans-2,3-enoyl-CoA reductase [Source:MGI

00000031708

Symbol;Acc:MGI:1915408]

ENSMUSG
Gipc1
protein_coding
GIPC PDZ domain containing family, member 1

00000019433

[Source:MGI Symbol;Acc:MGI:1926252]

ENSMUSG
Ptger1
protein_coding
prostaglandin E receptor 1 (subtype EP1) [Source:MGI

00000019464

Symbol;Acc:MGI:97793]

ENSMUSG
Ddx39
protein_coding
DEAD (Asp-Glu-Ala-Asp) box polypeptide 39

00000004815

[Source:MGI Symbol;Acc:MGI:1915528]

ENSMUSG
Lphn1
protein_coding
latrophilin 1 [Source:MGI Symbol;Acc:MGI:1929461]

00000013033

ENSMUSG
Gm10644
protein_coding
predicted gene 10644 [Source:MGI

00000074219

Symbol;Acc:MGI:3704314]

ENSMUSG
Samd1
protein_coding
sterile alpha motif domain containing 1 [Source:MGI

00000079003

Symbol;Acc:MGI:2142433]

ENSMUSG
Rfx1
protein_coding
regulatory factor X, 1 (influences HLA class II

00000031706

expression) [Source:MGI Symbol;Acc:MGI:105982]

ENSMUSG
Dcaf15
protein_coding
DDB1 and CUL4 associated factor 15 [Source:MGI

00000037103

Symbol;Acc:MGI:2684420]

ENSMUSG
Mri1
protein_coding
methylthioribose-1-phosphate isomerase homolog (S.

00000004996

cerevisiae) [Source:MGI Symbol;Acc:MGI:1915123]

ENSMUSG
Ier2
protein_coding
immediate early response 2 [Source:MGI

00000053560

Symbol;Acc:MGI:104815]

ENSMUSG
Nacc1
protein_coding
nucleus accumbens associated 1, BEN and BTB (POZ)

00000001910

domain containing [Source:MGI

Symbol;Acc:MGI:1914080]

ENSMUSG
Nfix
protein_coding
nuclear factor I/X [Source:MGI

00000001911

Symbol;Acc:MGI:97311]

ENSMUSG
Lyl1
protein_coding
lymphoblastomic leukemia 1 [Source:MGI

00000034041

Symbol;Acc:MGI:96891]

ENSMUSG
Calr
protein_coding
calreticulin [Source:MGI Symbol;Acc:MGI:88252]

00000003814

ENSMUSG
Farsa
protein_coding
phenylalanyl-tRNA synthetase, alpha subunit

00000003808

[Source:MGI Symbol;Acc:MGI:1913840]

ENSMUSG
Prdx2
protein_coding
peroxiredoxin 2 [Source:MGI

00000005161

Symbol;Acc:MGI:109486]

ENSMUSG
2310036O22Rik
protein_coding
RIKEN cDNA 2310036O22 gene [Source:MGI

00000041203

Symbol;Acc:MGI:1922833]

ENSMUSG
Tnpo2
protein_coding
transportin 2 (importin 3, karyopherin beta 2b)

00000031691

[Source:MGI Symbol;Acc:MGI:2384849]

ENSMUSG
Man2b1
protein_coding
mannosidase 2, alpha B1 [Source:MGI

00000005142

Symbol;Acc:MGI:107286]

ENSMUSG
Zfp791
protein_coding
zinc finger protein 791 [Source:MGI

00000074194

Symbol;Acc:MGI:3648473]

ENSMUSG
Dnaja2
protein_coding
DnaJ (Hsp40) homolog, subfamily A, member 2

00000031701

[Source:MGI Symbol;Acc:MGI:1931882]

ENSMUSG
N4bp1
protein_coding
NEDD4 binding protein 1 [Source:MGI

00000031652

Symbol;Acc:MGI:2136825]

ENSMUSG
Adcy7
protein_coding
adenylate cyclase 7 [Source:MGI

00000031659

Symbol;Acc:MGI:102891]

ENSMUSG
Fto
protein_coding
fat mass and obesity associated [Source:MGI

00000055932

Symbol;Acc:MGI:1347093]

ENSMUSG
Amfr
protein_coding
autocrine motility factor receptor [Source:MGI

00000031751

Symbol;Acc:MGI:1345634]

ENSMUSG
Fam192a
protein_coding
family with sequence similarity 192, member A

00000031774

[Source:MGI Symbol;Acc:MGI:1919637]

ENSMUSG
Arl2bp
protein_coding
ADP-ribosylation factor-like 2 binding protein

00000031776

[Source:MGI Symbol;Acc:MGI:1349429]

ENSMUSG
Gpr56
protein_coding
G protein-coupled receptor 56 [Source:MGI

00000031785

Symbol;Acc:MGI:1340051]

ENSMUSG
Zfp319
protein_coding
zinc finger protein 319 [Source:MGI

00000046556

Symbol;Acc:MGI:1890618]

ENSMUSG
Cnot1
protein_coding
CCR4-NOT transcription complex, subunit 1

00000036550

[Source:MGI Symbol;Acc:MGI:2442402]

ENSMUSG
Got2
protein_coding
glutamate oxaloacetate transaminase 2,

00000031672

mitochondrial [Source:MGI Symbol;Acc:MGI:95792]

ENSMUSG
E2f4
protein_coding
E2F transcription factor 4 [Source:MGI

00000014859

Symbol;Acc:MGI:103012]

ENSMUSG
Fam65a
protein_coding
family with sequence similarity 65, member A

00000038604

[Source:MGI Symbol;Acc:MGI:1922937]

ENSMUSG
Ctcf
protein_coding
CCCTC-binding factor [Source:MGI

00000005698

Symbol;Acc:MGI:109447]

ENSMUSG
Ranbp10
protein_coding
RAN binding protein 10 [Source:MGI

00000037415

Symbol;Acc:MGI:1921584]

ENSMUSG
Thap11
protein_coding
THAP domain containing 11 [Source:MGI

00000036442

Symbol;Acc:MGI:1930964]

ENSMUSG
Nutf2
protein_coding
nuclear transport factor 2 [Source:MGI

00000008450

Symbol;Acc:MGI:1915301]

ENSMUSG
Pskh1
protein_coding
protein serine kinase H1 [Source:MGI

00000048310

Symbol;Acc:MGI:3528383]

ENSMUSG
Slc12a4
protein_coding
solute carrier family 12, member 4 [Source:MGI

00000017765

Symbol;Acc:MGI:1309465]

ENSMUSG
Slc7a6
protein_coding
solute carrier family 7 (cationic amino acid

00000031904

transporter, y+ system), member 6 [Source:MGI

Symbol;Acc:MGI:2142598]

ENSMUSG
Chtf8
protein_coding
CTF8, chromosome transmission fidelity factor 8

00000046691

homolog (S. cerevisiae) [Source:MG

Symbol;Acc:MGI:2443370]

ENSMUSG
Vps4a
protein_coding
vacuolar protein sorting 4a (yeast) [Source:MGI

00000031913

Symbol;Acc:MGI:1890520]

ENSMUSG
Cog8
protein_coding
component of oligomeric golgi complex 8

00000031916

[Source:MGI Symbol;Acc:MGI:2142885]

ENSMUSG
Terf2
protein_coding
telomeric repeat binding factor 2 [Source:MGI

00000031921

Symbol;Acc:MGI:1195972]

ENSMUSG
Cyb5b
protein_coding
cytochrome b5 type B [Source:MGI

00000031924

Symbol;Acc:MGI:1913677]

ENSMUSG
Nob1
protein_coding
NIN1/RPN12 binding protein 1 homolog (S. cerevisiae)

00000003848

[Source:MGI Symbol;Acc:MGI:1914869]

ENSMUSG
Psmd7
protein_coding
proteasome (prosome, macropain) 26S subunit, non-

00000039067

ATPase, 7 [Source:MGI Symbol;Acc:MGI:1351511]

ENSMUSG
Dhx38
protein_coding
DEAH (Asp-Glu-Ala-His) box polypeptide 38

00000037993

[Source:MGI Symbol;Acc:MGI:1927617]

ENSMUSG
Pkd1l3
protein_coding
polycystic kidney disease 1 like 3 [Source:MGI

00000048827

Symbol;Acc:MGI:2664670]

ENSMUSG
2400003C14Rik
protein_coding
RIKEN cDNA 2400003C14 gene [Source:MGI

00000031729

Symbol;Acc:MGI:1919205]

ENSMUSG
Atxn1l
protein_coding
ataxin 1-like [Source:MGI Symbol;Acc:MGI:3694797]

00000069895

ENSMUSG
Ap1g1
protein_coding
adaptor protein complex AP-1, gamma 1 subunit

00000031731

[Source:MGI Symbol;Acc:MGI:101919]

ENSMUSG
Hydin
protein_coding
HYDIN, axonemal central pair apparatus protein

00000059854

[Source:MGI Symbol;Acc:MGI:2389007]

ENSMUSG
Sf3b3
protein_coding
splicing factor 3b, subunit 3 [Source:MGI

00000033732

Symbol;Acc:MGI:1289341]

ENSMUSG
St3gal2
protein_coding
ST3 beta-galactoside alpha-2,3-sialyltransferase 2

00000031749

[Source:MGI Symbol;Acc:MGI:99427]

ENSMUSG
Ddx19a
protein_coding
DEAD (Asp-Glu-Ala-Asp) box polypeptide 19a

00000015023

[Source:MGI Symbol;Acc:MGI:99526]

ENSMUSG
Aars
protein_coding
alanyl-tRNA synthetase [Source:MGI

00000031960

Symbol;Acc:MGI:2384560]

ENSMUSG
Pdpr
protein_coding
pyruvate dehydrogenase phosphatase regulatory

00000033624

subunit [Source:MGI Symbol;Acc:MGI:2442188]

ENSMUSG
Znrf1
protein_coding
zinc and ring finger 1 [Source:MGI

00000033545

Symbol;Acc:MGI:2177308]

ENSMUSG
Kars
protein_coding
lysyl-tRNA synthetase [Source:MGI

00000031948

Symbol;Acc:MGI:1934754]

ENSMUSG
Mon1b
protein_coding
MON1 homolog b (yeast) [Source:MGI

00000078908

Symbol;Acc:MGI:1923231]

ENSMUSG
Wwox
protein_coding
WW domain-containing oxidoreductase [Source:MGI

00000004637

Symbol;Acc:MGI:1931237]

ENSMUSG
4933407C03Rik
protein_coding
RIKEN cDNA 4933407C03 gene [Source:MGI

00000034390

Symbol;Acc:MGI:1921690]

ENSMUSG
Plcg2
protein_coding
phospholipase C, gamma 2 [Source:MGI

00000034330

Symbol;Acc:MGI:97616]

ENSMUSG
Cdh13
protein_coding
cadherin 13 [Source:MGI Symbol;Acc:MGI:99551]

00000031841

ENSMUSG
Necab2
protein_coding
N-terminal EF-hand calcium binding protein 2

00000031837

[Source:MGI Symbol;Acc:MGI:2152211]

ENSMUSG
Mbtps1
protein_coding
membrane-bound transcription factor peptidase, site

00000031835

1 [Source:MGI Symbol;Acc:MGI:1927235]

ENSMUSG
Taf1c
protein_coding
TATA box binding protein (Tbp)-associated factor,

00000031832

RNA polymerase I, C [Source:MGI

Symbol;Acc:MGI:109576]

ENSMUSG
4632415K11Rik
protein_coding
RIKEN cDNA 4632415K11 gene [Source:MGI

00000034105

Symbol;Acc:MGI:1921597]

ENSMUSG
Cotl1
protein_coding
coactosin-like 1 (Dictyostelium) [Source:MGI

00000031827

Symbol;Acc:MGI:1919292]

ENSMUSG
Usp10
protein_coding
ubiquitin specific peptidase 10 [Source:MGI

00000031826

Symbol;Acc:MGI:894652]

ENSMUSG
Gm20388
protein_coding
predicted gene 20388 [Source:MGI

00000092329

Symbol;Acc:MGI:5141853]

ENSMUSG
Zdhhc7
protein_coding
zinc finger, DHHC domain containing 7 [Source:MGI

00000031823

Symbol;Acc:MGI:2142662]

ENSMUSG
Gse1
protein_coding
genetic suppressor element 1 [Source:MGI

00000031822

Symbol;Acc:MGI:1098275]

ENSMUSG
Gins2
protein_coding
GINS complex subunit 2 (Psf2 homolog) [Source:MGI

00000031821

Symbol;Acc:MGI:1921019]

ENSMUSG
Cox4i1
protein_coding
cytochrome c oxidase subunit IV isoform 1

00000031818

[Source:MGI Symbol;Acc:MGI:88473]

ENSMUSG
Map1lc3b
protein_coding
microtubule-associated protein 1 light chain 3 beta

00000031812

[Source:MGI Symbol;Acc:MGI:1914693]

ENSMUSG
Slc7a5
protein_coding
solute carrier family 7 (cationic amino acid

00000040010

transporter, y+ system), member 5 [Source:MGI

Symbol;Acc:MGI:1298205]

ENSMUSG
Banp
protein_coding
BTG3 associated nuclear protein [Source:MGI

00000025316

Symbol;Acc:MGI:1889023]

ENSMUSG
Zfpm1
protein_coding
zinc finger protein, multitype 1 [Source:MGI

00000049577

Symbol;Acc:MGI:1095400]

ENSMUSG
Zc3h18
protein_coding
zinc finger CCCH-type containing 18 [Source:MGI

00000017478

Symbol;Acc:MGI:1923264]

ENSMUSG
Fam38a
protein_coding
family with sequence similarity 38, member A

00000014444

[Source:MGI Symbol;Acc:MGI:3603204]

ENSMUSG
Cdt1
protein_coding
chromatin licensing and DNA replication factor 1

00000006585

[Source:MGI Symbol;Acc:MGI:1914427]

ENSMUSG
Aprt
protein_coding
adenine phosphoribosyl transferase [Source:MGI

00000006589

Symbol;Acc:MGI:88061]

ENSMUSG
Cbfa2t3
protein_coding
core-binding factor, runt domain, alpha subunit 2,

00000006362

translocated to, 3 (human) [Source:MGI

Symbol;Acc:MGI:1338013]

ENSMUSG
Ankrd11
protein_coding
ankyrin repeat domain 11 [Source:MGI

00000035569

Symbol;Acc:MGI:1924337]

ENSMUSG
Rpl13
protein_coding
ribosomal protein L13 [Source:MGI

00000000740

Symbol;Acc:MGI:105922]

ENSMUSG
Fanca
protein_coding
Fanconi anemia, complementation group A

00000032815

[Source:MGI Symbol;Acc:MGI:1341823]

ENSMUSG
Tcf25
protein_coding
transcription factor 25 (basic helix-loop-helix)

00000001472

[Source:MGI Symbol;Acc:MGI:1914105]

ENSMUSG
Rab4a
protein_coding
RAB4A, member RAS oncogene family [Source:MGI

00000019478

Symbol;Acc:MGI:105069]

ENSMUSG
Abcb10
protein_coding
ATP-binding cassette, sub-family B (MDR/TAP),

00000031974

member 10 [Source:MGI Symbol;Acc:MGI:1860508]

ENSMUSG
Taf51
protein_coding
TAF5-like RNA polymerase II, p300/CBP-associated

00000038697

factor (PCAF)-associated factor [Source:MGI

Symbol;Acc:MGI:1919039]

ENSMUSG
Urb2
protein_coding
URB2 ribosome biogenesis 2 homolog (S. cerevisiae)

00000031976

[Source:MGI Symbol;Acc:MGI:2681124]

ENSMUSG
Galnt2
protein_coding
UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-

00000089704

acetylgalactosaminyltransferase 2 [Source:MGI

Symbol;Acc:MGI:894694]

ENSMUSG
Exoc8
protein_coding
exocyst complex component 8 [Source:MGI

00000074030

Symbol;Acc:MGI:2142527]

ENSMUSG
Egln1
protein_coding
EGL nine homolog 1 (C. elegans) [Source:MGI

00000031987

Symbol;Acc:MGI:1932286]

ENSMUSG
BC021891
protein_coding
cDNA sequence BC021891 [Source:MGI

00000031853

Symbol;Acc:MGI:2385307]

ENSMUSG
Irf2bp2
protein_coding
interferon regulatory factor 2 binding protein 2

00000051495

[Source:MGI Symbol;Acc:MGI:2443921]

ENSMUSG
Tomm20
protein_coding
translocase of outer mitochondrial membrane 20

00000058779

homolog (yeast) [Source:MGI

Symbol;Acc:MGI:1915202]

ENSMUSG
4930433N12Rik
protein_coding
RIKEN cDNA 4930433N12 gene [Source:MGI

00000042360

Symbol;Acc:MGI:2149746]

ENSMUSG
Cwf19l2
protein_coding
CWF19-like 2, cell cycle control (S. pombe)

00000025898

[Source:MGI Symbol;Acc:MGI:1918023]

ENSMUSG
Kbtbd3
protein_coding
kelch repeat and BTB (POZ) domain containing 3

00000025893

[Source:MGI Symbol;Acc:MGI:1916399]

ENSMUSG
Amotl1
protein_coding
angiomotin-like 1 [Source:MGI

00000013076

Symbol;Acc:MGI:1922973]

ENSMUSG
Taf1d
protein_coding
TATA box binding protein (Tbp)-associated factor,

00000031939

RNA polymerase I, D [Source:MGI

Symbol;Acc:MGI:1922566]

ENSMUSG
5830418K08Rik
protein_coding
RIKEN cDNA 5830418K08 gene [Source:MGI

00000046111

Symbol;Acc:MGI:2442521]

ENSMUSG
Zfp266
protein_coding
zinc finger protein 266 [Source:MGI

00000060510

Symbol;Acc:MGI:1924769]

ENSMUSG
Dnmt1
protein_coding
DNA methyltransferase (cytosine-5) 1 [Source:MGI

00000004099

Symbol;Acc:MGI:94912]

ENSMUSG
Raver1
protein_coding
ribonucleoprotein, PTB-binding 1 [Source:MGI

00000010205

Symbol;Acc:MGI:1919016]

ENSMUSG
Cdc37
protein_coding
cell division cycle 37 homolog (S. cerevisiae)

00000019471

[Source:MGI Symbol;Acc:MGI:109531]

ENSMUSG
Keap1
protein_coding
kelch-like ECH-associated protein 1 [Source:MGI

00000003308

Symbol;Acc:MGI:1858732]

ENSMUSG
Atg4d
protein_coding
autophagy-related 4D (yeast) [Source:MGI

00000002

Symbol;Acc:MGI:2444308]

820

ENSMUSG
Kri1
protein_coding
KRI1 homolog (S. cerevisiae) [Source:MGI

00000035

Symbol;Acc:MGI:2384899]

047

ENSMUSG
Ilf3
protein_coding
interleukin enhancer binding factor 3 [Source:MGI

00000032178

Symbol;Acc:MGI:1339973]

ENSMUSG
Carm1
protein_coding
coactivator-associated arginine methyltransferase 1

00000032185

[Source:MGI Symbol;Acc:MGI:1913208]

ENSMUSG
Smarca4
protein_coding
SWI/SNF related, matrix associated, actin dependent

00000032187

regulator of chromatin, subfamily a, member 4

[Source:MGI Symbol;Acc:MGI:88192]

ENSMUSG
Ldlr
protein_coding
low density lipoprotein receptor [Source:MGI

00000032193

Symbol;Acc:MGI:96765]

ENSMUSG
Prkcsh
protein_coding
protein kinase C substrate 80K-H [Source:MGI

00000003402

Symbol;Acc:MGI:107877]

ENSMUSG
Bmper
protein_coding
BMP-binding endothelial regulator [Source:MGI

00000031963

Symbol;Acc:MGI:1920480]

ENSMUSG
Opcml
protein_coding
opioid binding protein/cell adhesion molecule-like

00000062257

[Source:MGI Symbol;Acc:MGI:97397]

ENSMUSG
Aplp2
protein_coding
amyloid beta (A4) precursor-like protein 2

00000031996

[Source:MGI Symbol;Acc:MGI:88047]

ENSMUSG
Fli1
protein_coding
Friend leukemia integration 1 [Source:MGI

00000016087

Symbol;Acc:MGI:95554]

ENSMUSG
Kirrel3
protein_coding
kin of IRRE like 3 (Drosophila) [Source:MGI

00000032036

Symbol;Acc:MGI:1914953]

ENSMUSG
Fez1
protein_coding
fasciculation and elongation protein zeta 1 (zygin I)

00000032118

[Source:MGI Symbol;Acc:MGI:2670976]

ENSMUSG
Pknox2
protein_coding
Pbx/knotted 1 homeobox 2 [Source:MGI

00000035934

Symbol;Acc:MGI:2445415]

ENSMUSG
Nrgn
protein_coding
neurogranin [Source:MGI Symbol;Acc:MGI:1927184]

00000053310

ENSMUSG
Gramd1b
protein_coding
GRAM domain containing 1B [Source:MGI

00000040111

Symbol;Acc:MGI:1925037]

ENSMUSG
Ubash3b
protein_coding
ubiquitin associated and SH3 domain containing, B

00000032020

[Source:MGI Symbol;Acc:MGI:1920078]

ENSMUSG
Grik4
protein_coding
glutamate receptor, ionotropic, kainate 4

00000032017

[Source:MGI Symbol;Acc:MGI:95817]

ENSMUSG
Cbl
protein_coding
Casitas B-lineage lymphoma [Source:MGI

00000034342

Symbol;Acc:MGI:88279]

ENSMUSG
H2afx
protein_coding
H2A histone family, member X [Source:MGI

00000049932

Symbol;Acc:MGI:102688]

ENSMUSG
Bcl9l
protein_coding
B cell CLL/lymphoma 9-like [Source:MGI

00000063382

Symbol;Acc:MGI:1933114]

ENSMUSG
Ddx6
protein_coding
DEAD (Asp-Glu-Ala-Asp) box polypeptide 6

00000032097

[Source:MGI Symbol;Acc:MGI:104976]

ENSMUSG
Sidt2
protein_coding
SID1 transmembrane family, member 2 [Source:MGI

00000034908

Symbol;Acc:MGI:2446134]

ENSMUSG
Pafah1b2
protein_coding
platelet-activating factor acetylhydrolase, isoform 1b,

00000003131

subunit 2 [Source:MGI Symbol;Acc:MGI:108415]

ENSMUSG
Sik3
protein_coding
SIK family kinase 3 [Source:MGI

00000034135

Symbol;Acc:MGI:2446296]

ENSMUSG
Cadm1
protein_coding
cell adhesion molecule 1 [Source:MGI

00000032076

Symbol;Acc:MGI:1889272]

ENSMUSG
Zbtb16
protein_coding
zinc finger and BTB domain containing 16

00000066687

[Source:MGI Symbol;Acc:MGI:103222]

ENSMUSG
Ppp2r1b
protein_coding
protein phosphatase 2 (formerly 2A), regulatory

00000032058

subunit A (PR 65), beta isoform [Source:MGI

Symbol;Acc:MGI:1920949]

ENSMUSG
Rdx
protein_coding
radixin [Source:MGI Symbol;Acc:MGI:97887]

00000032050

ENSMUSG
Atm
protein_coding
ataxia telangiectasia mutated homolog (human)

00000034218

[Source:MGI Symbol;Acc:MGI:107202]

ENSMUSG
C230081A13Rik
protein_coding
RIKEN cDNA C230081A13 gene [Source:MGI

00000074305

Symbol;Acc:MGI:2442366]

ENSMUSG
Hmg20a
protein_coding
high mobility group 20A [Source:MGI

00000032329

Symbol;Acc:MGI:1914117]

ENSMUSG
Imp3
protein_coding
IMP3, U3 small nucleolar ribonucleoprotein, homolog

00000032288

(yeast) [Source:MGI Symbol;Acc:MGI:1916119]

ENSMUSG
1700017B05Rik
protein_coding
RIKEN cDNA 1700017B05 gene [Source:MGI

00000032300

Symbol;Acc:MGI:1921461]

ENSMUSG
Csk
protein_coding
c-src tyrosine kinase [Source:MGI

00000032312

Symbol;Acc:MGI:88537]

ENSMUSG
Neo1
protein_coding
neogenin [Source:MGI Symbol;Acc:MGI:1097159]

00000032340

ENSMUSG
Arih1
protein_coding
ariadne ubiquitin-conjugating enzyme E2 binding

00000025234

protein homolog 1 (Drosophila) [Source:MGI

Symbol;Acc:MGI:1344363]

ENSMUSG
Pkm2
protein_coding
pyruvate kinase, muscle [Source:MGI

00000032294

Symbol;Acc:MGI:97591]

ENSMUSG
Tle3
protein_coding
transducin-like enhancer of split 3, homolog of

00000032280

Drosophila E(spl) [Source:MGI

Symbol;Acc:MGI:104634]

ENSMUSG
Rplp1
protein_coding
ribosomal protein, large, P1 [Source:MGI

00000007892

Symbol;Acc:MGI:1927099]

ENSMUSG
Fem1b
protein_coding
feminization 1 homolog b (C. elegans) [Source:MGI

00000032244

Symbol;Acc:MGI:1335087]

ENSMUSG
Rpl4
protein_coding
ribosomal protein L4 [Source:MGI

00000032399

Symbol;Acc:MGI:1915141]

ENSMUSG
Tipin
protein_coding
timeless interacting protein [Source:MGI

00000032397

Symbol;Acc:MGI:1921571]

ENSMUSG
Rab11a
protein_coding
RAB11a, member RAS oncogene family [Source:MGI

00000004771

Symbol;Acc:MGI:1858202]

ENSMUSG
2010321M09Rik
protein_coding
RIKEN cDNA 2010321M09 gene [Source:MGI

00000034263

Symbol;Acc:MGI:1917132]

ENSMUSG
Parp16
protein_coding
poly (ADP-ribose) polymerase family, member 16

00000032392

[Source:MGI Symbol;Acc:MGI:2446133]

ENSMUSG
Plekho2
protein_coding
pleckstrin homology domain containing, family O

00000050721

member 2 [Source:MGI Symbol;Acc:MGI:2143132]

ENSMUSG
Zfp609
protein_coding
zinc finger protein 609 [Source:MGI

00000040524

Symbol;Acc:MGI:2674092]

ENSMUSG
Csnk1g1
protein_coding
casein kinase 1, gamma 1 [Source:MGI

00000032384

Symbol;Acc:MGI:2660884]

ENSMUSG
Herc1
protein_coding
hect (homologous to the E6-AP (UBE3A) carboxyl

00000038664

terminus) domain and RCC1 (CHC1)-like domain (RLD)

1 [Source:MGI Symbol;Acc:MGI:2384589]

ENSMUSG
Tpm1
protein_coding
tropomyosin 1, alpha [Source:MGI

00000032366

Symbol;Acc:MGI:98809]

ENSMUSG
Tln2
protein_coding
talin 2 [Source:MGI Symbol;Acc:MGI:1917799]

00000052698

ENSMUSG
Rora
protein_coding
RAR-related orphan receptor alpha [Source:MGI

00000032238

Symbol;Acc:MGI:104661]

ENSMUSG
Myo1e
protein_coding
myosin IE [Source:MGI Symbol;Acc:MGI:106621]

00000032220

ENSMUSG
Adam10
protein_coding
a disintegrin and metallopeptidase domain 10

00000054693

[Source:MGI Symbol;Acc:MGI:109548]

ENSMUSG
Polr2m
protein_coding
polymerase (RNA) II (DNA directed) polypeptide M

00000032199

[Source:MGI Symbol;Acc:MGI:107282]

ENSMUSG
Gcom1
protein_coding
GRINL1A complex locus [Source:MGI

00000092137

Symbol;Acc:MGI:5141967]

ENSMUSG
Myzap
protein_coding
myocardial zonula adherens protein [Source:MGI

00000041361

Symbol;Acc:MGI:2142908]

ENSMUSG
Tcf12
protein_coding
transcription factor 12 [Source:MGI

00000032228

Symbol;Acc:MGI:101877]

ENSMUSG
Rfx7
protein_coding
regulatory factor X, 7 [Source:MGI

00000037674

Symbol;Acc:MGI:2442675]

ENSMUSG
Nedd4
protein_coding
neural precursor cell expressed, developmentally

00000032216

down-regulated 4 [Source:MGI

Symbol;Acc:MGI:97297]

ENSMUSG
Unc13c
protein_coding
unc-13 homolog C (C. elegans) [Source:MGI

00000062151

Symbol;Acc:MGI:2149021]

ENSMUSG
Arpp19
protein_coding
CAMP-regulated phosphoprotein 19 [Source:MGI

00000007656

Symbol;Acc:MGI:1891691]

ENSMUSG
Myo5a
protein_coding
myosin VA [Source:MGI Symbol;Acc:MGI:105976]

00000034593

ENSMUSG
Mapk6
protein_coding
mitogen-activated protein kinase 6 [Source:MGI

00000042688

Symbol;Acc:MGI:1354946]

ENSMUSG
2310046A06Rik
protein_coding
RIKEN cDNA 2310046A06 gene [Source:MGI

00000032355

Symbol;Acc:MGI:1916892]

ENSMUSG
Eef1a1
protein_coding
eukaryotic translation elongation factor 1 alpha 1

00000037742

[Source:MGI Symbol;Acc:MGI:1096881]

ENSMUSG
Senp6
protein_coding
SUMO/sentrin specific peptidase 6 [Source:MGI

00000034252

Symbol;Acc:MGI:1922075]

ENSMUSG
Mei4
protein_coding
meiosis-specific, MEI4 homolog (S. cerevisiae)

00000043289

[Source:MGI Symbol;Acc:MGI:1922283]

ENSMUSG
Phip
protein_coding
pleckstrin homology domain interacting protein

00000032253

[Source:MGI Symbol;Acc:MGI:1932404]

ENSMUSG
Fam46a
protein_coding
family with sequence similarity 46, member A

00000032265

[Source:MGI Symbol;Acc:MGI:2670964]

ENSMUSG
Ibtk
protein_coding
inhibitor of Bruton agammaglobulinemia tyrosine

00000035941

kinase [Source:MGI Symbol;Acc:MGI:1918677]

ENSMUSG
Gm20537
protein_coding
predicted gene 20537 [Source:MGI

00000092541

Symbol;Acc:MGI:5142002]

ENSMUSG
Syncrip
protein_coding
synaptotagmin binding, cytoplasmic RNA interacting

00000032423

protein [Source:MGI Symbol;Acc:MGI:1891690]

ENSMUSG
2610101N10Rik
protein_coding
RIKEN cDNA 2610101N10 gene [Source:MGI

00000032407

Symbol;Acc:MGI:1915208]

ENSMUSG
Tfdp2
protein_coding
transcription factor Dp 2 [Source:MGI

00000032411

Symbol;Acc:MGI:107167]

ENSMUSG
Atp1b3
protein_coding
ATPase, Na+/K+ transporting, beta 3 polypeptide

00000032412

[Source:MGI Symbol;Acc:MGI:107788]

ENSMUSG
Rasa2
protein_coding
RAS p21 protein activator 2 [Source:MGI

00000032413

Symbol;Acc:MGI:2149960]

ENSMUSG
Msl2
protein_coding
male-specific lethal 2 homolog (Drosophila)

00000066415

[Source:MGI Symbol;Acc:MGI:1925103]

ENSMUSG
Cdv3
protein_coding
carnitine deficiency-associated gene expressed in

00000032803

ventricle 3 [Source:MGI Symbol;Acc:MGI:2448759]

ENSMUSG
Cpne4
protein_coding
copine IV [Source:MGI Symbol;Acc:MGI:1921270]

00000032564

ENSMUSG
Pik3r4
protein_coding
phosphatidylinositol 3 kinase, regulatory subunit,

00000032571

polypeptide 4, p150 [Source:MGI

Symbol;Acc:MGI:1922919]

ENSMUSG
Wdr82
protein_coding
WD repeat domain containing 82 [Source:MGI

00000020257

Symbol;Acc:MGI:1924555]

ENSMUSG
Twf2
protein_coding
twinfilin, actin-binding protein, homolog 2

00000023277

(Drosophila) [Source:MGI Symbol;Acc:MGI:1346078]

ENSMUSG
Rad54l2
protein_coding
RAD54 like 2 (S. cerevisiae) [Source:MGI

00000040661

Symbol;Acc:MGI:1933196]

ENSMUSG
Manf
protein_coding
mesencephalic astrocyte-derived neurotrophic factor

00000032575

[Source:MGI Symbol;Acc:MGI:1922090]

ENSMUSG
Rbm15b
protein_coding
RNA binding motif protein 15B [Source:MGI

00000045365

Symbol;Acc:MGI:1923598]

ENSMUSG
Cacna2d2
protein_coding
calcium channel, voltage-dependent, alpha 2/delta

00000010066

subunit 2 [Source:MGI Symbol;Acc:MGI:1929813]

ENSMUSG
Gnai2
protein_coding
guanine nucleotide binding protein (G protein), alpha

00000032562

inhibiting 2 [Source:MGI Symbol;Acc:MGI:95772]

ENSMUSG
Rbm5
protein_coding
RNA binding motif protein 5 [Source:MGI

00000032580

Symbol;Acc:MGI:1933204]

ENSMUSG
Traip
protein_coding
TRAF-interacting protein [Source:MGI

00000032586

Symbol;Acc:MGI:1096377]

ENSMUSG
Cdhr4
protein_coding
cadherin-related family member 4 [Source:MGI

00000079323

Symbol;Acc:MGI:1916648]

ENSMUSG
6230427J02Rik
protein_coding
RIKEN cDNA 6230427J02 gene [Source:MGI

00000042106

Symbol;Acc:MGI:1915426]

ENSMUSG
Dag1
protein_coding
dystroglycan 1 [Source:MGI Symbol;Acc:MGI:101864]

00000039952

ENSMUSG
Rhoa
protein_coding
ras homolog gene family, member A [Source:MGI

00000007815

Symbol;Acc:MGI:1096342]

ENSMUSG
Gpx1
protein_coding
glutathione peroxidase 1 [Source:MGI

00000063856

Symbol;Acc:MGI:104887]

ENSMUSG
Ccdc71
protein_coding
coiled-coil domain containing 71 [Source:MGI

00000049305

Symbol;Acc:MGI:1919704]

ENSMUSG
Qrich1
protein_coding
glutamine-rich 1 [Source:MGI

00000006673

Symbol;Acc:MGI:1916482]

ENSMUSG
Wdr6
protein_coding
WD repeat domain 6 [Source:MGI

00000066357

Symbol;Acc:MGI:1930140]

ENSMUSG
Mtap4
protein_coding
microtubule-associated protein 4 [Source:MGI

00000032479

Symbol;Acc:MGI:97178]

ENSMUSG
Dhx30
protein_coding
DEAH (Asp-Glu-Ala-His) box polypeptide 30

00000032480

[Source:MGI Symbol;Acc:MGI:1920081]

ENSMUSG
Smarcc1
protein_coding
SWI/SNF related, matrix associated, actin dependent

00000032481

regulator of chromatin, subfamily c, member 1

[Source:MGI Symbol;Acc:MGI:1203524]

ENSMUSG
Nbeal2
protein_coding
neurobeachin-like 2 [Source:MGI

00000056724

Symbol;Acc:MGI:2448554]

ENSMUSG
Pdcd6ip
protein_coding
programmed cell death 6 interacting protein

00000032504

[Source:MGI Symbol;Acc:MGI:1333753]

ENSMUSG
Fbxl2
protein_coding
F-box and leucine-rich repeat protein 2 [Source:MGI

00000032507

Symbol;Acc:MGI:1919429]

ENSMUSG
Cmtm7
protein_coding
CKLF-like MARVEL transmembrane domain containing

00000032436

7 [Source:MGI Symbol;Acc:MGI:2447166]

ENSMUSG
Stt3b
protein_coding
STT3, subunit of the oligosaccharyltransferase

00000032437

complex, homolog B (S. cerevisiae) [Source:MGI

Symbol;Acc:MGI:1915542]

ENSMUSG
Rbms3
protein_coding
RNA binding motif, single stranded interacting protein

00000039607

[Source:MGI Symbol;Acc:MGI:2444477]

ENSMUSG
Ctdspl
protein_coding
CTD (carboxy-terminal domain, RNA polymerase II,

00000047409

polypeptide A) small phosphatase-like [Source:MGI

Symbol;Acc:MGI:1916524]

ENSMUSG
Cx3cr1
protein_coding
chemokine (C-X3-C) receptor 1 [Source:MGI

00000052336

Symbol;Acc:MGI:1333815]

ENSMUSG
Rpsa
protein_coding
ribosomal protein SA [Source:MGI

00000032518

Symbol;Acc:MGI:105381]

ENSMUSG
Eif1b
protein_coding
eukaryotic translation initiation factor 1B

00000006941

[Source:MGI Symbol;Acc:MGI:1916219]

ENSMUSG
Ctnnb1
protein_coding
catenin (cadherin associated protein), beta 1

00000006932

[Source:MGI Symbol;Acc:MGI:88276]

ENSMUSG
Nktr
protein_coding
natural killer tumor recognition sequence

00000032525

[Source:MGI Symbol;Acc:MGI:97346]

ENSMUSG
Zdhhc3
protein_coding
zinc finger, DHHC domain containing 3 [Source:MGI

00000025786

Symbol;Acc:MGI:1926134]

TABLE S4

Table S4. Percentages of human donor derived

chimerism used to calculate CRU

Percentage of hCD45 + GFP + cells in TNC

Primary LDA

Control
1.20%

50K
1.11%

0.85%

4.29%

Control
1.66%

20K
1.51%

1.47%

2.10%

YTHDF2
7.37%

KD 50K
67.90%

22.80%

21.00%

YTHDF2
12.30%

KD 20K
8.23%

4.57%

1.53%

Control
0.17%

10K *
0.46%

1.00%

0.65%

1.23%

YTHDF2
2.51%

KD 20K
1.60%

1.28%

1.40%

1.66%

3.07%

0.95%

Secondary LDA

Control
0.27%

12E+6
0.26%

0.35%

Control
0.02%

8E+6
0.01%

7.57E−05

Control
0.03%

4E+6
0.02%

0.01%

0.02%

YTHDF2
0.92%

KD 12E+6
1.13%

1.45%

YTHDF2
0.38%

KD 8E+6
0.37%

0.72%

YTHDF2
0.27%

KD 4E+6
0.29%

0.19%

0.44%

* Control 10K group also transplanted 7 mice. However, 2 mice were dead before the time point of analysis partially due to the deficiency in hematopoietic recovery.

TABLE S5

qPCR primers used to verify the

expressional levels of transcription factors

in wt and Ythdf2 KO HSPCs.

mouse Tal1 qPCR primer F
CGCTGCTCTATAGCCTTAGCC

(SEQ ID NO: 10)

mouse Tal1 qPCR primer R
CTCTTCACCCGGTTGTTGTT

(SEQ ID NO: 11)

mouse Gata2 qPCR primer F
CAACCCTTACTACGCCAACC

(SEQ ID NO: 12)

mouse Gata2 qPCR primer R
GCTGTGCAACAAGTGTGGTC

(SEQ ID NO: 13)

mouse Runx1 qPCR primer F
CCAGCCTCTCTGCAGAACTT

(SEQ ID NO: 14)

mouse Runx1 qPCR primer R
GGAGATGGACGGCAGAGTAG

(SEQ ID NO: 15)

mouse Stat5a qPCR primer F
AGAAGCAAGTGTCCCTGGAG

(SEQ ID NO: 16)

mouse Stat5a qPCR primer R
GTCGTCCAGGATGATGGTCT

(SEQ ID NO: 17)

mouse Actb qPCR primer F
TGTCACCAACTGGGACGATA

(SEQ ID NO: 18)

mouse Actb qPCR primer R
ACCCTCATAGATGGGCACAG

(SEQ ID NO: 19)

Number	Name	Date	Kind
9439928	Kasahara et al.	Sep 2016	B2
10787643	Perry	Sep 2020	B2
20160264934	Giallourakis et al.	Sep 2016	A1
20170037396	Lee	Feb 2017	A1

Number	Date	Country
2023171952	Dec 2023	JP
2012027376	Mar 2012	WO

	Number	Date	Country
	62695820	Jul 2018	US
	62570076	Oct 2017	US

Methods and compositions for expansion of cell population

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