Patent Application
20210261923
A sequence listing accompanies this application and is submitted as an ASCII text file of the sequence listing named “960296_04145_ST25.txt” which is 6.38 kb in size and was created on Apr. 16, 2021. The sequence listing is electronically submitted via EFS-Web with the application and is incorporated herein by reference in its entirety.
Generating autologous hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) that can be precisely genetically modified with designer endonucleases, and subsequently clonally selected, represents a promising approach for novel patient-specific gene therapies. Although multiple studies were able to generate hematopoietic progenitors (HPs) with a HSC phenotype and limited engraftment potential from pluripotent stem cells (PSCs) 1-4, the robust and consistent engraftment with recapitulation of the full spectrum of terminally differentiated hematopoietic cells, including lymphoid cells has not been achieved. Thus, identifying key cellular and molecular programs required for proper HSC specification in vitro is essential to overcome the current roadblocks.
The present disclosure provides methods of producing arterial type hemogenic endothelial cells (AHE) which are CD144+CD43−CD73−DLL4+ HE that express high level of EFNB2 and NOTCH1 arterial markers and MYB gene required for definitive hematopoiesis. These cells have broad lympho-myeloid and definitive erythroid potentials.
In one aspect, the disclosure provides method of inducing an arterial-type hemogenic endothelium (AHE) cell population, comprising the steps of (a) obtaining CD144+CD43−CD73-hemogenic endothelial cells on day 4 of differentiation (D4), and (b) exposing the D4 HE cells to a sufficient amount of a NOTCH activation agent, such that arterial-type cells (AHE cells) are created, wherein the AHE cells are detected as CD144+CD43−CD73−DLL4+ HE that express high level of EFNB2 and NOTCH1 arterial markers and MYB gene. In some aspects, the method additionally comprises the step of exposing the AHE created in step (b) to a sufficient amount of a NOTCH activation agent, such that the AHE undergo endothelial-to-hematopoietic transition and produce definitive-type hematopoietic progeny with adult-like characteristics.
In another aspect, the disclosure provides a method of inducing an arterial-type hemogenic endothelium (AHE) cell population, comprising the steps of exposing immature CD144+CD43−CD73″ hemogenic endothelial (HE) cells which express HAND1 to a sufficient amount of a NOTCH activation agent, such that AHE cells are obtained, wherein the AHE cells are detected as CD144+CD43−CD73−DLL4+ HE that express EFNB2 and NOTCH1 arterial markers and MYB gene.
In another aspect, the disclosure provides a cell population comprising at least 90% AHE cells produced by the methods described herein. In another aspect, the disclosure provides a cell population comprising at least 95% AHE-cells produced by the methods described herein.
In another aspect, the disclosure provides a method of inducing a population of differentiated hematopoietic cells, comprising the steps of creating the AHE cells of claim 1 and further differentiating the cells into a cell type selected from the group of platelet-producing megakaryocytes, adult-globin expressing erythrocytes, and T-lymphocytes.
In yet another aspect, the disclosure provides a method of differentiating T cells from CD144+CD43−CD73− hemogenic endothelial cells, the method comprising: (a) culturing CD144+CD43−CD73− hemogenic endothelial cells in a sufficient amount of a NOTCH activation agent to produce hematopoietic progenitors (HPs) with increased T-cell potential compared to cells not cultured with NOTCH activation agent, (b) culturing the hematopoietic progenitors in a sufficient amount of NOTCH activation agent with T-cell differentiation conditions for a sufficient time to produce T cells.
In yet another aspect, the disclosure provides a method of isolating an arterial-type hemogenic endothelium (AHE) cell population, comprising the steps of detecting and isolating DLL4+ AHE cells in day 5 of differentiation (D5), wherein the DLL4+ AHE detected are CD144+CD43−CD73−DLL4+ HE that express high level of EFNB2 and NOTCH1 arterial markers and MYB gene.
In yet another aspect, the disclosure provides a method of obtaining a cellular composition comprising more than 95% arterial-type hemogenic endothelium (AHE) cell population, comprising the steps of a. differentiating human pluripotent stem cells (hPSCs) for five days in defined conditions to induce formation of CD144+CD43−CD73-Dll4+ arterial HE; and b. detecting and isolating a cell fraction being characterized by CD144+CD43−CD73−DLL4+ phenotype.
The patent or patent application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The present disclosure demonstrates methods that allow for the promoting of arterial hemogenic progenitors by NOTCH activation from immature CD144+CD43−CD73− HE and post-transition expansion of blood cells.
CD144+CD43−CD73− hemogenic endothelial (HE) cells on day 4 of differentiation are immature or primordial hemogenic endothelial cells which express HAND1. The immature CD144+CD43−CD73− hemogenic endothelial (HE) cells are also referred to herein as D4 HE cells. Methods of producing and obtaining D4 HE are described in the Examples and description herein. This cell population of immature HE can be seen in
Generating autologous hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) that can be precisely genetically modified with designer endonucleases, and subsequently clonally selected, represents a promising approach for novel patient-specific gene therapies. Although multiple studies were able to generate hematopoietic progenitors with HSC phenotype from PSCs, these cells failed to produce multilineage engraftment. By “failure to produce multilineage engraftment,” we mean that the cells did not have the capacity to reconstitute the hematopoietic system when transplanted into immunocompromised murine host (i.e. to repopulate bone marrow and produce lymphoid, myeloid and erythromegakaryocytic cells for more than 6 weeks post-transplantation). Thus, identification of key elements of cellular and molecular programs that reproduce in vitro the proper specification of HSCs would be essential to overcome current roadblocks on the way to de novo HSC generation.
We use the term “arterial specification” and “arterial type” interchangeably herein. The term arterial type hemogenic endothelial cells (AHE) of the present invention are CD144+CD43−CD73−DLL4+ HE that express high level of EFNB2 and NOTCH1 arterial markers and MYB gene required for definitive hematopoiesis. These cells have broad lympho-myeloid and definitive erythroid potentials.
During development, HSCs emerge by budding from hemogenic endothelium (HE) lining arterial vessels, most robustly from the ventral wall of the dorsal aorta. (See Bertrand, J. Y., Chi, N. C., Santoso, B., Teng, S., Stainier, D. Y., and Traver, D. (2010); Haematopoietic stele cells derive directly from aortic endothelium during development. Nature 464, 108-111; Dzierzak, E., and Speck, N. A. (2008)); Of lineage and legacy: the development of mammalian hematopoietic stem cells. Nat Immunol 9, 129-136; Medvinsky, A., Rybtsov, S., and Taoudi, S. (2011); Embryonic origin of the adult hematopoietic system: advances and questions. Development 138, 1017-1031.)
In the present invention, we disclose that NOTCH activation promotes EHT (endothelial to hematopoietic transition) from CD144+CD43−CD73− HE and post-transition expansion of blood cells. We have also found that NOTCH induces the arterial type CD144+CD43−CD73-DLL4+ HE (AHE) that express high level of EFNB2 and NOTCH1 arterial markers and MYB gene required for definitive hematopoiesis.
Definitive hematopoiesis produces the entire spectrum of adult-type erythro-myeloid progenitors (EMP), lymphoid cells and cells capable of limited engraftment and HSCs with capacity of long-term repopulation of an adult recipient. Definitive-type hematopoietic progeny with adult-like characteristics are CD144+CD43−CD73−DLL4+ HE that express high level of EFNB2 and NOTCH1 arterial markers and MYB gene. These definitive-type hematopoietic progeny with adult-like characteristics are cells able to give rise to hematopoietic progeny, such as platelet-producing megakaryocytes, adult-globin expressing erythrocytes, multipotential granulocyte/erythrocyte/megakaryocyte/macrophage colony forming cells (CFC-GEMM) and T-lymphocytes.
As described in the Examples, using transgenic reporter H1 human embryonic stem cell (hESC) line in which RUNX1+23 enhancer mediates GFP expression, we found that only DLL4+ HE demonstrated enhancer activity which is typically found in HE at sites of definitive hematopoiesis in mouse and zebra fish embryos (Swiers et al 2013, Tamplin et al 2015s). Hematopoiesis from CD144+CD43−CD73−DLL4+ AHEs requires stroma and is strictly dependent on NOTCH activation.
It is important to note that one aspect of the present invention comprises exposing the CD144+CD43−CD73−DLL4+ AHE to a sufficient amount of a NOTCH activation agent such that the AHE undergo endothelial-to-hematopoietic transition and produce definitive-type hematopoietic progeny with adult-like characteristics. Without sufficient NOTCH activation, the AHE cannot undergo endothelial-to-hematopoietic transition. In one embodiment of the present invention, one may wish to collect the hematopoietic progenitors and place them into specialized differentiation conditions to generate hematopoietic progeny, such as platelet-producing megakaryocytes, adult-globin expressing erythrocytes, CFC-GEMM and T-lymphocytes.
The present invention allows clear commercial advantages. Current methods of generating hematopoietic progenitors from human PSCs do not efficiently produce adult-type hematopoietic progenitors. Many of the hematopoietic progeny are not adult-type and have limited lymphoid potential and maintain embryonic-globin expression in erythrocytes. Here, we describe a method that generates definitive-type (adult-type) hematopoietic progenitors that give rise to progeny with increased T-lymphocyte potential and erythrocytes that express adult-globins. This technology allows us to derive the arterial hemogenic endothelial precursor to facilitate the production of definitive hematopoietic stem cells from human PSCs.
In summary, our disclosure reveals that the activation of NOTCH allows for specification of the arterial type of definitive HE that is the proper precursor for HSC formation in the embryo.
Cells of the Present Invention
In one embodiment, the present invention is a population of arterial hemogenic endothelium cells (AHE) that are CD144+CD43−CD73−DLL4+ HE. Preferably, the cells express high level of EFNB2 and NOTCH1 arterial markers and MYB gene required for definitive hematopoiesis. These cells have broad lympho-myeloid and definitive erythroid potentials.
The present invention involves the creation of cells with definitive potential. Definitive erythroid potential includes the ability to generate red blood cells that express increased levels of adult-type alpha- and beta-globin expression, while hematopoietic progenitors with only primitive erythroid potential only generate erythrocytes that express embryonic (zeta and epsilon) globins. This invention discloses that AHE-derived hematopoietic progenitors have increased potential to generate erythrocytes with increased adult-type alpha- and beta-globins.
Preferably, the population is at least 90%, at least 95% or at least 99% pure.
The ability to specifically derive arterial hemogenic endothelial precursors also allows for the increase in the ability to in vitro differentiate the AHEs into T cells. AHEs derived by the present methods have at least a four (4)-fold increase in T cell potential than prior methods of in vitro differentiation.
Methods of the Present Invention
In one embodiment, the present invention is a method of creating AHE cells. In another embodiment, the present invention is a method of creating various kinds of hematogenic cells by differentiation of AHE cells. The AHE cells in these embodiments may be differentiated from pluripotent stem cells (PSCs) or from AHE isolated from mammalian tissues. Preferred examples of differentiated cells include platelet-producing megakaryocytes, adult-globin expressing erythrocytes, or T-lymphocytes.
The Example below describes exemplary methods to create the AHE of the present invention. However, these methods may be modified, with one or more of the modifications listed below, and still be within the scope of the invention.
As Example 1 discloses, we utilized a modified version of the serum- and feeder-free differentiation system described previously (Uenishi et al., 2014) where we identified developmental stage equivalencies to in vivo development that can be identified by cell-surface antigens and functional assays on specific days of differentiation: Day 2 APLNR+PDGFRα+ Primitive Mesoderm (D2 PM), Day 4 KDRhiPDGFRαlow/−CD31− Hematovascular Mesoderm Precursors (D4 HVMP), Day 4 and 5 CD144+CD43−CD73− Hemogenic Endothelial cells (D4 or D5 HE), and Day 8 CD34+CD43+ Hematopoietic Progenitors (D8 HP) (Choi et al., 2012b). During differentiation, we found that the Notch1 receptor is first expressed at high levels uniquely on D4 HEPs while the Notch ligand, DLL4, is first expressed on D5 within the CD144+ (VE-Cadherin) population (
Therefore, in one embodiment of the present invention, one will isolate D4 HE, preferably by simple magnetic enrichment of CD31+ cells since at this stage, the CD31+ population is entirely CD144+CD43−CD73− (Choi et al., 2012b; Uenishi et al., 2014)). D4 HEs can be isolated by the way disclosed in Example 1 and other equivalent ways, such as FACS.
In some embodiments, the defined conditions comprise culturing the cells with stromal cells, preferably OP9 cells.
In another embodiment, the defined conditions in which PSCs are differentiated to the immature HE cells include the conditions described in Uenishi et al. 2014, incorporated by reference in its entirety. In brief, in one embodiment, the defined conditions and differentiating step comprises (1) exposing the stem cells to a xenogen-free and serum albumin-free mixture comprising components of about 25 ng/ml to about 50 ng/ml FGF2, high levels of BMP4 of at least 50 ng/ml, low levels of Activin A of less than 15 ng/ml, and about 1 mM to about 2 mM LiCl under hypoxic conditions for a period of about two days to form a population of EMHlin-KDR+APLNR+PDGFRalpha+ primitive mesoderm cells without the formation of embryoid bodies or coculture with stromal cell lines and (2) exposing the cells at the hematovascular mesoderm stage of step (1) to a mixture comprising components FGF2, VEGF, IL6, SCF, TPO, and IL3 for about one day to achieve formation of CD144+CD73-CD235a/CD43− immature hemogenic endothelial, and (3) detecting and isolating the CD144+CD73-CD235a/CD43− HE from culture of step (2).
The isolated D4 HE cells may be plated onto an NOTCH activation agent, such as immobilized Notch ligands, to activate NOTCH signaling (Hadland et al., 2015; Ohishi et al., 2002) (See
Examples of suitable Notch ligands include DLL1-Fc (which has been described in other papers as Deltalext-IgG), Jag1 ligand, and DLL4 (see Example 1)). Other examples would include an immobilized synthetic molecule that can bind to NOTCH and sufficiently activate the NOTCH receptor and the ectopic expression of the active, intracellular domain of NOTCH1 (Notch-ICD).
We confirmed by western blot analysis of the active form of Notch1, Notch-ICD, and qPCR analysis of the downstream Notch1 target gene, HES1, by qPCR, these respective conditions efficiently activated NOTCH signaling (
In another embodiment of the present invention, one would differentiate AHE cells into another hematopoietic cell type. Suitable hematopoietic cell types include, T lymphocytes, B-cell, definitive (adult-type) erythrocytes, myeloid progenitors and mature myelomonocytic cells. There are numerous prior art examples of differentiation protocols.
Another embodiment provides a method of differentiating the AHE cells into T cells by culturing the AHEs in T cell differentiation medium with sufficient amount of NOTCH activating agent in order to differentiate the cells into T lymphocytes (T cells). Suitable conditions for differentiating T cells are known in the art. The T cells can be identified as CD4+CD8+. In some embodiments, the T cells are identified as CD7+CD5+, CD8+CD4+, or a combination thereof (CD7+CD5+ and CD8+/CD4+).
In yet another embodiment, the disclosure provides a method of obtaining a cellular composition comprising more than 95% arterial-type hemogenic endothelium (AHE) cell population, comprising the steps of a. differentiating human pluripotent stem cells (hPSCs) for five days in defined conditions to induce formation of CD144+CD43−CD73-Dll4+ arterial HE; and b. detecting and isolating a cell fraction being characterized by CD144+CD43−CD73−DLL4+ phenotype. The defined conditions necessary to differentiate the hPSCs are known in the art, for example, as described in Vodyanik et al. 2005 and Uenishi et al. 2014, the contents of which are incorporated by reference and detailed above. However, other suitable methods known in the art can be used.
In some embodiments, the defined conditions comprise culturing the cells with stromal cells, preferably OP9 cells.
In another embodiment, the defined conditions include the conditions described in Uenishi et al. 2014, incorporated by reference in its entirety. In brief, in one embodiment, the defined conditions and differentiating step comprises (1) exposing the stem cells to a xenogen-free and serum albumin-free mixture comprising components of about 25 ng/ml to about 50 ng/ml FGF2, high levels of BMP4 of at least 50 ng/ml, low levels of Activin A of less than 15 ng/ml, and about 1 mM to about 2 mM LiCl under hypoxic conditions for a period of about two days to form a population of EMHlin-KDR+APLNR+PDGFRalpha+ primitive mesoderm cells without the formation of embryoid bodies or coculture with stromal cell lines and (2) exposing the cells at the hematovascular mesoderm stage of step (1) to a mixture comprising components FGF2, VEGF, IL6, SCF, TPO, and IL3 for about one day to achieve formation of CD144+CD73-CD235a/CD43− immature hemogenic endothelial, and (3) detecting and isolating the CD144+CD73-CD235a/CD43− HE from culture of step (2).
In some embodiments, after step (a), the cells are combined with a detecting agent specific for different cell surface markers, for example, CD144, CD43, CD73 and DLL4, and wherein the detecting agents with different labels are used to separate the cell fraction characterized by CD144+CD43−CD73−DLL4+ phenotype. In a preferred embodiment, the detecting agents are antibodies, for example, monoclonal antibodies with different labels that are specific to the cell surface markers. In an embodiment, the monoclonal antibodies are labeled with different fluorescent labels.
In some embodiments, the different labels are different fluorescent labels or fluorophores. Suitable fluorescent labels or fluorophores are known in the art and include, but are not limited to, for example, dyes green fluorescent protein (GFP), red fluorescent protein (RFP), CFP, Alexa Fluor (available from ThermoFisherScientific, Waltham Mass.), including Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750, BODIPY FL, Coumarin, Cyanine 3 (Cy3), Cyanine 5 (Cy5), Fluorescein (FITC), Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, Tetramethylrhodamine (TRITC), Texas Red, Super Bright dyes including Super Bright 436, Super Bright 600, Super Bright 645, Super Bright 702, among others. Suitable fluorescently labeled detecting agents (including antibodies and monoclonal antibodies) are known in the art and not limited herein. Suitable methods of detection and isolation are known in the art and include, but are not limited to, FACSorting.
In another embodiment of the present invention, one would isolate AHE cells from mammalian cells and further differentiate the AHE as described above.
It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as “comprising” certain elements are also contemplated as “consisting essentially of” and “consisting of” those elements. The term “consisting essentially of” and “consisting of” should be interpreted in line with the MPEP and relevant Federal Circuit's interpretation. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. “Consisting of” is a closed term that excludes any element, step or ingredient not specified in the claim.
The following non-limiting examples are included for purposes of illustration only, and are not intended to limit the scope of the range of techniques and protocols in which the compositions and methods of the present invention may find utility, as will be appreciated by one of skill in the art and can be readily implemented. The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.
This Example demonstrates that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144+CD43−CD73−DLL4+Runx1+23-GFP+ arterial-type HE which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs.
During in vivo development, HSCs emerge by budding from hemogenic endothelium (HE) lining arterial vessels, primarily from the ventral wall of the dorsal aorta 5-7. NOTCH signaling is essential for arterial specification and generation of HSCs 8-11. Notch1−/−, Dll4−/− and Rbpjk−/− mice, which are embryonic lethal, have severe impairment in arterial vasculogenesis, fail to develop the dorsal artery 10, 12, 13 and lack intra-embryonic hematopoiesis. NOTCH signaling is also required for the acquisition of arterial identity in extraembryonic vessels, including the yolk sac vasculature 14, 15. Interestingly, definitive hematopoietic progenitors with lymphoid potential in the yolk sac, umbilical cord and vitelline vessels only emerge within the arterial vasculature 16, 17. In contrast, the primitive extraembryonic wave of erythropoiesis and the first wave of definitive yolk sac erythro-myelopoiesis (EMP), which lack lymphoid potential, are not NOTCH-dependent or specific to arterial vessels 10, 13, 16, 18-20. The lack of venous contribution to HSCs along with the shared requirements of Notch, VEGF, and Hedgehog signaling for both arterial fate acquisition and HSC development 21-25 led to the hypothesis that arterial specification could be a critical prerequisite for HSC formation. However, a direct progenitor-progeny link between arterial specification and definitive hematopoiesis has never been demonstrated. Moreover, demonstration in recent studies that HE represents a distinct CD73− lineage of endothelial cells 26, 27 and that hematopoietic specification is initiated at the HE stage 28-30 raises the question whether NOTCH signaling at arterial sites creates a permissive environment for HSC development following endothelial-to-hematopoietic transition (EHT), or that arterial specification per se is required for HE to become HSCs. Although, recent studies have demonstrated that NOTCH activation induces arterialization of CD73+ non-HE 27, and that NOTCH inhibition with DAPT reduces production of CD45+ cells from CD34+CD43−CD73− HE progenitors 27, 31, the effect of NOTCH signaling on HE specification has never been explored.
Here, using a chemically defined human pluripotent stem cell (hPSC) differentiation system combined with the use of DLL1-Fc and the small molecule DAPT to manipulate NOTCH signaling following the emergence of the well-defined CD144+CD43−CD73− population of HE during EHT, the inventors discovered that NOTCH activation leads to the formation of arterial-type CD144+CD43−CD73−DLL4+ HE (AHE) that expresses arterial markers and possesses definitive lympho-myeloid and erythroid potentials. Using a transgenic reporter H1 hESC line in which the Runx1+23 enhancer mediates eGFP expression, the inventors found that only DLL4+, and not DLL4− HE cells, demonstrated enhancer activity that is typically found in HE at sites of definitive hematopoiesis in mouse and zebra fish embryos.
Hematopoiesis from CD144+CD43−CD73−DLL4+ AHE required stroma and was strictly dependent on NOTCH activation. In contrast, NOTCH modulation has limited effect on EHT from the HE fraction that remains DLL4− following NOTCH activation, indicating that definitive hematopoietic activity segregates to AHE. Together, this Example established a direct progenitor-progeny link between arterialization of HE and embryonic definitive hematopoiesis and revealed that NOTCH-mediated induction of AHE is an important prerequisite for establishing the definitive hematopoietic program from hPSCs.
Immobilized DLL1-Fc Increases NOTCH Signaling in Hemogenic Endothelial Cells and Increases Hematopoietic Activity
In order to determine the direct effect of NOTCH signaling on hematoendothelial differentiation from hPSCs, we utilized a modified version of the serum- and feeder-free differentiation system described previously 35 where the inventors identified developmental stage equivalencies to in vivo development that can be identified by cell-surface antigens and functional assays on specific days of differentiation: Day 2-3 APLNR+PDGFRα+ Primitive Mesoderm (D2 or D3 PM), Day 4 KDRhiPDGFRαlow/− CD31− Hematovascular Mesoderm Precursors (D4 HVMP), Day 4 and 5 CD144+CD43−CD73− Hemogenic Endothelial Cells (D4 or D5 HE), and Day 8 CD34+CD43+ Hematopoietic Progenitors (D8 HP) 26, 35. During differentiation, the inventors found that the NOTCH1 receptor is first highly expressed on D4 HE cells while the NOTCH ligand, DLL4, is first expressed on D5 within the CD144+ (VE-Cadherin) population (
Following the establishment of optimal conditions for EHT culture in defined feeder- and serum-free conditions, the inventors isolated D4 HE by magnetic enrichment of CD31+ cells, since at this stage (
NOTCH Activation Facilitates Endothelial-to-Hematopoietic Transition in Hemogenic Endothelium
The increase in hematopoiesis due to increased NOTCH signaling can be attributed to three reasons: 1) increased EHT, 2) increased hematopoietic expansion or 3) increased survival post-EHT. To evaluate these possibilities, the inventors isolated D4 HE cells and cultured them with DAPT for either 1 day during initiation of EHT (from D4 to D4+1), or throughout the entire culture (D4 to D4+4), followed by kinetic analysis of CD43 and CD144 expression on each day of the culture period (
To further verify that NOTCH activation affects EHT, the inventors also performed a single cell deposition assay of the D4 HE using the OP9 stromal cells and serum-containing medium which support hematoendothelial development from single cells. Using a DOX-inducible DLL4 OP9 cell line (OP9-iDLL4), D4 HE were deposited onto 96-well plates at three different conditions; OP9-iDLL4 with DAPT without DOX-pretreatment (NOTCH inhibition condition), OP9-iDLL4 with DMSO without DOX-pretreatment (control condition), and OP9-iDLL4 with DMSO with pretreatment of DOX (NOTCH activation condition). The inventors found that D4 HE in the NOTCH inhibition condition had a markedly decreased ratio of hematopoietic/endothelial colonies compared to D4 HE cells in the control condition. In contrast, the D4 HE in the NOTCH activation condition had substantially increased ratio of hematopoietic colonies compared to D4 HE in the NOTCH inhibition condition, and a slight increase compared to D4 HE in the control condition (
The inventors also stained the purified D4 HE before plating with CellTracer to track cell proliferation. When analyzed, the cells in each of the three NOTCH conditions on D4+1 showed a significant increase in the proportion of CD144+CD43+ to CD144+CD43″ cells within the first generation of cells in the NOTCH activation condition (+DLL-Fc), when compared to the NOTCH inhibition (+DAPT) condition. This result, in combination with the absence of a second generation on D4+1, suggests that the activation of NOTCH signaling at HE stage potentiate EHT initiation, but not proliferation (
To evaluate whether NOTCH signaling affects apoptosis, the inventors performed Annexin V flow cytometric analysis of HE cultured with DAPT, DMSO or on DLL1-Fc on D4+4. As shown in
Together, these results suggest that NOTCH activation at the HE stage facilitates EHT, but has minimal effect on expansion or survival of blood cells at post-EHT stage.
NOTCH Activation Maintains Multilineage Potential and Increases Definitive Characteristics of Hematopoietic Progenitors Emerging from HE
Next, the inventors determined whether NOTCH has an effect on HPs emerging through the EHT. While NOTCH1 expression decreases among the CD144+ endothelial population from D4+1 to D4+4, CD144−CD43+ blood cells increase and maintain expression of NOTCH1 post-transition from D4+2 to D4+4, notably among the CD34+ subpopulation (
Next, whether increased NOTCH activation increases definitive-type hematopoiesis was determined. Previously, the Runx1+23 enhancer was found to be active in all hematopoietic progenitors, including yolk sac. HE found in regions where definitive hematopoiesis emerges have also been found to activate Runx1+23, including the para-aortic splanchnopleura, AGM region, vitelline and umbilical arteries 32-34, 39, 40. The inventors generated a hESC reporter line with Runx1+23 enhancer driving eGFP expression knocked into the AAVS1 locus (
T cell potential is another hallmark of definitive hematopoiesis (Kennedy et al., 2012a). Comparative analysis of T-cell potential of the D4+4 CD43+ cells from DAPT, DLL1-Fc and control conditions revealed that HPs from the NOTCH inhibition condition had no T-cell potential while HPs from the NOTCH activation condition had significantly increased T-cell potential (
In a separate assay, the inventors collected floating HPs on D4+4 and continued culture in a modified erythrocyte expansion condition (Dias et al., 2011). After 10 days, the inventors collected the cells and isolated mRNA to analyze their globin expression. The inventors found that erythrocytes generated from HPs from the NOTCH activation condition have significantly increased ratios of adult-type β-globin expression to embryonic ε-globin and fetal γ-globin expression, and the ratio of adult-type α-globin expression to embryonic ζ-globin expression, when compared to the erythrocytes generated from HPs from both the NOTCH inhibition condition and the control condition (
NOTCH Activation of Day 4 HE Increases a Transient Population of DLL4+ HE Cells with Arterial Identity
Previously, the inventors identified CD73 expression to demark the loss of hemogenic potential within the D5 CD144+ endothelial population 26. As demonstrated above, D4 HE cells lacked the expression of the arterial marker, DLL4. However, when the inventors analyze CD73 and DLL4 expression within the D4+1 and D4+2 CD144+ populations in each of the three NOTCH conditions, a significant increase in a unique transient population of CD73−DLL4+ endothelial cells in the NOTCH activation condition was found, and a delayed upregulation of CD73 expression on DLL4+ endothelial cells was found, compared to the NOTCH inhibition and control conditions (
To corroborate this hypothesis, the inventors evaluated the expression of arterial, venous and definitive hematopoietic markers by real-time qPCR analysis of sorted D4 CD144+CD43−CD73− HE that are DLL4− by default (D4 HE) and D5 CD144+ endothelial subpopulations CD144+CD43−CD73−DLL4+ (D5 HE:DLL4+), CD144+CD43−CD73−DLL4− (D5 HE:DLL4−), and CD144+CD43−CD73+DLL4− (D5 nonHE:DLL4−), (
Definitive-type Hematopoietic Progenitors Emerge from Arterial-Type Hemogenic Endothelium Upon NOTCH Activation
To determine the hematopoietic potential of endothelium with arterial identity, the inventors continued differentiation of hPSCs to D5 and then sorted the D5 CD144+CD43−CD73−DLL4− (HE:DLL4−) and D5 CD144+CD43−CD73−DLL4+ (HE:DLL4+) endothelial subpopulations (
Next, the inventors determined whether the HPs from each of the D5 HE subsets have differential definitive hematopoietic potential. When the HPs from the D5 HE subpopulations were plated in colony forming medium, the HPs which emerged from the HE:DLL4+ subpopulation cultured on OP9-DLL4 had increased colony forming cells, particularly of GEMM-CFCs compared to the HPs from D5 HE:DLL4− on OP9 and OP9-DLL4 (
When we collected the floating HPs derived from D5 HE:DLL4− on OP9 and OP9-DLL4, and HPs derived from D5 HE:DLL4+ on OP9-DLL4, and continued to grow them in the aforementioned erythrocyte expansion and maturation culture 42, the inventors found that erythrocytes generated from HPs derived from the D5 HE:DLL4+ on OP9-DLL4 have significantly increased ratios of β-globin expression to ε-globin and γ-globin expression, and an increased ratio of α-globin expression to ζ-globin expression, when compared to the erythrocytes generated from HPs derived from D5 HE:DLL4− on OP9 and OP9-DLL4 (
A limiting dilution assay (LDA) for lymphoid potential was also performed and we found that 1 in 14 HPs derived from D5 HE:DLL4+ on OP9-DLL4 have T-cell potential, while 1 in 44 HPs derived from D5 HE:DLL4− on OP9-DLL4 have lymphoid potential. HPs derived from D5 HE:DLL4− on OP9 and D5 HE:DLL4− on OP9 with DAPT had only 1 in 10,706 and 1 in 10,895 cells had T-cell potential, respectively (
In order to determine whether there are any molecular differences between HPs derived from HE:DLL4+ and HE:DLL4− cells, the inventors performed RNA-seq analysis of CD235a/CD41a−CD34+CD43+CD45+ cells generated from these two different hemogenic endothelial cells following tertiary culture on either OP9 or OP9-DLL4 (
RNAseq analysis of NOTCH ligands, receptors and their downstream targets in D5 DLL4+ and DLL4− HE, and HPs obtained from these populations, revealed D5 DLL4+ AHE express greater levels of NOTCH1, NOTCH4, DLL4, and JAG2 as compared to DLL4− HE. However, expression of NOTCH associated molecules and SOX17 was substantially lower in HPs, including HPs generated from DLL4+ AHE on OP9-DLL4, suggesting a downregulation of NOTCH signaling and arterial program following EHT (
Together, these results imply that arterial-type CD144+CD43−CD73−DLL4+ HE represents the precursor of definitive NOTCH-dependent hematopoiesis with broad lympho-myeloid and definitive erythroid potential, while the CD144+CD43−CD73−DLL4− phenotype is associated with emerging immature HE endothelium (D4) or HE that has primitive NOTCH-independent hematopoietic potential (D5).
Discussion
In the current Example, the inventors revealed that NOTCH signaling is essential for specification of definitive lympho-myeloid hematopoiesis by eliciting arterial specification of HE from hPSCs. The inventors demonstrated that NOTCH activation promotes formation of transient CD144+CD43−CD73−DLL4+ HE population with high expression of arterial genes and active Runx1+23 enhancer that mark arterial type HE in AGM, umbilical and vitelline arteries 32, 33, 39, 40, 43. Although CD144+CD43−CD73−DLL4+ AHE have lower hemogenic capacity compared to DLL4− HE, the hematopoietic potential of AHE is strictly NOTCH dependent. AHE is specified from CD144+CD43−CD73−DLL4− immature HE cells emerging on D4 of differentiation in a NOTCH-dependent manner following acquisition of an arterial CD144+CD43−CD73−DLL4+ phenotype, while CD144+CD43−CD73−DLL4− HE cells that failed to undergo arterial specification on day 5 of differentiation retained mostly primitive hematopoietic potential and were minimally affected by NOTCH activation (
In the present study, we provided evidence that NOTCH has several effects on hematopoiesis from HE. First, the inventors demonstrated that NOTCH signaling is important for the specification of arterial-type HE cells with definitive hematopoietic program. In addition, NOTCH activation also potentiates the EHT from these cells, while having little effect on expansion and survival of blood cells at post-EHT stage.
Overall, this Example indicate that regulation of NOTCH signaling would be important to mimic the arterial HE, definitive lympho-myeloid hematopoiesis and HSC specification in hPSC culture.
Human Pluripotent Stem Cell Maintenance and Differentiation
Human pluripotent stem cells, H1 hESC line, DF19-9-7T fibroblast-hiPSC line, IISH2i-BM9 bone marrow-iPSC line, and IISH3i-CB6 cord blood-iPSC line, were maintained and passaged in chemically defined conditions using vitronectin and E8 medium, as previously described 85. The human PSCs were differentiated into hematoendothelial lineages using a modified protocol previously described 35. On Day −1, hPSCs were singularized and plated on collagen IV-coated plates (0.5 μg/cm2) at a cell density of 7,500 cells/cm2 in E8 medium supplemented with 10 uM Rock inhibitor (Y-27632, Cayman Chemicals). On Day 0, the medium was changed to IF9S medium supplemented with BMP4, FGF2 (50 ng/ml), Activin A (15 ng/ml, Peprotech), LiCl (2 mM, Sigma), and ROCK inhibitor (0.5 μM, Cayman Chemicals) and cultured in hypoxia (5% O2, 5% CO2). On day 2, the medium was changed to IF9S medium supplemented with FGF2, VEGF (50 ng/ml, Peprotech), and 2.5 μM TGFβ inhibitor (SB-431542, Cayman Chemicals). On day 4, cell cultures were singularized and stained with anti-CD31 microbeads (Miltenyi) for 15 minutes. Cells were washed and HE were purified using CD31 antibody and MACS LS columns (Miltenyi). Purified CD31+ HE were then plated at a density of 20,000 to 30,000 cells/cm2 on collagen IV-coated plates (1 μg/cm2) that were either co-coated with IgG-Fc fragments or human DLL1-Fc (made in-house), in IF9S medium supplemented with FGF2, VEGF, EGF, IGF-I, IGF-II, TPO, IL-6 (50 ng/ml), SCF (20 ng/ml), IL-3, FLT3L (10 ng/ml, Peprotech), and ROCK inhibitor (5 μM, Cayman Chemicals), and where specified, DMSO (1:1000, Fisher Scientific) or DAPT (10 μM, Cayman Chemicals), and cultured in normoxia (20% O2, 5% CO2). In some experiments, HE was cultured on plates co-coated with human JAG1-Fc (R&D Systems). A sample of the purified cells was analyzed by flow cytometry, and experiments were continued only if the purity of the HE was over 95% CD144+. On Day 4+1, the medium was replaced with fresh medium containing the same supplements without ROCK inhibitor. On day 4+3, extra medium with the same supplements was added to the culture.
OP9 Maintenance and Co-Culture
OP9, OP9-DLL4, and the inducible OP9-iDLL4 (made in-house) cell lines were maintained in αMEM with 20% FBS (GE) on gelatin-coated plates in normoxia as previously described 86. Using TrypLE (Thermo), OP9 were passaged at a 1:8 ratio every 3-4 days when they were 80% confluent. One day before co-culture with differentiated human HE cells, OP9 lines were treated with mitomycin C (1 mg/ml) for 2 hours and then plated at a density of 12,500 cells/cm2 as previously described 87. D4 HE cells or D5 CD144+ subsets were plated onto OP9 lines at a density between 1000 to 2000 cells/cm2 in medium containing αMEM with 10% FBS (GE), TPO, SCF, IL-6 (50 ng/ml), IL-3, and FLT3L (10 ng/ml). Medium was changed after 24 hours, and extra medium added 2 days later. Experiments conducted with DAPT were treated with 20 μM, while corresponding control conditions had DMSO added at a 1:500 dilution.
Generation of OP9-DLL4, OP9-JAG1 and DOX-Inducible OP9-iDLL4
Human DLL4 gene fragment was amplified by PCR from a vector previously used to establish the OP9-DLL4 cell line, and the JAG1 gene was amplified by PCR from cDNA of D5 differentiation cultures that were treated with Sonic Hedgehog from D2-5, which has been found to increase Jag1 expression (data not shown). The DLL4 and JAG1 gene fragments were subsequently cloned into a pSIN-EF1a-DLL4-IRES-Puro and p SIN-EF1a-JAG1-IRES-Puro lentiviral expression vector for the constitutively expressed OP9-DLL4 and JAG1 lines, respectively. Virus production and concentration was carried out by calcium phosphate transfection of Lenti-X 293T cells (Clonetech, Mountain View, Calif.). After 12 hours, virus-containing medium was replaced with fresh OP9 culture medium. After 3 days, cells were treated with Puromycin for 2 weeks. For dox-inducible OP9-DLL4, the DLL4 gene fragment was subsequently cloned into a pPB-TRE-DLL4-P2A-Venus-EF1α-Zeo∥EF1a-M2rtTA-T2A-Puro PiggyBac vector made in house. OP9 cells were then transfected with pPB vector. 3 days later the transfected OP9 cells were treated with Puromycin/Zeocin for 2 weeks. Samples of the OP9-iDLL4 cells were treated with doxycycline for 24 hours, then DLL4 and Venus expression were confirmed by flow cytometry.
Single-Cell Deposition Assay for Endothelial-to-Hematopoietic Transition\
One day before single-cell deposition, the OP9-iDLL4 cell line was treated with mitomycin C as described above, and passaged into 96-well plates at a density of 12,500 cells/cm2. OP9-iDLL4 used for the NOTCH activation condition was incubated with doxycycline for 24 hours after passaging into 96-well plates. On the day of single-cell sorting, OP9-iDLL4 medium was changed to αMEM with 10% FBS (GE), TPO, SCF, IL-6 (50 ng/ml), IL-3, FLT3L (10 ng/ml), and DMSO (1:500) for the control, and NOTCH activation conditions, or DAPT (20 μM) for the NOTCH inhibition condition. Day 4 differentiated human pluripotent stem cells were singularized, stained for CD309-PE and CD144-APC (Miltenyi Biotech), and single-cell sorted into individual wells of the 96-well plates using a FACS Aria II. To exclude possibility of doublets, we used a low density (less than 1 million cells/ml) cells suspension, sorting speed less than 1000 cellular events/per second and stringent gating on single cells using both FSC-A vs FSC-H and SSC-A vs SSC-H. One day after sorting, the medium was changed to fresh medium without DMSO or DAPT, and extra medium was added every 3 days. Seven days later, the plates were fixed and stained for immunofluorescent staining with anti-CD144 (rabbit, eBioscience) and anti-CD43 (mouse, BD Biosciences) primary antibodies and anti-rabbit AlexaFluor488 and anti-mouse AlexaFluor594 secondary antibodies (Jackson Immunology) in order to score the hematopoietic/endothelial colonies.
CellTracer Proliferation Assay and Cell Cycle Analysis
D4 CD31+ HE cells were incubated in PBS with CellTracer (1 μg/ml, Thermo) for 20 minutes at 37° C. After washing, the cells were plated on collagen IV-coated plates with either Fc-IgG or DLL1-Fc and the modified Day 4 medium, as described above, at a higher density of 30,000 to 40,000 cells/cm2 due to toxicity from the CellTracer. Aliquots of the purified cells were analyzed by flow cytometry to determine the purity of the MACS cells and establish the Generation 0 peak for the proliferation assay. Secondary cultures were collected every day after plating for flow cytometry analysis, and calibration beads were used to generate compatible CellTracer results. After D4+4, FlowJo™ Analysis software was used to concatenate the data from each day. The average number of cell divisions was calculated based on the number of cells on each day (
T-Cell Differentiation and T-Cell Limiting Dilution Assay
Total D4+4 cultures were singularized, strained, and cultured in T-cell differentiation conditions on OP9-DLL4 for 3 weeks as described 35. For D5+4 cultures, only the floating hematopoietic cells were collected and cultured in T-cell differentiation conditions. Limiting Dilution Assays were conducted with the floating cells collected from D5+4 cultures (HE:DLL4− on OP9+DAPT, OP9+DMSO, and OP9-DLL4, and HE:DLL4+ on OP9-DLL4). Row A of a 96-well plate received 500 cells/well, and each subsequent row afterwards had half the previous row (Row B contained 250, Row C contained 125 . . . Row H contained 3-4 cells). The wells were scored 2 weeks later by eye and flow-cytometry for CD5+CD7+ containing cells. Positive threshold was set at 167 CD5+CD7+ cells/well. Extreme limiting dilution analysis was conducted using the previously established algorithm 88.
Red Blood Cell Differentiation and Maturation of D4+4 Cultures
In order to assess the definitive erythropoietic potential of hematopoietic progenitor cells, we adopted our previously describe red blood cell differentiation protocol 42 to become chemically defined and feeder- and serum-free. Floating cells were collected, washed, and plated back into their respective cultures for D4+5 cells, or plated onto collagen IV-coated plates for D5+4 cells, with IF9S supplemented with dexamethasone (10 μM), EPO (2 U/ml), SCF, FLT3L, TPO, IL-6 (100 ng/ml), and IL-3 (10 ng/ml). Extra medium with the same supplements was added 2 days later. An additional 2 days later, the cultures were treated with half-medium changes every 2 days with IF9S supplemented with dexamethasone (10 μM), SCF (100 ng/ml), and EPO (2 U/ml). The floating cells were collected 10 days later to analyze by flow cytometry and RNA isolated for qPCR analysis.
Generating Runx1+23 Enhancer Reporter Cell Line
Runx1+23 enhancer fragment 33 was amplified by PCR and subsequently cloned into the AAVS-SA-2A-PURO vector (gift from Gadue Lab, The Children's Hospital of Philadelphia). Human ESCs were transfected with zinc-finger nuclease vectors and later puromycin-resistant individual cells were clonally expanded and on-targeted clones were selected, as previously described 38. Southern Blot (SB) analysis was performed by DIG-labeling hybridization (Roche). Briefly, 10 μg genomic DNA was digested using a EcoRV restriction enzyme for overnight, separated on a 0.7% agarose gel for 6 hours, transferred to a nylon membrane (Amersham), and incubated with DIG-labeling probes. The external probe is a DIG-labeled 600 nucleotide fragment that binds to the EcoRV-digested fragment of the 5′ external region. The internal probe is a DIG-labeled 700 nucleotide fragment that binds to the EcoRV-digested fragment of the of the eGFP region.
Hematopoietic Colony Forming Unit Assay
Hematopoietic colony forming unit assay was conducted in serum-containing H4436 Methocult (Stem Cell Technologies) as previously described 26, 35.
Flow Cytometry and FACS-Sorting
Flow Cytometry was conducted using the MACSQuant 10 (Miltenyi Biotech).
FACS-sorting was conducted on a FACS Aria II (BD) as previously described 26, 35, 86.
Western Blot
Cell extracts were prepared by adding IP Lysis buffer (Thermo Scientific) with protease inhibitor cocktail (Sigma). Cell lysates (10 μg) were separated by 6% SDS-PAGE. Separated proteins were transferred to a PVDF membrane, and were stained with Notch1 and Notch1-ICD antibody (Cell Signaling Technology) and GAPDH (Santa Cruz). Immunoblots were visualized using the ECL PLUS detection kit (Amersham Pharmacia).
qPCR Analysis
Cells were differentiated for the respective days and sorted on a FACS Aria II. RNA was collected using RNA MiniPrep Plus (Invitrogen) and quantified on a NanoDrop (GE Healthcare). Equal amounts of RNA were used for cDNA synthesis using SuperScript III First-Strand Synthesis System (Life Technologies). qPCR was conducted using Platinum SYBR Green qPCR SuperMix (Life Technologies). The reactions were run on a Mastercycler RealPlex Thermal Cycler (Eppendorf) and the expression levels were calculated by minimal cycle threshold values (Ct) normalized to the reference expression of RPL13a. The qPCR products were run on an agarose gel and stained with ethidium bromide to confirm specificity of the primers. Primer sequences can be found in
RNA-Seq Data Processing and Analysis
Total RNA was isolated from the D4 HE, D5 HE:DLL4+ and HE:DLL4− and CD235a/CD41a−CD34+CD45+ derived from HE:DLL4+ and HE:DLL4− cells using the RNeasy mini Plus Kit (Qiagen). RNA purity and integrity was evaluated by capillary electrophoresis on the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, Calif.). One hundred nanograms of total RNA was used to prepare sequencing libraries using the TruSeq RNA Sample Preparation kit (Illumina, San Diego, Calif.). Final cDNA libraries were quantitated with the Qubit Fluorometer (Life Technologies, Carlsbad, Calif.) and multiplexed with eighteen total indexed libraries per lane. Sequencing was performed using the HiSeq 3000 (Illumina, San Diego, Calif.) with a single read of 64 bp and index read of 7 bp.
Base-calling and demultiplexing were completed with the Illumina Genome Analyzer Casava Software, version 1.8.2. Following quality assessment and filtering for adapter molecules and other sequencing artifacts, the remaining sequencing reads were aligned to transcript sequences corresponding to hg19 human genome annotation. Bowtie v 1.1.2 was used, allowing two mismatches in a 25 bp seed, and excluding reads with more than 200 alignments 89. RSEM v 1.3.0 was used to estimate isoform or gene relative expression levels in units of “transcripts per million” (tpm), as well as posterior mean estimate of the “expected counts” (the non-normalized absolute number of reads assigned by RSEM to each isoform/gene) 90, 91. R statistical environment (R core team, 2014) was used at all of the stages of downstream data analysis. The entire set of libraries was pre-normalized as a pool using median normalization routine from EBSeq package 92. EBSeq with 10 iterations was applied to call for differential expression. The EBSeq's default procedure of filtering low-expressed genes was suppressed by setting the QtrmCut parameter to zero. Genes with assigned value of Posterior Probability of Differential Expression above 0.95 were preliminary selected. Subsequently, only genes demonstrating the Critical Coefficent 93 value above 1.5 were retained as differentially expressed.
Statistical Analysis
Statistical analysis was performed in PRISM software. Data obtained from multiple experiments were reported as mean+/−standard error. Where appropriate, either a 1-way ANOVA or 2-way ANOVA were utilized with a Bonferroni post-hoc test. Differences were considered significant when *p<0.05, **p<0.01, or ***p<0.001.
Additional Information
Accession codes: The RNAseq data has been deposited in Gene Expression Omnibus under accession number GSE95028 and GSE96815.
This application is a divisional of U.S. application Ser. No. 15/932,317 filed on Feb. 16, 2018, which claims priority to U.S. Provisional Application No. 62/460,348 filed on Feb. 17, 2016, the contents of which are incorporated by reference in their entireties.
This invention was made with government support under HL099773, HL116221 and OD011106 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62460348 | Feb 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15932317 | Feb 2018 | US |
| Child | 17233643 | US |
