Patent Application
20220090023
The present invention relates to a method for regulating potency of pluripotent stem cells (PSCs) by modulating expression of podocalyxin-like protein 1 (PODXL) and cholesterol and applications thereof.
Human embryonic stem cells (hESCs), generated from the inner cell mass of the early embryo have the ability to unlimited proliferation and differentiate into endoderm, mesoderm, and ectoderm, and potentially into all cell types except placenta (Thomson et al., 1998). hESCs behave like epiblast cells and claimed as the primed state (Brons et al., 2007; Kumari, 2016; Nichols and Smith, 2009; Tesar et al., 2007). Switching the culture medium can change the primed state ESCs into naive-like state. Naïve stem cells are less differentiated and able to form chimeras in mouse (Chan et al., 2013; Gafni et al., 2013; Guo et al., 2016; Takashima et al., 2014; Takeda et al., 2000; Theunissen et al., 2014; Wang et al., 2014; Ware et al., 2014). Two papers published in 2017 in Cell and Nature claimed the extended pluripotent stem cells (EPSCs) by culturing cells with the presence of 4 to 7 chemicals 1 (Yang et al., 2017a; Yang et al., 2017b). EPSCs behave like two to four cell stage of the embryos. They contribute to inner cell mass with much higher efficiency compare to naïve stem cells and can also distribute to trophoectoderm in the mouse model (Yang et al., 2017a; Yang et al., 2017b).
The potential of ESCs in regenerative medicine is enormous, but it rises immunorejection problems. Induced pluripotent stem cells (iPSCs), which turn somatic cells into ESC-like cells by Oct4, Sox2, Myc and Klf4 (or Oct4, Nanog, Sox2, and Lin28) becomes a promising approach for regenerative medicine. (Okita et al., 2007; Park et al., 2008; Takahashi et al., 2007; Wernig et al., 2007; Yu and Thomson, 2008; Zhao and Daley, 2008). IPSCs have the same characters while compare to ESCs, it proliferates unlimited, has pluripotency, and forms teratoma upon ectopic injection. iPSCs undergoing clinical trial for the patients of macular dystrophy, Parkinson disease, and heart disease.
With respect to PSC renewal, in numerous papers, the transcription factor have been studied like Oct4, Sox2, Nanog, Klf4, and c-Myc (Dunn et al., 2014; Hu et al., 2009; Jaenisch and Young, 2008; Jiang et al., 2008; Kagey et al., 2010; Leeb et al., 2010; Silva et al., 2009; van den Berg et al., 2010; Young, 2011). However, the transmembrane proteins have not been studied in detail. Only a few factors like EpCAM (Kuan et al., 2017) and E-cadherin (Chen et al., 2011), and C90RF135 have been studied in mouse ESCs or hESCs (Zhou et al., 2017).
TRA-1-60 and TRA-1-81 are widely used and are the golden standard markers of undifferentiated hESCs (Andrews, 2011; Muramatsu and Muramatsu, 2004). TRA-1-60 and TRA-1-81 are the glycan epitopes of podocalyxin protein (PODXL, also designated as podocalyxin like protein-1, MEP21, PCLP1, Gp200/GCTM-2, and Thrombomucin). TRA-1-60, of note, can be used to recognize the fully reprogrammed iPSCs from partially reprogrammed cells (Chan et al., 2009). In contrast, the well-known transcriptional factor NANOG cannot use to mark the fully reprogramming cells (Chan et al., 2009). PODXL is highly expressed in hESCs at the undifferentiated state (Brandenberger et al., 2004; Cai et al., 2006; Kang et al., 2016). The expression levels are high as the house keeping genes actin (Kang et al., 2016). PODXL expression levels are higher than the core transcriptional factor and OCT4, SOX2, and NANOG. Cytotoxic antibody to PODXL can kill the oncogenic undifferentiated ESCs/iPSCs (Choo et al., 2008; Kang et al., 2016; Tan et al., 2009).
However, the importance of cholesterol in human pluripotent stem cell (hPSCs) remain elusive.
In this present invention, it is unexpectedly found that the potency of pluripotent stem cells (PSCs) can be regulated via modulating expression of podocalyxin-like protein 1 (PODXL). PODXL is essential for the EPSC and iPSC reprogramming. Through microarray results, we found cholesterol biosynthesis pathway, is the downstream of PODXL to maintain hESC/iPSC/EPSC renewal. ESCs are more sensitive to cholesterol inhibitor simvastatin/AY9944/MβCD while compare to fibroblasts, bone marrow mesenchymal stem cells (BMMSCs), and hESC derived neural stem cells (NSCs), which are three differentiated cell types. The PODXL-cholesterol pathway is the upstream of oncogene c-MYC and an immortalize gene telomerase (TERT). PODXL and cholesterol also regulated the lipid raft formation. These data point out PODXL is a protein that orchestrates the cholesterol metabolism transmitting from the membrane in ESCs/iPSC renewal.
Therefore, in one aspect, the present invention provides a method for regulating potency of pluripotent stem cells, comprising exposing the stem cells to an effective amount of a PODXL modulator.
In some embodiments, the modulator is a PODXL antagonist. Specifically, a PODXL antagonist as described herein is effective in downregulating the potency of the pluripotent stem cells.
In some embodiments, the PODXL antagonist is anti-PODXL antibody, an interfering nucleic acid targeting PODXL, or a small molecule that inhibits PODXL.
In some embodiments, the PODXL antagonist is an inhibitor of cholesterol synthesis.
In some embodiments, the stem cells are cultured in a culture medium free of cholesterol.
In some other embodiments, the modulator is a PODXL agonist. Specifically, a PODXL agonist as described herein is effective in upregulating the potency of pluripotent stem cells e.g. the ESCs/iPSCs/EPSCs.
In one further aspect, the present invention provides a method for preparing differentiated cells, comprising
(a) subjecting undifferentiated pluripotent stem cells to a condition suitable for differentiation to produce a cell population that comprises differentiated cells and undifferentiated pluripotent stem cells;
(b) removing the undifferentiated pluripotent stem cells by exposing the cell population to an effective amount of a PODXL antagonist or an inhibitor of cholesterol synthesis; and
(c) optionally culturing the remaining differentiated cells.
In some embodiments, the undifferentiated pluripotent stem cells are selected from the group consisting of embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), and extended pluripotent stem cells (EPSCs).
In some embodiments, the differentiated cells are selected from the group consisting of osteoblasts, adipocytes, chondrocytes, endothelial cells, neuron cells, oligodendrocytes, astrocytes, microglial cells, hepatocytes, heart cells, lung cells, intestine cells, blood cells, gastric cells, ovary cells, uterus cells, bladder cells, kidney cells, eye cells, ear cells, mouth cells, and adult stem cells (all the differentiated cell type).
Also provided is use of a PODXL modulator as described herein for performing the method of the present invention, including a method for regulating potency of pluripotent stem cells and a method for preparing differentiated cells. Further provided is a composition comprising a PODXL modulator as described herein for performing the method of the present invention, including a method for regulating potency of pluripotent stem cells and a method for preparing differentiated cells
The present invention also provides a method for treating teratoma in a subject in need, comprising administering to the subject an effective amount of a PODXL antagonist or an inhibitor of cholesterol synthesis.
The present invention further provides a method for upregulating potency of pluripotent stem cells, comprising inducing expression of podocalyxin-like protein 1 (PODXL) in the stem cells.
In some embodiments, the expression of PODXL is induced by (a) introducing to the stem cells a recombinant nucleic acid sequence comprising a gene encoding PODXL and (b) culturing the stem cells under conditions which allows expression of PODXL.
In some embodiments, PODXL agonist such as chemicals, growth factor, intracellular protein can upregulate the expression of PODXL.
In some embodiments, the pluripotent stem cells as described herein can be EPSC, ESC and/or iPSC.
In another aspect, the present invention provides a method for promoting the efficiency of chimerism in the embryo, comprising contacting a fertilized embryo of a non-human host with a human extended pluripotent cell (hEPSCs) that comprises a recombinant polynucleotide encoding PODXL and culturing the host embryo in contact with the hEPSCs wherein the PODXL is overexpressed to form a chimeric embryo.
In some embodiments, the contact is performed by injecting the hEPSCs into the host embryo.
In some embodiments, the host embryo generates from animals such as dogs, cats and the like), farm animals (such as cows, sheep, pigs, horses and the like), or laboratory animals (such as rats, mice, guinea pigs and the like).
In some embodiments, the method further comprises transplanting the chimeric embryo to a pseudopregnant non-human female recipient animal of the same species as the non-human host to allow an offspring to be produced, and optionally obtaining a humanized organ from the offspring.
Further, it is found in the present invention that cholesterol can boost the reprogramming efficiency of somatic cells, such as skin cells e.g. fibroblast. Therefore, the present invention provides a method for generating pluripotent stem cells (iPSCs) comprising culturing somatic cells in a condition which allows a proportion of the skin cells to dedifferentiate into iPSCs, wherein the condition comprises a culture medium which comprises cholesterol. In some embodiments, the somatic cells are genetic engineered, for example, by introduced with a recombinant nucleic acid, to overexpress one or more reprograming factors, for example, OSKM including Oct4, Sox2, Klf4, and cMyc. Also provided is use of cholesterol for treating somatic cells for generating pluripotent stem cells (iPSCs) therefrom via reprograming. Further provided is a composition e.g. medium composition comprising cholesterol and a basic medium which is useful in treating somatic cells for generating pluripotent stem cells (iPSCs) therefrom via reprograming.
The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.
In the drawings:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.
As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a component” includes a plurality of such components and equivalents thereof known to those skilled in the art.
The term “comprise” or “comprising” is generally used in the sense of include/including which means permitting the presence of one or more features, ingredients or components. The term “comprise” or “comprising” encompasses the term “consists” or “consisting of.”
The term “about” as used herein means plus or minus 5% of the numerical value of the number with which it is being used.
As used herein, the term “pluripotent stem cells” or “undifferentiated pluripotent stem cells” refer to cells that are capable of self-renewal and pluripotent. The term “pluripotent” means the ability of a cell to differentiate into all cell lineages. Specifically, pluripotent cells include those that can differentiate into the three main germ layers: endoderm, ectoderm, and mesoderm. In general, undifferentiated pluripotent stem cells are embryonic stem cells (ESCs), which may be derived from embryonic sources e.g. pre-embryonic, embryonic before day 8 of embryo after fertilization. Undifferentiated pluripotent stem cells can also include induced pluripotent stem cells (IPSCs) that are artificially derived from non-pluripotent cells (e.g., somatic cells) by insertion of one or more specific genes or by stimulation with chemicals. The induced pluripotent stem cells are considered the same as pluripotent stem cells (e.g., embryonic stem cells) in that the induced pluripotent stem cells have the two unique characteristics i.e. self-renewal capacity and pluripotency as well. Undifferentiated pluripotent stem cells also included extended pluripotent stem cells (EPSCs). EPSCs can differentiate into trophoectoderm and inner cell mass upon embryo injection. ESCs and IPSCs are capable of forming teratoma. Human ESCs, IPSCs, or EPSCs are further known to express certain cell markers such as Nanog, Oct4, Sox2, TRA-1-60, TRA-1-81, alkaline phosphatase.
As used herein, the term “potency” may typically include a cell's ability to differentiate into other cell types. The more cell types a cell can differentiate into, the greater its potency. In some instances, the term “potency” may also generally include a cell's self-renewal capacity and/or growth/proliferation/survival ability.
As used herein, the term “extended cell potency” refers to the ability of a stem cell to differentiate into at least one cell type more that of a corresponding cell.
As used herein, the term “extended pluripotent stem cells (EPSCs)” may refer to a pluripotent stem cell with an improved ability to generate extraembryonic lineages in vivo, when compared to ESCs and iPSCs from which it is derived (Yang et al., 2017a; Yang et al., 2017b). EPSCs are generated from the treatment of ESCs/iPSCs with 4 to 7 chemicals (Yang et al., 2017a; Yang et al., 2017b). Specifically, EPSCs mimic the two to four cell stage of embryos and can contribute to both inner cell mass and trophectoderm (placenta). The EPSCs have superior ability to form chimeras in inner cell mass compare to naïve stem cells. Human naïve stem cells can be generated with naïve induction medium (Chan et al., 2013; Gafni et al., 2013; Guo et al., 2016; Takashima et al., 2014; Takeda et al., 2000; Theunissen et al., 2014; Wang et al., 2014; Ware et al., 2014). Both naïve and EPSC can contribute to chimerism in the mouse model, but not prime human ESCs/iPSCs that culture in regular medium.
As used herein, the phrase “regulating potency” of stem cells may include upregulating or downregulating one or more particular features of a cells' potency. For example, upregulating potency of stem cells may include enhancing pluripotency and/or promoting self-renewal capacity/growth/proliferation/survival of the cells via an upregulating approach (e.g. contacting with cells with a PODXL agonist), and downregulating potency of stem cells may include decreasing pluripotency and/or inhibiting self-renewal capacity/growth/proliferation/survival of the cells, via an downregulating approach (e.g. contacting the cells with a PODXL antagonist), when compared to the same cells without such approach.
As used herein, the term “differentiation” refers to a process for differentiating pluripotent stem cells into progeny that are enriched for cells of a particular form or function. Differentiation is a relative process. Mature somatic cells e.g. osteoblasts (bone), chondrocytes (cartilage), adipocytes (fat), hepatocytes (liver), endothelial cells, neuron cells, oligodendrocytes, astrocytes, microglial cells, hepatocytes, heart cells, lung cells, intestine cells, blood cells, gastric cells, ovary cells, uterus cells, bladder cells, kidney cells, eye cells, ear cells, mouth cells, adult stem cells, (all the differentiated cell type) can be terminally differentiated that already lose the ability to differentiate into different cell types under spontaneous condition.
As used herein, the term “remove” or “eliminate” when used with respect to undifferentiated pluripotent stem cells, refers to isolation or separation of such cells from other components in the original sample or from components in the sample that are remaining after one or more steps of processing. The other components for example can include other cells, particularly differentiated cells. The removal or elimination of a target cells may include kill, suppress or deplete the target cells in the samples by applying the compound as used herein, for example, such that other components such as differentiated cells are enriched in the sample. Killing a target cell can include causing apoptosis or cytotoxicity to the cells. Suppressing or depleting a target cell may include a decrease in the number, proportion, proliferation or activity (pluripotent ability or tumor formation activity) by a measurable amount. The removal can be partial or complete. As used herein, a sample or a culture that are substantially free of undifferentiated pluripotent stem cells, for example, can contain less than about 10%, about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or undetectable undifferentiated pluripotent stem cells.
As used herein, the term “culture” refers to a group of cells incubated with a medium. The cells can be passaged. A cell culture can be primary culture which has not been passaged after being isolated from the animal tissue, or can be passaged multiple times (subculture one or more times).
As used here, the term “subject” as used herein includes human and non-human animals such as companion animals (such as dogs, cats and the like), farm animals (such as cows, sheep, pigs, horses and the like), or laboratory animals (such as rats, mice, guinea pigs and the like).
As used herein, the term “treating” as used herein refers to the application or administration of a composition including one or more active agents to a subject afflicted with a disorder, a symptom or conditions of the disorder, or a progression of the disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptoms or conditions of the disorder, the disabilities induced by the disorder, or the progression of the disorder.
As used herein, the term “effective amount” used herein refers to the amount of an active ingredient to confer a biological effect in a treated cell or subject. The effective amount may change depending on various reasons, such as treatment route and frequency, body weight and species of the cells or individuals receiving said active ingredient.
Podocalyxin-like protein 1 (PODXL) is a cell surface glycoprotein belonging to the CD34 family that is encoded by a PODXL gene. Specifically, a human PODXL comprises the amino acid sequences as set forth in SEQ ID NO: 1 and a PODXL gene encoding the human PODXL comprises the nucleic acid sequence of SEQ ID NO: 2.
As used herein, a modulator of PODXL refers to an agent, a substance or a molecule when treating a cell can upregulate or downregulate the PODXL expression in the cell. Specifically, a PODXL agonist includes an agent, a substance or a molecule when treating a cell can upregulate (enhance) the PODXL expression level in the cell, as compared to that of a control cell (without treatment of the agonist). A PODXL antagonist includes an agent, a substance or a molecule when treating a cell can downregulate (reduce) the PODXL expression level in the cell, as compared to that of a control cell (without treatment of the antagonist).
According to the present invention, it is found for the first time that a PODXL modulator can be used to regulate the potency of pluripotent stem cells. In some embodiments, a PODXL agonist is used to upregulate (enhance) the potency of pluripotent stem cells. In some embodiments, a recombinant nucleic acid molecules encoding PODXL is introduced into stem cells to overexpress PODXL in the cells which then exhibit upregulated (enhanced) potency of pluripotent stem cells.
In other embodiments, a PODXL antagonist is used to downregulate (reduce) the potency of pluripotent stem cells. Specifically, a PODXL antagonist can be anti-PODXL antibody, an interfering nucleic acid targeting PODXL, or a compound that inhibits PODXL. In some particular instances, a PODXL antagonist as used herein is an inhibitor of cholesterol synthesis.
In particular embodiments, the method of the present invention is to remove undifferentiated pluripotent stem cells from a culture sample by exposing said sample to an effective amount of a PODXL antagonist.
In particular embodiments, the method of the present invention is to prepare differentiated cells where undifferentiated pluripotent stem cells are cultured in a condition suitable for differentiation to produce a cell population that comprises differentiated cells and undifferentiated pluripotent stem cells, and the undifferentiated pluripotent stem cells are removed/killed by exposing the cell population to an effective amount of a PODXL antagonist or an inhibitor of cholesterol synthesis; and optionally the remaining differentiated cells are cultured in a suitable condition, for example, allowable to achieve a sufficient cell number for cell therapy.
In some embodiments, undifferentiated pluripotent stem cells are selected from the group consisting of embryonic stem cells (ESCs) and induced pluripotent stem cells (IPSCs). Preferably, the pluripotent stem cells are sourced from humans. Human ESCs can be obtained from human blastocyst cells using the techniques known in the art. Hunan IPSCs can be prepared by isolating and culturing suitable somatic donor cells, for example, human fibroblasts or blood cells, and subjected to genetic engineering using techniques known in the art.
In some embodiments, the culture medium suitable for culturing undifferentiated pluripotent stem cells and/or differentiated cells according to the present invention are available in this art, such as DMEM, MEM, DMEM/F12, or IMEM medium with 20% fetal bovine serum or 20% knockout serum. The culture can be carried out at in a normal condition, for example, 37° C. under 1-5% CO2. Differentiation may be promoted by adding a medium component which promotes differentiation towards the desired cell lineage. In certain embodiments, a proper culture medium as used herein is a commercial medium free of cholesterol.
In some embodiments, the culture medium contains DMEM/F12, AlbuMAX I, N2 supplement, nonessential amino acids (NEAA).
In some embodiments, the culture medium can comprise one or more growth factors and/or culture supplements in favor of EPSC induction. Examples of culture supplements include but are not limited to N2, B27, DMEM/F12, Neurobasal medium, GlutaMAX, nonessential amino acids, β-mercaptoethanol and knockout serum replacement, recombinant human LIF, CHIR 99021, IWR-1-endo, (S)-(+)-Dimethindene maleate, Minocycline hydrochloride, and Y-27632.
By treatment with a PODXL antagonist, residual undifferentiated pluripotent stem cells can be selectively killed and removed from their differentiated progenies, so that a sample comprising the differentiated progenies after removing residual undifferentiated pluripotent stem cells can be applied in cell therapy with reduced tumorigenic risk. Particularly, alive undifferentiated pluripotent stem cells after treatment with a PODXL antagonist is in an amount less than that of a control (e.g. the same cells without such treatment) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. More particularly, the removal is complete; namely, undifferentiated pluripotent stem cells after such treatment are completely killed and no residual undifferentiated pluripotent stem cells are detectable.
In addition, the present invention also provides a method for treating teratoma in a subject in need, comprising administering to the subject an effective amount of a PODXL antagonist or an inhibitor of cholesterol synthesis.
In some embodiments, a PODXL antagonist or the inhibitor of cholesterol synthesis is selected from the group consisting of simvastatin [(1S,3R,7S,8S,8aR)-1,2,3,7,8,8a-Hexahydro-3,7-dimethyl-8-[2-[(2R,4R)-tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl]ethyl]-1-naphthalenyly-2,2-dimethyl butanoate], AY9944 (trans-N,N-bis[2-Chlorophenylmethyl]-1,4-cyclohexanedimethanamine dihydrochloride), MBCD (Methyl-β-cyclodextrin methyl-β-cyclodextrin cyclomaltoheptaose, methylether), pracastatin, atorvastatin, pitavastatin, rovasimibe, VULM 1457, YM750, U 18666A, CI 976, Ro 48-8071 fumarate, AK 7, BMS 795311, Lalistat 1, Atorvastatin, rosuvastatin, fluvastatin, Lovastatin, SB 204990, Filipin III, GGTI 298, Torcetrapib, Orli stat, ezetimibe, Alirocumab, Evolocumab, Bococitumab, niacin, amlodipine.
According to the present invention, it is found that activation of PODXL can enhance the potency of stem cells, especially extended pluripotent stem cell (EPSC) and thus a chimeric embryo can be prepared in a more efficient manner.
In particular embodiments, the method of the present invention is to prepare a chimeric embryo comprising contacting a fertilized embryo of a non-human host with a human EPSC that comprises a recombinant polynucleotide encoding PODXL, and culturing the host embryo in contact with the hEPSCs wherein the PODXL is overexpressed to form a chimeric embryo. Specifically, the human EPSC is injected into the host fertilized embryo. The chimeric embryo as prepared can be transplanted into a pseudopregnant non-human female recipient animal of the same species as the host to allow an offspring to be produced, and an organ can be collected from the offspring which can be transplanted to a subject in need for purpose of therapy.
The present invention also provides use of a PODXL modulator e.g. a PODXL agonist or a PODXL antagonist or a composition e.g. a medium composition for performing the method of the present invention, including a method for regulating potency of pluripotent stem cells and a method for preparing differentiated cells.
The present invention further provides a method for generating pluripotent stem cells (iPSCs) comprising culturing somatic cells in a condition which allows a proportion of the skin cells to dedifferentiate into iPSCs, wherein the condition comprises a culture medium which comprises cholesterol. In some embodiments, the somatic cells are genetic engineered, for example, by introduced with a recombinant nucleic acid, to overexpress one or more reprograming factors, for example, OSKM including Oct4, Sox2, Klf4, and cMyc. Also provided is use of cholesterol for treating somatic cells for generating pluripotent stem cells (iPSCs) therefrom via reprograming. Further provided is a composition e.g. medium composition comprising cholesterol and a basic medium which is useful in treating somatic cells for generating pluripotent stem cells (iPSCs) therefrom via reprograming. In particular, the cholesterol is present in the composition in an amount effective in reprograming somatic cells to iPSCs
The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Except for the well characterized function in tumor metastasis, transmembrane glycoprotein podocalyxin-like protein 1 (PODXL, also named as podocalyxin like protein-1, PCLP1, MEP21, Gp200/GCTM-2, and Thrombomucin), function in hPSCs is not known. Here, we demonstrate the knockdown of PODXL in undifferentiated hPSCs significantly inhibited the self-renewal abilities that currently block the expression of c-MYC and telomerase proteins. Of note, the induction or reprogramming of induced pluripotent stem cells (iPSCs) and extended pluripotent stem cells (EPSCs) were severely blocked upon the knockdown of PODXL. Consistently, upregulation of PODXL facilitated hPSC renewal, enhance the expressions of c-MYC and telomerase, and promote iPSC/EPSC formation. In a microarray analysis, overexpression of PODXL activate HMGCR expression, which control the cholesterol biosynthesis. We found that PODXL also upregulates SREBP1/2 expression. Of note, hPSCs are more sensitive to cholesterol inhibitor and lipid raft disruption that results in the inhibition of self-renewal and survival abilities. Cholesterol can fully rescue shPODXL knockdown-mediated pluripotency loss in a dose-dependent manner. Cholesterol also obviously rescue the expression of TERT, c-MYC, and HMGCR that downregulated by shRNA. Our data highlight the importance of PODXL in regulating cholesterol metabolism to control hPSC self-renewal.
1. Material and Methods
1.1 Culture of Primed hPSCs
The HUES6 (S6) cell line was a gift kindly obtained from Dr. Douglas A. Melton's laboratory (Harvard University, Boston, Mass., USA) (Cowan et al., 2004). The WA09 (H9) was obtanined from WiCells (Madison, Wis., USA) (Thomson et al., 1998). The iPSC-0102 and iPSC-0207 cell lines were brought from Food Industry Research and Development Institute (Taiwan).
For feeder-free experiments, cells were cultured in a chemically defined medium (Essential 8 medium).
1.2 Culture of Human EPSCs
Human EPS cells were maintained in N2B27-LCDM medium under 5% CO2 at 37° C. For 400 mL of N2B27-LCDM, it includes 193 mL DMEM/F12 (Thermo Fisher Scientific, 11330-032), 193 mL Neurobasal (ThermoFisher Scientific, 21103-049), 2 mL N2 supplement (Thermo Fisher Scientific, 17502-048), 4 mL B27 supplement (Thermo Fisher Scientific, 12587-010), 1% GlutaMAX (Thermo Fisher Scientific, 35050-061), 1% nonessential amino acids (Thermo Fisher Scientific, 11140-050), 0.1 mM mercaptoethanol (Sigma, M3148), and 5% knockout serum replacement (Thermo Fisher Scientific, A3181502) recombinant human LIF (10 ng/ml, Peprotech, 300-05), CHIR 99021 (1□M; LC laboratories, C-6556), IWR-1-endo (1□M; Abmole, M2782), (S)-(+)-Dimethindene maleate (DiM, 2QM; Tocris, 1425) and Minocycline hydrochloride (MiH, 2QM; Tocris, 3268), Y-27632 (2 uM, LC laboratories, Y-5301). Human EPSCs were passaged on mitomycin C inactivated mouse embryonic fibroblasts (MEF) (3*104 cells per cm2).
As for feeder-free condition, hPSC s were cultured in N2B27-LCDM medium in the absence of 5% KSR for one day before lentivirus transduction.
1.3 Embryoid Body Formation
To form embryoid bodies (EBs), hESCs were detached and the cell clumps were passaged in hPSC medium without bFGF for 13 days.
1.4 Alamar Blue Assay and Trypan Blue Exclusion Assay
hESCs were cultured with Essential 8 medium (Thermo Fisher, A1517001) which containing 15% Alamar blue at 37° C. for 5 hours. The activities were calculated by measuring absorbance at 570 nm and 600 nm. To count the cell numbers with trypan blue assay, cells were treated with trypsin and the suspended cells were mixed 0.2% trypan blue (1:1) and counted with a hemocytometer.
1.5 Crystal Violet Staining Assay
hESCs were fixed with 4% (v/v) paraformaldehyde for 10 minutes at room temperature. Cells were stained with 0.1% crystal violet for 10 min. After washing with PBS, extraction solution were added. The absorbance was measured at 590 nm.
1.6 Alkaline Phosphatase Activity and Staining Assay
Alkaline phosphatase (ALP) activities were calculated by adding the substrate of ALP, p-nitrophenyl phosphate (pNPP) (N7653, sigma), in the culture medium. The plate were incubated at 37° C. less than 5 minutes, then the absorbance was measured at 405 nm. For Alkaline phosphatase (ALP) staining, hPSCs were first washed with PBS and used 4% formaldehyde as a fixative. After fixing for 3 minutes, the cells were washed with 1×PBS and were stained by ALP staining reagent (Sigma). Then the cells were further washed by PBS.
1.7 Lentivirus Production and hESC Transduction
Lentivirus production was executed as previously described with some modifications (Huang et al., 2014). In brief, HEK293T cells were seeded (7.5 million per 10-cm dish). Then cells were transfected with the following plasmids (19.2 μg). cDNA of PODXL, shPODXL (shPODXL #1: TRCN0000310117, 5′-ACGAGCGGCTGAAGGACAAAT-3′ (SEQ ID NO: 3); shPODXL #2: TRCN0000117019, 5′-GTCGTCAAAGAAATCACTATT-3′(SEQ ID NO: 4)) (National RNAi Core Facility, Taipei, Taiwan) and the vector controls. 15.6 μg helper plasmids (pCMV-dR8.91: pMD. G=10:1 (w/w) was added. After 24 hours, medium was changed with fresh medium that contains 1% BSA. The supernatant was collected and filtered through a 0.45 m filter. For lentivirus transduction, cells were seeded on matrigel precoated plates, incubated with the lentivirus with the presence of 8 ug/ml protamine sulfate.
1.8 Reprogramming Somatic Cells to Generate hiPSC
Human foreskin fibroblasts (ATCC® CRL-2097™) were co-infected with pRRL. PPT. SF.hOKSM.idTomato.preFRT lentivirus and which is obtained from Dr. Axel Schambach (Warlich et al., 2011), and with PODXL overexpression or shRNA lentivirus. On days 1-3 post-infection, cells were changed daily with induction media (DMEM, 10% FBS, 250 uM sodium butyrate, and 50 ug/ml ascorbic acid). On day 4 post-infection, cells were passaged onto Matrigel-coated plates. Cells were cultured with induction media for 6 days and changed to half of induction media and half of mTeSR1 (STEM CELL, 85850) with 250 uM sodium butyrate and 50 ug/ml ascorbic acid. For day 7 to 16, transfected cells were changed daily with mTeSR1.
1.9 sgRNA Design and Subclone
MIT CRISPR design (http://crispr.mit.edu) was performed to design the sgRNA that has less off-target effect. sgRNAs were designed to target the sequence at 5′UTR and intron 1 of PODXL locus. sgRNA1 is located at −205 from TSS site. sgRNA2 is located −58 from TSS site and sgRNA3 is located at +460 from TSS site. Cas9 sgRNA vector (Addgene #68463) was cut with BbsI and gel purified. A pair of oligo nucleotides including targeting sgRNA sequences was denatured, annealed and ligated into the Cas9 sgRNA vector.
1.10 Genomic Deletion Assay
HEK293T cells were co-transfected with sgRNA pairs (sgNRA1+sgRNA3) and (sgRNA2+sgRNA3) and with wild type Cas9 plasmid. After transfection for 3 days, genomic DNA were collected. For genotyping, 100 ng of genomic DNA were added into a 25 ul of PCR reaction mix (KAPA HiFi Hotstart PCR).
1.11 Inducible CRISPR Line Production in iPSCs
The inducible iPSC lines with a Doxycyline inducible Cas9 stably integrated at the AAV site (CRISPRn Gen 1C iPSC lines) were generated and obtained from Bruce R. Conklin's lab (Mandegar et al., 2016). After 24 hours, fresh StemFlex medium with (2 uM) or without doxycycline (as solvent control group) were added for 24 hours to induce Cas9 gene expression. Then iPSC lines were co-transfected with different pairs of sgRNAs (sgRNA1+sgRNA3, or sgRNA2+sgRNA3) and a Blasticidin expressing vector (pLAS3W-GFP-Blasticidin) using TransIT®-LT1 Transfection Reagent (Mirus Bio, MIR 2304). After 24 hour of transfection, medium were switch to E8 medium. The cells were selected with 2.5 ug/ml Blasticidin for 1 days, and then refreshed the medium every day with 5 ug/ml Blasticidin in the presence or absence of doxycycline.
1.12 RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
Total RNA was purified with TOOLSmart RNA Extractor (Biotools, DPT-BD24). Reverse transcription was performed with Super Script III System (Invitrogen, 18080051). Quantitative real-time PCR was performed using KAPA SYBR FAST PCR Master Mix (KAPA Biosystems, KR0389) with an ABI7900 Sequence Detection System. The data were quantified using the delta-delta CT method. The samples were normalized against HPRT mRNA levels control.
1.13 Western Blot Analysis
Whole-cell protein extracts were purified from hPSCs or using RIPA lysis buffer (1% NP40, 50 mM Tris, pH 8.0, 150 mM NaCl, 2 mM EDTA) with the presence of protease inhibitor cocktail (Roche, 04693132001). Protein concentration was quantified by Bio-Rad Bradford Protein Assay. Equal amounts of protein were subjected to a 10% SDS-PAGE gel and blotted onto 0.22 um PVDF membrane (Millipore, ISEQ00010). Blots were blocked in 5% BSA/TBST at room temperature for 1 hr. The blots were incubated with the primary antibodies in 5% BSA/TBST at 4° C. overnight. These antibodies include: anti-PODXL (1:1000; Santa Cruz, sc-23904), anti-TRA-1-60 (1:1000, Santa Cruz, sc-21705), anti-TRA-1-81 (1:500, Santa Cruz, sc-21706), anti-c-MYC (1:1000; Abcam, ab32072), anti-OCT4 (1:1000; Cell Signaling Technology), anti-KLF4 (1:1000; Abcam, ab72543), anti-TERT (1:1000; Abcam, ab183105), anti-HMGCR (1:1000; Abcam, ab174830), anti-SREBP1 (1:500; Santa Cruz, sc-13551), anti-SREBP2 (1:1000; Abcam, ab30682), anti-FlOTILLIN-1 (1:1000; BD Biosciences, 610821), anti-CD49B (1:1000; Abcam, ab133557), anti-CD49F(1:500; Millipore, 217657), anti-Intergrin β1 (1:500; Santa Cruz, sc-13590), anti-Histone 3 (1:1000; Abcam, ab1791), anti-HDAC2 (1:1000, Santa Cruz, sc-81599), anti-GAPDH (1:5000; Abcam, ab9485), anti-β-Tubulin (1:5000; Sigma, SAB4200715), anti-β-Actin (1:5000; Sigma, A1978). The blots were washed three times with TBS/0.2% Tween-20. The blots were reacted with the specific secondary antibodies: anti-rabbit IgG, HRP-linked antibody (1:10000; Jackson Immuno Research, 711-036-150), anti-mouse IgG, HRP-linked antibody (1:10000; Jackson Immuno Research, 711-036-152), anti-mouse IgM, HRP-linked antibody (1:1000; Millipore, AP128P) at 4° C. overnight. After washing three times with TBS/0.2% Tween-20, the membranes were then developed with ECL solution (Thermo Fisher Scientific, 34095).
1.14 Cholesterol Quantification
Cholesterol levels were measured by the Amplex Red cholesterol assay (Molecular Probes). Samples were diluted in reaction buffer, then further reacted with Amplex Red working solution (1:1) (300 μM Amplex Red, 2 U/ml cholesterol oxidase, 2 U/ml cholesterol esterase, and 2 U/ml horseradish peroxidase). The samples were reacted at 37° C. for 30 min. The absorbance was detected at 590 nm. Cholesterol values were calculated using standard cholesterol solutions, and the normalization by protein content that was performed by the Bradford Protein Assays (Bio-Rad).
1.15 Flow Cytometry
hESCs were dissociated by accutase. The cells were stained according to manufactures' instructions (eBioscience, 88-8005-72). In brief, cells (5×105) were suspended at in 100 μl 1× binding buffer, and then stained with 2.5 μl of Annexin V-FITC. After reaction for 20 min at room temperature, the cells were incubated with 2.5 μl of PI solution for 10 min. Then the cells were diluted with PBS and analyzed with a flow cytometer.
1.16 Microarray and GO-Term Analysis
Published data array (listed in Table 2) and GFP and PODXL overexpression arrays were analyzed according with GeneSpring GX 11. The candidate genes those have over 2 fold-change and below 0.5 fold-change were listed. GO-term analysis was performed with DAVID program.
1.17 Culture of BMMSCs and NSCs
Human BMMSCs (Lonza) were cultured in MSC NutriStem XF Medium (Defined, xeno-free, serum-free medium) and grown on Corning CellBIND Surface plates with inhibitor treatments for 3 days. Human neural stem cells (NSCs) were differentiated from H9 hESCs with Gibco PSC Neural Induction Medium (serum-free medium) for 7 days. And the NSCs were replated on matrigel-coated plate and supplied with each inhibitors for 3 days.
1.18 Treatment with Cholesterol
CRL2097 (passage 9) were seeded and infected with lentivirual vector (OSKM) with final concentration of cholesterol (0, 0.5×, 1×, 2×, 5×, 8×) which was diluted from 500× concentrated SyntheChol® NSO Supplement (S5442, Sigma). After 4 days viral transduction, cell were replated on matrigel-coated 6-well plates as cell number 27, 000 per well. 2 days later for cell attachment, cholesterol were supplied continually during reprogramming procedure. To better evaluate cholesterol effect to iPSC generation, serum-free defined E8 medium (containing 250 μM sodium butyrate, 50 μg/ml Vitamin C) was used for iPSC generation.
1.19 Statistical Analysis
Data is presented as mean±SD/mean±SEM. P values were calculated using two-tailed Student's unpaired t test or one way Anova and P<0.05 implies the data have significant difference. All figures and statistically analyses were established using GraphPad Prism 5.
2. Results
2.1 PODXL is Required for hPSC Growth and Pluripotency
To investigate expression pattern of PODXL in human early embryo, we checked the relative amounts of PODXL mRNAs during pre-implantation stage. The dataset we used is different from the previous study (Kang et al., 2016). We also found PODXL transcripts were enriched from 1-cell stage to 4-cell stage (bar,
To reveal PODXL expression pattern in PSCs and differentiated cells, we analyzed a global transcriptomic expression pattern with dozens of arrays. The hierarchical clustering heat map showed that PODXL transcripts were abundantly expressed in PSCs, and the expression levels were a lot lower in the differentiated cells (data not shown). Similarly, in protein levels, PODXL expression is enriched in in two undifferentiated hESC lines, HUES6 and H9. The expression levels decreased in in multipotent mesenchymal stem cells, and were much lower expressed in fibroblasts (
To examine the function of PODXL in hPSCs, we used two different shRNAs to knockdown PODXL. In HUES6 cells, the cell differentiated after two shRNA knockdown (
To study the functional roles of PODXL in iPSC reprogramming, human primary foreskin fibroblasts CRL2097 were co-infected with shPODXL and four factors (OKSM). The iPSC colony were calculated on 16 days post-transduction (
In previous data, PODXL expression was enriched in zygote to 4-cell embryo in the embryo (
2.2 Overexpression of PODXL Restores the Decrease of Pluripotency and c-MYC and Telomerase Expression that was Induced by the shPODXL Treatment
To exclude the off-target effect of shRNA, we overexpressed PODXL in shPODXL expressing cells. Overexpression of PODXL in shPODXL expressing cells rescued the decrease of relative cell numbers and the stem cell marker (
2.3 PODXL is Sufficient for Primed State and Extended State hPSC Renewal
PODXL overexpression in HUES6 was proved by the Western blot analysis (
Yang et al. reported derivation of the extended pluripotent stem cells (EPSCs) from primed ESCs with four chemicals that enable cells to develop into both embryonic and extraembryonic lineage (Yang et al., 2017b). In the transcriptomic profile, these EPSCs partially mimic the embryo at 4-cell stage (Yang et al., 2017b). Thus, to test the function of PODXL in the EPSC reprogramming, we cultured hPSCs in N2B27-LCDM medium, the cocktail to derive EPSCs (Yang et al., 2017b). Upon PODXL overexpression, we found an increase in domed shape colony numbers while compared to GFP control (
2.4 PODXL Regulates Cholesterol Levels and c-MYC Levels Through HMGCR and SREBPs
To map the early signals triggered by PODXL, cDNA microarray is performed in cells that overexpressed PODXL for after 3 days. By David functional tool (Huang da et al., 2009a, b), in upregulated gene set, cholesterol biosynthesis pathways were significantly enriched (data not shown). In the downregulated gene set regulation of RNA metabolic process and morphogenesis were enriched (data not shown). We found that 38 genes were upregulated more than two fold, while 26 genes were downregulated more than two fold (data not shown). Among upregulated genes, it contains six cholesterol related genes-3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), 7-dehydrocholesterol reductase (DHCR7), squalene epoxidase (SQLE), protein convertase subtilisin/kexin type 9 (PCSK9), insulin induced gene 1 (INSIG1), hydroxymethylglutaryl-CoAreductases (HMGCR) (up 1.6-fold change) (data not shown). At the same time, downregulated gene set includes the differentiation related genes-TBX3, TGFB2, ZEB2, GATA6, GATA3, FOXE1 (data not shown). This result strongly suggests that PODXL may positively regulate cholesterol biosynthesis pathway.
To understand how PODXL affects cholesterol homeostasis pathway, we performed qRT-PCR. Several cholesterol-related genes were downregulated upon PODXL knockdown (
SREBP2 is the master regulator of endogenous cholesterol biosynthesis. It activates the expression of multiple cholesterol synthesize gene such as HMGCR, HMGCS1, mevalonate kinase (MVK) (Horton et al., 2002; Madison, 2016). SREBP1a also can drive cholesterol synthesis pathways in all tissues (Horton et al., 2002; Madison, 2016). HMGCR is the rate-limiting enzyme in cholesterol biosynthesis. HMGCR expression are regulated by SREBP2 and SREBP1 in the previous paper. We next like to check whether PODXL can regulate that SREBP2 or SREBP1 expression levels. By mRNA levels, SREBP1 and SREBP2 were decreased in shPODXL transductants (
Next, we check if transcriptional factor SREBP2 and SREBP1 binds to DNA which implies its activity. In shPODXL hESCs, clearly, both SREBP2 and SREBP1 was decreased in chromatin-bound fraction, indicating reduced SREBP2 and SREBP1 binding to DNA (
2.5 Cholesterol is Essential to hPSC Pluripotency and Survival
To check the functional roles of cholesterol on pluripotency, cholesterol inhibitor simvastatin, AY9944, Methyl-β-cyclodextrin (MBCD) were used to inhibit cholesterol biosynthesis (
These results showed that hPSCs are much more sensitive to the inhibition of cholesterol synthesis while compared to somatic fibroblasts.
To reveal whether cholesterol is the downstream target of PODXL, we first overexpressed PODXL for one day. Then the cells were treated with cholesterol inhibitors simvastatin, AY9944 and MBCD, separately. Overexpression of PODXL in hESCs enhanced cell growth and ALP activity (
2.6 Cholesterol can Rescue the shPODXL Phenotype and Boost the iPSC Reprogramming Efficiency
To examine if cholesterol is the major downstream of PODXL, rescue experiment with cholesterol was performed. Surprisingly, cholesterol supplement prevent morphological changes, relative cell number loss, and reduction in ALP activity from PODXL knockdown (
In addition, cholesterol can boost the reprogramming efficiency (total AP positive, 7.62-fold) with OSKM 4 factors. See
2.7 Inducible CRISPR/Cas9 Knockout of PODXL Inhibits Self-Renewal of hPSCs
To exclude the off-target of shRNA, we knocked out PODXL in hPSC genome using inducible CRISPR/Cas9 editing method (
3. Discussion
Besides the well-studied multiple transcription regulators and evidences support epigenetic regulators of chromatin states are important for maintaining distinct status of self-renewal of PSCs (Jaenisch and Young, 2008), very few functional roles of transmembrane proteins in hPSC renewal have been discovered. Here, we provide evidences that the surface marker, PODXL, plays an important role in self-renewing primed PSCs and EPSCs. To the best of our knowledge, this is the first study highlight the importance of cholesterol signals in PSCs and defines its molecular mechanisms.
c-MYC is crucial for proliferation, anti-apoptosis and stem cell renewal (Chappell and Dalton, 2013; Scognamiglio et al., 2016; Varlakhanova et al., 2011; Varlakhanova et al., 2010; Wilson et al., 2004). Interestingly, human iPSC generation is inhibited by the presence of MYC inhibitor (Asaf Zviran, 2019), it suggests Myc is essential for iPSC reprogramming Although there is functional redundancy between MYC family members during early development, simultaneous knockout of c-MYC and N-MYC in PSCs results in self-renewal impairment and loss of pluripotency due to cell cycle blockage and cell differentiation toward primitive endoderm and mesoderm lineages (Smith et al., 2010). In addition, c-MYC can activate telomerase reverse transcriptase (TERT) which is crucial for the maintenance of telomere lengthen and immortalized properties of PSCs (Wu et al., 1999). We note that PODXL particularly regulates c-MYC and TERT expression in hPSCs (
PODXL knockdown impaired the human iPSC generation (
In order to exclude the concern of off-target of shRNA, forcing ectopic PODXL expression can rescue shPODXL induced phenotypes (
Cholesterol plays an important role not only in sterol hormone and vitamin D production, but also in signaling transduction and lipid raft formations. But, limited data are available on grasping the relationship between cholesterol metabolism and renewal in PSCs. One paper reported that simvastatin impaired mouse ESC self-renewal by modulating RhoA/ROCK-dependent cell signaling and was cholesterol independent (Lee et al., 2007). Strikingly, in our study, we found PODXL can regulate cholesterol levels and lipid raft formations by regulating the master regulator SREBP1/SREBP2, and the rate-limiting enzyme of cholesterol biosynthesis pathway HMGCR (
Taken together, our data suggest that PODXL is abundantly expressed in human primed and extended PSCs functions as a transmembrane protein to promote self-renewal through SREBP1/SREBP2-HMGCR-c-MYC-TERT signaling. Given the potent ability of PODXL to activate c-MYC, TERT, cholesterol pathway, promote growth, and prevent apoptosis, it is tempting to speculate that cancer stem cells may also display a similar dependence on PODXL for tumor initiation and progression. Also, due to its properties in supporting primed and extended pluripotency, PODXL harbors ideally infinite potential in regenerative medicine and provides an effective target for anti-cancer therapy in the future.
This application claims the benefit of U.S. provisional application No. 62/784,942, filed Dec. 26, 2018 under 35 U.S.C. § 119, the entire content of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/068528 | 12/26/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62784942 | Dec 2018 | US |
