Compositions and methods for amplifying pluripotent stem cells based on small molecules

Abstract

Methods and compositions for growing and maintaining Oct3 / 4 + SOX2 + NANOG + pluripotent stem cells (PSC) in cell culture, or generating and maintaining CD7 + CD75 + CD77 + CD130 + F11R + initial PSC, comprising the use of a small molecule-based culture medium containing an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, and an AMPK pathway agonist.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 414,265, filed Oct. 7, 2022, the entire content of which is incorporated herein by reference. Background of the Invention

[0003] Pluripotent stem cells have the ability to differentiate into all cell types of an adult, making them an important part of regenerative medicine technologies. Therefore, large-scale production of pluripotent cells with highly consistent phenotypes is of great significance. Several methods for culturing pluripotent stem cells have been established previously. Some of the most common methods include co-culturing with mouse embryonic fibroblasts (MEFs), or using conditioned media that has been previously exposed to MEFs. Both methods rely on using another cell type to secrete components into the culture medium to maintain the pluripotent phenotype, which, by definition, are complex media with unknown components.

[0004] Fully defined media, such as commercially available mTeSR and Essential 8 (E8) media, are the most commonly used media for maintaining pluripotency in cell culture. Both media rely on using high concentrations of FGF2 and protein components in the presence of low concentrations of TGF-β to maintain the naive phenotype in human pluripotent cell culture. However, the protein components are prone to degradation, which can lead to variability within their short shelf life.

[0005] In addition, these media maintain human pluripotent cells in a naive phenotype that more closely represents an ectodermal population with an ectodermal bias, rather than the initial state of pluripotent cells, which represents an earlier developmental state consisting of a cell population similar to the inner cell mass of a pre-implantation blastocyst. Pluripotent cells in the naive state have a lower differentiation potential than pluripotent cells in the initial state.

[0006] Accordingly, although there are currently some methods available for maintaining and expanding pluripotent stem cells in culture, these methods still have limitations, and thus there is a need in the art for additional methods and compositions for maintaining and expanding pluripotent stem cells in culture, particularly those that can maintain the cells in an initial state with high differentiation potential. Summary of the Invention

[0008] The present disclosure provides a culture medium composition of fully defined components that is capable of maintaining the pluripotent phenotype of stem cells in culture, such as in the naive (as opposed to primed) state with high differentiation potential. Accordingly, the compositions and methods of the present disclosure do not rely on complex culture medium formulations with unknown components. Since the culture medium composition consists of defined components, it is less susceptible to degradation and batch variability. Specifically, the present disclosure describes compositions and methods for using a defined component culture medium consisting of small molecule agonists and antagonists to continuously maintain the pluripotent state of stem cells. The advantage of this is that the pluripotent state can be maintained in the absence of protein components that are vulnerable to variation and degradation. In one embodiment, the culture medium of the present application comprises an Akt agonist, an FGF agonist, a JAK / STAT antagonist, a PKC antagonist, and an AMPK agonist.

[0009] In one aspect, a method of maintaining and expanding Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs) in cell culture comprises: culturing the pluripotent stem cells (PSCs) in a culture medium comprising an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, and an AMPK pathway agonist such that the culture medium maintains the PSCs in a primed or naive state comprising Oct3 / 4, SOX2, and NANOG markers.

[0010] In some embodiments, the PSCs are human PSCs (hPSCs). In other embodiments, the PSCs are induced PSCs (iPSCs). In other embodiments, the PSCs are human embryonic stem cells (hESCs). Generally, the PSCs are in a primed state (“primed PSCs”), a naive state (“naive PSCs”), or a combination of both. In one embodiment, the PSCs are human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells. In another embodiment, the PSCs express KLF2 / 4 / 5, ZFP42, ESRRB, DAPP3 / 5, TFCP2L1, FGF4, TBX3, CDH1, PECAM, CD31, NR5A2, and IDID1

[0011] In one embodiment, the Akt pathway agonist is selected from the group consisting of: SC79, demethylcoclaurine, LM22B-10, YS-49, YS-49 monohydrate, demethylasterriquinone B1, recilisib, N-oleoyl glycine, NSC45586 sodium, periplocin, CHPG sodium salt, bilobalide, 6-hydroxyflavone, muscone, SEW2871, 8-isoprenyl, razuprotafib, and combinations thereof. In a more specific embodiment, the Akt pathway agonist is SC79, and its concentration in the culture medium is 1 ng / ml.

[0012] In one embodiment, the FGFR agonist is FGF2 or SUN11602. In a more specific embodiment, the FGFR agonist is SUN11602, and its concentration in the culture medium is 5 μM.

[0013] In one embodiment, the JAK / STAT signal transduction antagonists are selected from the group consisting of: tofacitinib, ruxolitinib, baricitinib, filgotinib, upadacitinib, peficitinib, orantinib, soctinib, desatinib, digalectinib, deucravicitinib, abrocitinib, lestaurtinib, pacritinib, fedratinib, momelotinib, gandotinib, cerdulatinib, GS-829845, GSK2586184, AZD1480, R348, VX-509, GLPG0634, JSI-124, TG101348, AC-430, NS-018, CHZ868, SHR0302, INCB039110, BMS-911543, BMS-986165, PF-04965841, PF-04965842, PF-06263276, PF-06651600, and combinations thereof. In a more specific embodiment, the JAK / STAT antagonist is tofacitinib, and its concentration in the culture medium is 100 nM.

[0014] In one embodiment, the PKC pathway antagonists are selected from the group consisting of: Go6983, sotrastaurin, enzastaurin, staurosporine, LY31615, Go 6976, GF 109203X, Ro31-8220 mesylate, and combinations thereof. In a more specific embodiment, the PKC pathway antagonist is Go6983, and its concentration in the culture medium is 5 nM.

[0015] In one embodiment, the AMPK agonist is selected from the group consisting of metformin, AICAR, Kazinol B, maritimoside, amarogentin, A 769662, PF 06409577, metformin hydrochloride, ZLN 024, ZLN024 hydrochloride, nilotinib, phenformin, nilotinib hydrochloride monohydrate, adenosine 5'-monophosphate monohydrate, hispidulin, MK 8722, euphorbiasteroid, ASP4132, GSK621, EX229 (Compound 991), trans-ferulic acid, O-304, MK3903, BAM 15, ligustroside, ETC-1002, BC1618, IMM-H007, IM156, japonicoside IVa, pachymic acid A, 7-methoxyisoflavone, urolithin B, danthron, demethyleneberberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, malvidin chloride-3-O-arabinoside, RSVA 405, etilefrine, COH-SR4, butformin, butformin hydrochloride, PT1, bempedoic acid, 3a-hydroxy mogrosol, ampkinone, and combinations thereof. In one embodiment, the AMPK pathway agonist is metformin or AICAR. In a more specific embodiment, the AMPK pathway antagonist is metformin at a concentration of 500 μM in the culture medium.

[0016] In some embodiments, the culture medium is used in combination with the protein components FGF2 and / or TGF-β to increase the growth rate of the pluripotent culture.

[0017] In another embodiment, the culture medium further comprises a ROCK inhibitor, a TGF-β1 agonist, or both. In one embodiment, the culture medium comprises a ROCK inhibitor and a TGF-β1 agonist. In one embodiment, the ROCK inhibitor is selected from the group consisting of Y27632, H1152, GSK429286A, RKI-1447, DJ4, thiazovivin, besirudin, fasudil, hydroxyfasudil, ripasudil, netarsudil, and verasudil. In a more specific embodiment, the ROCK inhibitor is Y27632 at a concentration of 10 μM in the culture medium.

[0018] In one embodiment, the TGF-β1 agonist is selected from the group consisting of TGF-β1, SRI-011381, activin A, Nodal, DPS-1, and combinations thereof. In one embodiment, the TGF-β1 agonist is TGF-β1 or SRI-011381. In a more specific embodiment, the TGF-β1 agonist is TGF-β1, which is present in the culture medium at a concentration of 2 ng / ml.

[0019] In one embodiment, the culture medium contains SC79, SUN11602, tofacitinib, Go6983, and metformin. In another embodiment, the culture medium further contains selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

[0020] In one embodiment, the culture medium contains a basal medium composition selected from the group consisting of DMEM, F12, IMDM, CDM2, and combinations thereof. In another embodiment, the basal medium composition is further supplemented with ascorbic acid and transferrin. In another embodiment, the basal medium composition contains F12 or IMDM medium supplemented with ascorbic acid, transferrin, and penicillin-streptomycin. In a more specific embodiment, the basal medium composition contains a 1:1 F12 / IMDM medium supplemented with 20 μg / ml ascorbic acid, 10 μg / ml transferrin, and 1% penicillin-streptomycin. In another embodiment, the basal medium composition contains selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

[0021] In one embodiment, the TB5i formulation (as described herein) is supplemented to a commonly used basal medium. In another embodiment, the TB5i formulation is supplemented to a developed culture medium formulation. In another embodiment, the TB5i formulation contains the addition of a ROCK inhibitor and / or a cAMP pathway activator.

[0022] In another aspect, a method for generating and maintaining human CD7+CD75+CD77+CD130+F11R+ naïve pluripotent cells in cell culture comprises: culturing pluripotent stem cells (PSCs) in a culture medium containing an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, an AMPK pathway agonist, a ROCK inhibitor, and a TGF-β1R agonist, thereby generating and maintaining human CD7+CD75+CD77+CD130+F11R+ naïve pluripotent cells in the culture medium.

[0023] In some embodiments, PSCs grow in adherent culture, such as on tissue culture plates. In one embodiment, the tissue culture plates are coated with gelatin. In another embodiment, the tissue culture plates are coated with vitronectin. In additional embodiments, the tissue culture plates are coated with or In one embodiment, the TB5i formulation is used to culture pluripotent stem cells in adherent culture.

[0024] In other embodiments, PSCs grow as cell aggregates in suspension culture. In one embodiment, the TB5i formulation is used to culture pluripotent cells as cell aggregates in suspension culture.

[0025] In other embodiments, PSCs grow in a bioreactor. In another embodiment, the TB5i formulation is used to culture pluripotent cells in a bioreactor. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a schematic diagram of fertilized egg development for determining the state of pluripotency. All genes monitored in the series of HD-DoE experiments described herein are shown in this schematic diagram. Gene markers for specific lineages are indicated. The optimization strategies in the present disclosure focus on minimizing the primordial and lineage-specific genes while optimizing the initial gene expression.

[0027] Figures 2A - 2C Shows the results of the HD-DoE experiment for the E8 critical process parameter (CPP) medium assay. Figure 2A Shows the conditions for each of the 96 reactions conducted in the HD-DoE run. Figure 2B Shows the effectors and maximum concentrations used in the experiment. Figure 2C Depicts the basal medium used in all experimental reactions.

[0028] Figures 3A - 3D Shows the results of determining the CPP of the basal medium formulation. Figures 3A - 3C Shows the measured genes representing the initial state ( Figure 3A ), pluripotent state ( Figure 3B ), and primordial state ( Figure 3C ) after maximization in the MODDE software, and shows the overall contribution factors of the effectors. Figure 3D Shows the average contribution factors of all effectors used, which are averaged for each representative state.

[0029] Figures 4A - 4C Shows the experimental results of the HD-DoE for pluripotency maintenance. Figure 4AShows the reaction conditions for each of the 96 reactions conducted during the HD-DoE run. Figure 4B Shows the effectors and maximum concentrations used in the experiment. Figure 4C Shows the basal medium used in all experimental reactions.

[0030] Figures 5A - 5B Shows the results of revealing small molecule pluripotency maintenance formulations through NANOG optimization. Figure 5A Shows the application of MODDE software in optimizing key regulators of the pluripotent state, which are associated with NANOG gene expression. Figure 5B Shows the well-known medium additives for maintaining the pluripotent state in HD-DoE.

[0031] Figures 6A - 6B Shows the results of the combined effects of 5 additives on maintaining the pluripotent state. Figure 6A Shows all the genes marking the initial, pluripotent, and primed states, where the optimization of the relative contribution factors of the effectors is shown in the heat map as presented. The 5 most important components for maintaining the pluripotent state are indicated. Figure 6B Shows bright-field images depicting the adaptation of pluripotent cultures to the TB5i formulation.

[0032] Figures 7A - 7B Shows the comparison between TB7i and TB5i media. Figure 7A Shows the comparison of the daily growth of PSCs when using the TB7i and TB5i medium formulations. FGF2 and TGF-β1 were added to the control cultures. Figure 7B Shows individual colonies monitored over consecutive days.

[0033] Figures 8A - 8C Indicates that adding FGF2 and TGF-β to TB5i enhances the initial phenotype. Figure 8A Shows the effects of adding the protein components FGF2, TGF-β, and insulin to the medium in the presence and absence of TB5i medium, where T represents TGF-β, F represents FGF2, and I represents insulin. Figure 8B Shows the IHC verification of the pluripotent state. Figure 8C Shows the rationale behind using the TB5i medium formulation.

[0034] Figures 9A - 9D Shows that TB5i medium can maintain pluripotent cells in suspension within a bioreactor. Figure 9A Shows a table of the two medium formulations used in a 100 ml PBS vertical wheel bioreactor. Figure 9B Is shown related to STEMSCALE TMGraph showing the overall growth of pluripotent cells in a bioreactor compared to (a commercial medium for suspension culture). Figure 9C Shows the average aggregate size during a 4-day bioreactor run. Figure 9D Shows aggregates removed from the bioreactor, immunofluorescently stained for pluripotency markers the next day after being seeded onto a vitronectin-coated plate and incubated overnight.

[0035] Figure 10 Shows the results of suspension culture validation with TB5i pluripotency maintenance medium. The results show that aggregates formed and grew in the PBS bioreactor with different medium formulations during the 4-day period shown. DETAILED DESCRIPTION OF THE INVENTION

[0037] Aspects of the present application will be described in further detail in the following subsections.

[0038] I. Cells

[0039] The starting cells in culture are pluripotent stem cells (PSCs), including human pluripotent stem cells (hPSCs). Generally, PSCs or hPSCs are defined as stem cells capable of differentiating into all cell types in an adult organism, including the characteristic cells of each germ cell layer (endoderm, mesoderm, and ectoderm). As used herein, the pluripotent state refers to PSCs or hPSCs that express specific key markers, such as induced pluripotent stem cells (iPSCs), human embryonic stem cells (hESCs, such as hESC cell lines), human primed pluripotent stem cells (hpPSCs), or human naive pluripotent stem cells (hnPSCs).

[0040] As used herein, the terms "induced pluripotent cells" and "iPSC" refer to cells obtained from a late stage of development that, upon induction, have an expression pattern consistent with pluripotent cells. The cell source can be embryonic or adult. In one embodiment, the iPSC is the iPSC cell line CR01 (NIH). Other non-limiting examples of induced pluripotent stem cells (iPSC) include the 19-11-1, 19-9-7, or 6-9-9 cells (e.g., as described in Yu, J. et al. (2009) Science 324:797-801). Non-limiting examples of human embryonic stem cell lines include the ES03 cell line (WiCell Research Institute) and the H9 cell line (Thomson, J. A. et al. (1998) Science 282:1145-1147). Human pluripotent stem cells (PSC) express cell markers that can be used to identify the cells as PSC. Non-limiting examples of pluripotent stem cell markers include TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, and / or SOX2.

[0041] As used herein, the terms "human embryonic stem cells" and "hESC" refer to pluripotent cells derived from the inner cell mass of a human blastocyst. The term "inner cell mass" refers to the cell mass located in the anterior region of the early blastocyst that can develop into the entire embryo. Key markers of iPSC or hESC include, but are not limited to: Oct3 / 4, SOX2, NANOG, and SSEA4. In addition, these cells are in a proliferative self-renewing state, accompanied by the expression of TERT and MKi67.

[0042] Both the naive state and the primed state are considered pluripotent cell states. As used herein, the term "primed state" refers to pluripotent stem cells with an ectodermal bias. This is typically due to the growth of the culture in the presence of FGF2 and / or TGF-β. Most commercially available media for culturing human pluripotent maintenance cells are in this state. The term "naive state" is used to refer to cells with pluripotency, which are more representative in that they have a greater differentiation capacity in the inner cell mass and lack the ectodermal bias characteristic of the primed state in which normal human pluripotent cells are cultured.

[0043] Previous studies have shown that after initial derivation, human embryonic stem cells exhibit distinct genotypic and phenotypic differences from murine pluripotent cells. Among these differences, murine embryonic stem cells have a faster growth rate and colonies that are domed, while human embryonic colonies are flat. Subsequent studies have shown that these differences represent an initiating event in hESC culture, rendering these cultures more representative of an epiblast population with epiblast bias. Since the culture conditions used for hESC culture are employed for the expansion of iPSCs, induced pluripotent stem cell cultures follow a similar path. Thus, iPSC cultures exhibit an initial phenotype.

[0044] The naive state represents an early developmental state, consisting of cells similar to the inner cell mass of a pre-implantation blastocyst, which have a greater differentiation potential. The primed state, on the other hand, is more representative of a post-implantation epiblast population, which is more prone to differentiating into the epiblast lineage. Advantages of culturing iPSC cells in the naive state include a faster growth rate, a stronger differentiation potential, and single-cell clonality. The latter may eliminate the need for aggregate formation and the use of ROCK inhibitors during passaging. These factors highlight the benefits of developing a growth medium capable of expanding the naive state on a bioreactor-based platform.

[0045] Despite a growing number of studies demonstrating the beneficial properties of the naive pluripotent state, there is currently no commercially available medium for expanding and maintaining naive pluripotent cells. In addition, most commercially available pluripotency-maintaining media rely on the addition of protein components, which induce the primed phenotype while significantly increasing the cost of the medium. For these reasons, we designed experiments aimed at addressing the possibility of inducing and maintaining the naive pluripotent state, with a focus on identifying small molecules capable of mediating this transition. Using a novel systems biology platform-guided approach that can evaluate complex interactions in a multi-dimensional experimental space, we discovered a complex combinatorial interaction among multiple signal transduction pathways for maintaining pluripotency. Through a series of experimental analyses, a small molecule-based medium was developed that can generally maintain the pluripotency of PSCs or, specifically, maintain an enhanced naive phenotype.

[0046] II. Medium Components

[0047] In one aspect, the methods of the present disclosure relate to maintaining and expanding Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs) in a cell culture, including PSCs in a primed and / or naive state. The methods involve using a small molecule-based medium comprising specific agonists and / or antagonists of cell signal transduction pathways. In some embodiments, the medium is serum-free, free of exogenously added growth factors, free of animal products, and is serum-free, xeno-free, and / or feeder-free.

[0048] As used herein, an "agonist" of a cell signaling pathway refers to an agent that stimulates (upregulates) a cell signaling pathway. In some embodiments, stimulation of a cell signaling pathway can be initiated extracellularly, e.g., by using an agonist to activate a cell surface receptor involved in the signaling pathway (e.g., the agonist can be a receptor ligand). Additionally or alternatively, stimulation of cell signaling can be initiated intracellularly, e.g., by using a small molecule agonist that interacts intracellularly with one or more components of the signaling pathway.

[0049] As used herein, an "antagonist" of a cell signaling pathway refers to an agent that inhibits (downregulates) a cell signaling pathway. In some embodiments, inhibition of a cell signaling pathway can be initiated extracellularly, e.g., by using an antagonist to block a cell surface receptor involved in the signaling pathway. Additionally or alternatively, inhibition of cell signaling can be initiated intracellularly, e.g., by using a small molecule antagonist that interacts intracellularly with one or more components of the signaling pathway.

[0050] The agonists and antagonists used in the methods of the present disclosure are known and / or commercially available. They are used in the culture medium at effective concentrations to achieve the desired results, e.g., to generate, expand, and / or maintain PSCs in a primed or "naïve" state, each state being characterized by the specific corresponding markers described herein. Non-limiting examples of suitable agonists and antagonists and effective concentration ranges will be further described below.

[0051] In one embodiment, a method of maintaining and expanding Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs) in cell culture comprises: culturing the pluripotent stem cells (PSCs) in a culture medium containing an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, and an AMPK pathway agonist such that the culture medium maintains the PSCs in a primed or naïve state comprising the markers Oct3 / 4, SOX2, and NANOG.

[0052] In some embodiments, the PSC is a human PSC (hPSC). In other embodiments, the PSC is an induced PSC (iPSC). In other embodiments, the PSC is a human embryonic stem cell (hESC). Generally, the PSC is in a naïve state (“naïve PSC”), a primed state (“primed PSC”), or a combination of both. In one embodiment, the PSC is a human CD7+CD75+CD77+CD130+F11R+ primed pluripotent cell. In another embodiment, the PSC expresses one or more of KLF2 / 4 / 5, ZFP42, ESRRB, DAPP3 / 5, TFCP2L1, FGF4, TBX3, CDH1, PECAM, CD31, NR5A2, and IDID1.

[0053] An Akt pathway agonist includes a reagent, molecule, compound, or substance that can stimulate (upregulate) the signal transduction pathway of one or more members of the serine / threonine kinase Akt family, including Akt1 (also known as PKB or RacPK), Akt2 (also known as PKBβ or RacPK-β), and Akt3 (also known as PKBγ or thymoma viral oncogene 3). In one embodiment, the Akt pathway agonist is a pan-Akt activator. In one embodiment, the Akt pathway agonist is selected from the group consisting of: SC79, demethylcoclaurine, LM22B-10, YS-49, YS-49 monohydrate, demethylasindoline B1, relescib, N-oleoylglycine, NSC45586 sodium, periplocin, CHPG sodium salt, bilobalide, 6-hydroxyflavone, muskone, SEW2871, 8-isoprenyl, razuprotafib, and combinations thereof.

[0054] In one embodiment, the Akt pathway agonist is present in the culture medium at a concentration range of 0.2 - 5 ng / ml, 0.3 - 3 ng / ml, 0.5 - 2.0 ng / ml, or 0.75 - 1.5 ng / ml. In one embodiment, the Akt pathway agonist is SC79. In one embodiment, the Akt pathway agonist is SC79 and is present in the culture medium at a concentration of 0.2 - 5 ng / ml, 0.3 - 3 ng / ml, 0.5 - 2.0 ng / ml, or 0.75 - 1.5 ng / ml. In one embodiment, the Akt pathway agonist is SC79 and is present in the culture medium at a concentration of 1 ng / ml.

[0055] An FGFR pathway agonist includes a reagent, molecule, compound, or substance that can activate (upregulate) the signal through the fibroblast growth factor 2 (FGF2) signal transduction pathway. In one embodiment, the FGFR pathway agonist is FGF2 or SUN11602.

[0056] In one embodiment, the concentration range of the FGFR pathway agonist in the culture medium is 100 - 500 μM, 200 - 400 μM, or 250 - 350 μM. In another embodiment, the FGFR pathway agonist is SUN11602, and its concentration range in the culture medium is 1 - 15 μM, 2 - 10 μM, or 3 - 7 μM. In another embodiment, the FGFR pathway antagonist is SUN11602, and its concentration in the culture medium is 5 μM.

[0057] JAK / STAT pathway antagonists include reagents, molecules, compounds, or substances capable of inhibiting (downregulating) signal transduction through the JAK / STAT signal transduction pathway. In one embodiment, the JAK / STAT pathway antagonists are selected from the group consisting of: tofacitinib, ruxolitinib, baricitinib, filgotinib, upadacitinib, peficitinib, orantinib, soaciclib, desatinib, digalectinib, Deucravicitinib, abrocitinib, lestaurtinib, pacritinib, fedratinib, momelotinib, gandotinib, Cerdulatinib, GS-829845, GSK2586184, AZD1480, R348, VX-509, GLPG0634, JSI-124, TG101348, AC-430, NS-018, CHZ868, SHR0302, INCB039110, BMS-911543, BMS-986165, PF-04965841, PF-04965842, PF-06263276, PF-06651600, and combinations thereof.

[0058] In one embodiment, the concentration range of the JAK / STAT pathway antagonist in the culture medium is 25 - 250 nM, 50 - 150 nM, or 75 - 125 nM. In another embodiment, the JAK / STAT pathway antagonist is tofacitinib, and its concentration in the culture medium is 25 - 250 nM, 50 - 150 nM, or 75 - 125 nM. In another embodiment, the JAK / STAT pathway antagonist is tofacitinib, and its concentration in the culture medium is 100 nM.

[0059] PKC pathway antagonists include reagents, molecules, compounds, or substances capable of inhibiting (downregulating) signal transduction through the PKC signal transduction pathway. In one embodiment, the PKC pathway antagonists are selected from the group consisting of: Go6983, Sotrastaurin, Enzastaurin, staurosporine, LY31615, Go 6976, GF 109203X, Ro31-8220 mesylate, and combinations thereof.

[0060] In one embodiment, the concentration range of the PKC pathway antagonist in the culture medium is 2-10 nM, 2.5-7.5 nM, 3-6.50 nM, or 4-6 nM. In another embodiment, the PKC pathway antagonist is Go6983, and its concentration in the culture medium is 2-10 nM, 2.5-7.5 nM, 3-6.50 nM, or 4-6 nM. In another embodiment, the PKC pathway antagonist is Go6983, and its concentration in the culture medium is 5 nM.

[0061] AMPK pathway agonists include reagents, molecules, compounds, or substances capable of activating (upregulating) signal transduction through the AMPK signal transduction pathway. In one embodiment, the AMPK pathway agonists are selected from the group consisting of metformin, AICAR, Kazinol B, maritimoside, amarogentin, A769662, PF 06409577, metformin hydrochloride, ZLN 024, ZLN 024 hydrochloride, nilotinib, phenformin, nilotinib hydrochloride monohydrate, adenosine 5'-monophosphate monohydrate, hispidulin, MK 8722, euphorbiasteroid, ASP4132, GSK621, EX229 (compound 991), trans-ferulic acid, O-304, MK3903, BAM 15, ligustroside, ETC-1002, BC1618, IMM-H007, IM156, japonicoside IVa, pachymic acid A, 7-methoxyisoflavone, urolithin B, danthron, demethyleneberberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, malvidin chloride-3-O-arabinoside, RSVA 405, etilefrine, COH-SR4, butformin, butformin hydrochloride, PT1, bempedoic acid, 3a-hydroxy mogrosol, amperozide, and combinations thereof.

[0062] In one embodiment, the concentration range of the AMPK pathway antagonist in the culture medium is 200-1000 μM, 250-750 μM, 300-650 μM, or 400-600 μM. In another embodiment, the AMPK pathway antagonist is metformin, and its concentration range in the culture medium is 200-1000 μM, 250-750 μM, 300-650 μM, or 400-600 μM. In another embodiment, the AMPK pathway antagonist is metformin, and its concentration in the culture medium is 500 μM.

[0063] In one embodiment, the small molecule-based culture medium comprises a basal medium composition supplemented with SC79, SUN11602, tofacitinib, Go6983, and metformin. In a more specific embodiment, the culture medium comprises a basal medium composition supplemented with 1 ng / ml SC79, 5 μM SUN11602, 100 nM tofacitinib, 5 nM Go6983, and 500 μM metformin.

[0064] In one embodiment, the small molecule-based culture medium comprises a basal medium composition selected from the group consisting of DMEM, F12, IMDM, CDM2, and combinations thereof. In another embodiment, the small molecule-based culture medium comprises a basal medium composition supplemented with ascorbic acid and transferrin.

[0065] In one embodiment, the small molecule-based culture medium comprises a basal medium composition that comprises F12 or IMDM medium supplemented with ascorbic acid, transferrin, and penicillin-streptomycin. In a more specific embodiment, the basal medium composition in the culture medium comprises a 1:1 F12 / IMDM medium and is supplemented with 20 μg / ml ascorbic acid, 10 μg / ml transferrin, and 1% penicillin-streptomycin. In another embodiment, the small molecule-based culture medium comprises a basal medium composition that comprises a 1:1 F12 / IMDM medium supplemented with 20 μg / ml ascorbic acid, 10 μg / ml transferrin, and 1% penicillin-streptomycin, wherein the basal medium composition is further supplemented with 100 ng / ml of FGF2 and 2 ng / ml of TGF-β1 (hereinafter referred to as "TB5i culture medium formulation") or 10 μM Y27632 and 1 μM forskolin (hereinafter referred to as "TB7i culture medium formulation").

[0066] In some embodiments, the small molecule-based culture medium comprises: a basal medium composition containing selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

[0067] In some embodiments, the small molecule-based culture medium is used in combination with the protein components FGF2 and / or TGF-β1 to increase the growth rate of pluripotent stem cell cultures.

[0068] In some embodiments, the culture medium further comprises a Rho kinase inhibitor (i.e., a ROCK inhibitor), a TGF-β1 pathway agonist, or both. In one embodiment, the culture medium comprises a ROCK inhibitor and a TGF-β1 pathway agonist.

[0069] ROCK inhibitors include reagents, molecules, compounds or substances that can inhibit (down-regulate) signal transduction through the Rho kinase pathway. In one embodiment, the ROCK inhibitors are selected from the group consisting of: Y27632, H1152, GSK429286A, RKI-1447, DJ4, Thiazovivin, besulifloxacin, fasudil, hydroxyfasudil, ripasudil, netarsudil and verusudil.

[0070] In one embodiment, the concentration range of the ROCK inhibitor in the culture medium is 2 - 50 μM, 3 - 30 μM, 5 - 20 μM or 7.5 - 15 μM. In another embodiment, the ROCK inhibitor is Y27632, and its concentration in the culture medium is 2 - 50 μM, 3 - 30 μM, 5 - 20 μM or 7.5 - 15 μM. In another embodiment, the ROCK inhibitor is Y27632, and its concentration in the culture medium is 10 μM.

[0071] TGF-β1 pathway agonists include reagents, molecules, compounds or substances that can activate (up-regulate) signal transduction through the TGF-β1 signal transduction pathway. In some embodiments, the TGF-β1 agonists are selected from the group consisting of: TGF-β1, SRI-011381, activin A, Nodal, DPS-1 and combinations thereof. In one embodiment, the TGF-β1 agonist is TGF-β1 or SRI-011381.

[0072] In one embodiment, the concentration range of the TGF-β1 pathway agonist in the culture medium is 0.4 - 10 ng / ml, 0.6 - 6 ng / ml, 1 - 4 ng / ml or 1.5 - 3 ng / ml. In one embodiment, the TGF-β1 pathway agonist is TGF-β1. In one embodiment, the TGF-β1 pathway agonist is TGF-β1, and its concentration range in the culture medium is 0.4 - 10 ng / ml, 0.6 - 6 ng / ml, 1 - 4 ng / ml or 1.5 - 3 ng / ml. In one embodiment, the TGF-β1 pathway agonist is TGF-β1, and its concentration in the culture medium is 2 ng / ml.

[0073] In another aspect, a method for generating and maintaining human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells in cell culture comprises: culturing pluripotent stem cells (PSCs) in the culture medium of the present application, the culture medium comprising an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, an AMPK pathway agonist, a ROCK inhibitor, and a TGF-β1R agonist, such that the culture medium generates and maintains human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells in cell culture.

[0074] In another aspect, the present application provides a small molecule-based culture medium as described herein for the growth, maintenance, and expansion of Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs), and for the generation, growth, maintenance, and expansion of human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells in cell culture.

[0075] In one embodiment, the small molecule-based culture medium comprises an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, and an AMPK pathway agonist. As described above, the basal culture medium composition of the present application is supplemented with the foregoing agonists and antagonists at the above concentrations.

[0076] In one embodiment, the Akt pathway agonist is SC79, and its concentration in the culture medium is 1 ng / ml. In another embodiment, the FGFR agonist is SUN11602, and its concentration in the culture is 5 μM. In another embodiment, the JAK / STAT antagonist is tofacitinib, and its concentration in the culture medium is 100 nM. In another embodiment, the PKC pathway antagonist is Go6983, and its concentration in the culture medium is 5 nM. In another embodiment, the AMPK pathway agonist is metformin, and its concentration in the culture medium is 500 μM. In a preferred embodiment, the small molecule-based culture medium comprises a basal culture medium composition supplemented with 1 ng / ml SC79, 5 μM SUN11602, 100 nM tofacitinib, 5 nM Go6983, and 500 μM metformin.

[0077] In another embodiment, the small molecule-based culture medium comprises a basal culture medium composition, which is further supplemented with a ROCK inhibitor, wherein the ROCK inhibitor is Y27632, and wherein the concentration of Y27632 in the culture medium is 10 μM.

[0078] In another embodiment, the small molecule-based culture medium comprises a basal culture medium composition further supplemented with a TGF-β1 agonist, wherein the TGF-β1 agonist is TGF-β1 and wherein the concentration of TGF-β1 in the culture medium is 2 ng / ml.

[0079] In a preferred embodiment, the small molecule-based culture medium comprises a basal culture medium composition comprising a 1:1 F12 / IMDM medium supplemented with 20 μg / ml ascorbic acid, 10 μg / ml transferrin, and 1% penicillin-streptomycin, the small molecule-based culture medium being supplemented with 1 ng / ml SC79, 5 μM SUN11602, 100 nM tofacitinib, 5 nM Go6983, and 500 μM metformin, and further supplemented with 100 ng / ml FGF2 and 2 ng / ml TGF-β1 (i.e., the TB5i culture medium formulation) or 10 μM Y27632 and 1 μM forskolin (i.e., the TB7i culture medium formulation).

[0080] When an agonist or antagonist is used in multiple steps of the method, in one embodiment, the same agonist or antagonist is used in each step in which the reagent is present in the culture medium. In another embodiment, different agonists or antagonists that affect the same signal transduction pathway are used in different steps of the method.

[0081] When an agonist or antagonist is used in multiple steps of the method, in one embodiment, the same concentration of the agonist or antagonist is used in each step in which the reagent is present in the culture medium. In another embodiment, different concentrations of the same agonist or antagonist are used in different steps of the method.

[0082] III. Culture Conditions

[0083] In combination with the chemical definitions and optimized culture media described in subsection II above, the methods for maintaining, expanding, and generating PSC cells described above employ standard culture conditions for cell culture established in the art. For example, cells can be cultured at 37 °C and 5% CO2.

[0084] In some embodiments, the PSC is cultured in adherent culture using the culture media described herein, using standard culture vessels or plates, such as 6-well, 24-well, or 96-well tissue culture (TC) plates, and the culture medium is changed daily. In certain embodiments, the PSC is coated with an extracellular matrix material. In one embodiment, the TC plate is coated with gelatin. In another embodiment, the TC plate is coated with vitronectin. In another embodiment, the TC plate is coated with In another embodiment, the TC plate is coated with

[0085] The culture media described herein (e.g., the TB5i formulation) have been shown to be effective for growing and maintaining adherent cultures in tissue culture plates. In an exemplary embodiment, the culture media of the present application (such as TB5i medium) are used to grow and maintain PSC cultures (such as the CR01 iPSC line) on a vitronectin-coated 6-well TC plate. PSC cultures are typically passaged every 3 - 4 days and are treated with a reagent that disrupts cell-cell adhesion (such as EDTA) or a digestive enzyme (such as collagenase, Accutase, trypsin, or TyrPLE). The treatment method is as follows: Remove the culture media and wash each well of the TC plate with 2 ml of PBS. Then incubate at 37 °C for 3 minutes in the presence of 5 mM EDTA. Then aspirate the wells, wash the cells off the culture plate, and seed them into fresh culture media. Each well is typically passaged and seeded into 6 wells of a newly vitronectin-coated TC plate, resulting in the expansion of the iPSC line from 1 to 6.

[0086] In some embodiments, suspension cultures of PSCs can be grown as cell aggregates in a bioreactor, as further described in Example 3 below. In an exemplary embodiment, the TB5i culture media formulation can be used to culture PSCs continuously for 5 days in suspension culture at a speed of 60 RPM in a 100 ml PBS VW bioreactor, with half depletion on day 1 and then every 2 days thereafter.

[0087] IV. Uses

[0088] The culture media described herein (such as the TB5i culture media formulation) can be used to maintain and expand Oct3 / 4+SOX2+NANOG+PSCs in cell culture, including cells in a committed or naive state of differentiation. In addition, the culture media can be used to generate and maintain human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells from a committed PSC state in cell culture. The ability to maintain and expand pluripotent cells in culture using the compositions and methods of the present disclosure allows for the obtaining of large numbers of these cells, including for a variety of regenerative medicine purposes.

[0089] V. Compositions

[0090] In other aspects, the present disclosure provides compositions related to methods for maintaining and expanding Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs) in cell culture, including culture media and cell cultures, and compositions related to methods for generating and maintaining human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells in cell culture, including culture media and cell cultures.

[0091] Thus, in one aspect, the present disclosure provides a culture medium for maintaining and expanding Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs), which contains an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, and an AMPK pathway agonist.

[0092] In certain embodiments, the culture medium further contains a ROCK inhibitor, a TGF-β1 agonist, or both.

[0093] In certain embodiments, the culture medium further contains a basal medium composition. In certain embodiments, the basal medium composition contains a culture medium selected from the group consisting of DMEM, F12, IMDM, CDM2, and combinations thereof. In certain embodiments, the basal medium composition is further supplemented with ascorbic acid and transferrin. In certain embodiments, the basal medium composition contains F12 or IMDM medium supplemented with ascorbic acid, transferrin, and penicillin-streptomycin. In certain embodiments, the basal medium composition contains selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

[0094] In another aspect, the present disclosure provides an isolated cell culture containing Oct3 / 4+SOX2+NANOG+ PSCs cultured in a culture medium formulation disclosed herein. Thus, in one embodiment, the present disclosure provides an isolated cell culture containing Oct3 / 4+SOX2+NANOG+ PSCs cultured in a culture medium containing an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, and an AMPK pathway agonist. In certain embodiments, the culture medium further contains a ROCK inhibitor, a TGF-β1 agonist, or both. In certain embodiments, the culture medium further contains a basal medium composition. In certain embodiments, the basal medium composition contains a culture medium selected from the group consisting of DMEM, F12, IMDM, CDM2, and combinations thereof. In certain embodiments, the basal medium composition is further supplemented with ascorbic acid and transferrin. In certain embodiments, the basal medium composition contains F12 or IMDM medium supplemented with ascorbic acid, transferrin, and penicillin-streptomycin. In certain embodiments, the basal medium composition contains selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

[0095] This application is further illustrated by the following examples, which should not be construed as further limitations. The content of the drawings and all references, patents, and published patent applications cited in this application are hereby expressly incorporated by reference. Examples

[0096] Example 1: Current pluripotent media passively maintain pluripotency

[0097] To monitor the differentiation state of pluripotent cultures and to track the naive, primed states, and any early lineage commitment biases that may arise during culture, a network of 54 genes was selected ( Figure 1 ). Other genes monitored in this series of experiments included TERT and KI67. Maximal TERT expression is required to maintain the pluripotent state of naive and primed cells, while the proliferation marker KI67 is expected to increase in the primed state. Housekeeping genes measured in this QS chip design were used to normalize individual experimental runs. The gene network was then monitored using the HD-DoE method (Bukys et al. (2020) Iscience 23:101346), as further described in Example 2, to enable computational modeling of the pluripotent state.

[0098] An initial HD-DoE modeling experiment was designed to determine whether any of the components in Essential 8 (E8) medium are essential for pluripotency maintenance. The experimental results are shown in Figure 2. The E8 medium components include HEPES, bicarbonate, selenium, ascorbic acid, transferrin, insulin, FGF2, and TGF-β. This HD-DoE design did not consider HEPES, bicarbonate, and selenium because they are not additives specifically for maintaining the pluripotent state but are involved in promoting the overall growth of any cells in culture. Additionally, these components are present in most common medium formulations, and the initial HD-DoE experiment here used a 1:1 F12 / IMDM mixed medium that already contained HEPES, bicarbonate, and selenium. In addition to the E8 components, the design also evaluated human leukemia inhibitory factor (hLIF), AICAR (AMPK pathway agonist), CHIR 99021 (Wnt agonist / GSK-3β antagonist), Go6983 (PKC pathway antagonist), PD0325901 (MEK pathway antagonist), and Y27632 (ROCK inhibitor) (Figure 2). These components were selected based on previous experiments that suggested their potential contribution to maintaining the pluripotent state.

[0099] Examination of the overall contribution of the aforementioned effectors revealed that the only components in E8 that are beneficial for maintaining the pluripotent state are ascorbic acid and transferrin (Figure 3). In this design, the only other components determined to potentially contribute to pluripotency maintenance were the PKC inhibitor Go6983 and the ROCK inhibitor Y27632. Although the overall pluripotent state was driven by Go6983, the primed state was driven by Y27632. All other components in this design either had conflicting contributions to the pluripotent state or were shown to be definitive lineage drivers.

[0100] Example 2: Defining key signaling pathways for maintenance of pluripotency

[0101] Data-driven, high-dimensional experimental design (HD-DoE)-based perturbations of pluripotent cultures were used to analyze several cell signaling pathways known to function in the pluripotent state. The HD-DoE method was used to find conditions for directly inducing the initial state from the pluripotent stem cell state. This example uses the method previously described by Bukys et al. (2020) Iscience 23:101346, which uses computer-designed geometric configurations to test multiple process inputs simultaneously and provides mathematical modeling of deep effect / response space. This method allows the finding of combined signal transduction inputs that control complex differentiation processes and allows the testing of multiple reasonable key process parameters that affect output responses (such as gene expression). Since gene expression provides a hallmark feature of the phenotype, such as human cells, this method can be used to identify and understand signal transduction pathways that control cell fate.

[0102] In order to develop a cell culture formula for growing and maintaining PSC in cell culture and differentiating stem cells into initial state progenitor cells, the agonists and antagonists (referred to herein as effectors) of various signal transduction pathways were tested and modeled to the impact of preselected gene expression. The impact of each effector on gene expression level is defined by a parameter called factor contribution, which is calculated for each effector during modeling. These effectors are small molecules or proteins, which are generally used to promote stem cell differentiation to a specific fate. The selection of the effector is based on the current document about stem cell differentiation into initial state progenitor cells.

[0103] Both the PKC inhibitor GO6983 and the ROCK inhibitor Y27632 were included in the design of the HD-DoE ( Figures 4A - 4B Ascorbic acid and transferrin were added to the basal medium of the perturbation matrix ( Figure 4C ) and all subsequent validation experiments. Focusing on maximizing NANOG as a surrogate of the pluripotent state and as a key driver of the initial state, we identified a synergistic pathway drive to pluripotency that can be achieved through the combined effects of activating the Akt, FGF, AMPK, and cAMP pathways, while simultaneously antagonizing the Jak Stat, ROCK, and PKC pathways ( Figure 5A ).

[0104] Further analysis by sequence optimization of all measured genes representing the naive state, the priming state, or general pluripotency markers showed that neither the cAMP activator forskolin nor the ROCK pathway inhibitor was essential for this process ( Figure 6A Initial validation with TB5i medium showed that colonies quickly began to cluster tightly together and exhibited a mound-like phenotype, a well-known characteristic of the initial state (Figure 6B )。The direct comparison between TB5i and TB7i confirmed that neither forskolin nor Y27632 was beneficial for the overall growth of pluripotent cells ( Figures 7A - 7B )。

[0105] Next, the protein additives FGF2, TGF-β1, and insulin were assayed as additives in the TB5i formulation ( Figure 8A )。It was assayed that insulin had little to no beneficial effect on the culture, thus confirming the Figure 3D results of the preliminary HD-DoE analysis shown. FGF2 increased the growth rate of the culture, while TGF-β1 maintained the normal morphology of the pluripotent colony edges, thus confirming the passive nature of the Essential 8 formulation. Immunohistochemical validation of Oct3 / 4 and SOX2 expression confirmed that pluripotency could be maintained in the TB5i medium. Cultures maintained in TB5i and TB5i supplemented with TGF-β1 and FGF2 showed a denser phenotype than the control cultures ( Figure 8B )。

[0106] Example 3: TB5i-mediated bioreactor-based pluripotent aggregate growth

[0107] To determine whether the TB5i formulation could maintain pluripotency in suspension culture, the PBS VW bioreactor system was used. Three experimental conditions were implemented. The first bioreactor was used as a control, and the cells were grown in STEMSCALE TM , STEMSCALE TM is a commercially available proprietary suspension medium (Gibco) for the suspension growth of pluripotent cultures. The second and third bioreactor cells were grown in TB5i or TB5i medium supplemented with FGF2 and TGF-β1, respectively ( Figure 9A )。Consistent with previous observations (Figure 8), both cultures grown in TB5i medium showed growth throughout the bioreactor run ( Figure 9B ), and proliferation increased after supplementing FGF2 and TGF-β1 in TB5i. Aggregate growth peaked on day 3 ( Figure 9C and 10 A). The pluripotent state was verified by inoculating aggregates for IHC analysis. The pluripotency markers Oct3 / 4, SOX2, NANOG, and SSEA4 were expressed in the entire cultures grown under both conditions ( Figure 9D )。

[0108] Equivalents

[0109] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein. Such equivalents are intended to be encompassed by the appended claims.

Claims

1. A method for maintaining and expanding Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs) in cell culture, comprising: culturing pluripotent stem cells (PSCs) in a medium containing an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, and an AMPK pathway agonist, such that the medium maintains the PSCs in a naive or initial state comprising the markers Oct3 / 4, S0X2, and NANOG.

2. The method according to claim 1, wherein the PSCs are human PSCs (hPSCs).

3. The method according to claim 1, wherein the PSCs are human induced PSCs (hiPSCs).

4. The method according to claim 1, wherein the PSCs are human embryonic stem cells (hESCs).

5. The method according to any one of claims 1-4, wherein the Akt pathway agonist is selected from the group consisting of: SC79, demethylcoclaurine, LM22B-10, YS-49, YS-49 monohydrate, demethylasrindole B1, relesib, N-oleoyl glycine, NSC45586 sodium, periplocin, CHPG sodium salt, bilobalide, 6-hydroxyflavone, muskone, SEW2871, 8-isoprenyl, lasudefylline, and combinations thereof.

6. The method according to claim 5, wherein the Akt pathway agonist is SC79.

7. The method according to claim 6, wherein the Akt pathway agonist is SC79, and its concentration in the medium is 1 ng / ml.

8. The method according to any one of claims 1-7, wherein the FGFR agonist is FGF2 or SUN11602.

9. The method according to claim 8, wherein the FGFR agonist is SUN11602.

10. The method according to claim 8, wherein the FGFR agonist is SUN11602, and its concentration in the medium is 5 μM.

11. The method according to any one of claims 1-10, wherein the JAK / STAT signal transduction antagonist is selected from the group consisting of: tofacitinib, ruxolitinib, baricitinib, filgotinib, upadacitinib, peficitinib, orantinib, soctinib, desatinib, digalectinib, deucravacitinib, abrocitinib, lestaurtinib, pacritinib, fedratinib, momelotinib, gandotinib, cedazuridine, GS-829845, GSK2586184, AZD1480, R348, VX-509, GLPG0634, JSI-124, TG101348, AC-430, NS-018, CHZ868, SHR0302, INCB039110, BMS-911543, BMS-986165, PF-04965841, PF-04965842, PF-06263276, PF-06651600, and combinations thereof.

12. The method according to claim 11, wherein the JAK / STAT antagonist is tofacitinib.

13. The method according to claim 11, wherein the JAK / STAT antagonist is tofacitinib, and its concentration in the culture medium is 100 nM.

14. The method according to any one of claims 1-13, wherein the PKC pathway antagonist is selected from the group consisting of: Go6983, sotrastaurin, enzastaurin, staurosporine, LY31615, Go 6976, GF 109203X, Ro 31-8220 mesylate, and combinations thereof.

15. The method according to claim 14, wherein the PKC pathway antagonist is Go6983.

16. The method according to claim 14, wherein the PKC pathway antagonist is Go6983, and its concentration in the culture medium is 5 nM.

17. The method according to any one of claims 1-16, wherein the AMPK agonist is selected from the group consisting of: metformin, AICAR, broussochalcone B, maritimoside, amygdalin, A 769662, PF 06409577, metformin hydrochloride, ZLN 024, ZLN024 hydrochloride, nilotinib, phenformin, nilotinib hydrochloride monohydrate, adenosine 5'-monophosphate monohydrate, homoplantaginin, MK 8722, euphol, ASP4132, GSK621, EX229 (compound 991), trans-ferulic acid, O-304, MK3903, BAM 15, ligustroside, ETC-1002, BC1618, IMM-H007, IM156, japonicoside IVa, pachymic acid A, 7-methoxyisoflavone, urolithin B, danthron, demethyleneberberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, malvidin chloride-3-O-arabinoside, RSVA 405, etilefrine, COH-SR4, butformin, butformin hydrochloride, PT1, feniprazone, 3a-hydroxy mogrosol, amkinone, and combinations thereof.

18. The method according to claim 17, wherein the AMPK pathway agonist is metformin or AICAR.

19. The method according to claim 17, wherein the AMPK pathway agonist is metformin, and its concentration in the culture medium is 500 μM.

20. The method according to any one of claims 1-19, wherein the culture medium contains SC79, SUN11602, tofacitinib, Go6983, and metformin.

21. The method according to claim 20, wherein the culture medium contains 1 ng / ml SC79, 5 μM SUN11602, 100 nM tofacitinib, 5 nM Go6983, and 500 μM metformin.

22. The method according to any one of claims 1-21, wherein the culture medium further contains a TGF-β1 agonist.

23. The method according to claim 22, wherein the TGF-β1 agonist is selected from the group consisting of: TGF-β1, SRI-011381, activin A, Nodal, DPS-1, and combinations thereof.

24. The method according to claim 23, wherein the TGF-β1 agonist is TGF-β1 or SRI-011381.

25. The method according to claim 23, wherein the TGF-β1 agonist is TGF-β1, and its concentration in the culture medium is 2 ng / ml.

26. The method according to any one of claims 1-25, wherein the culture medium further comprises a basal medium composition selected from the group consisting of DMEM, F12, IMDM, CDM2, and combinations thereof.

27. The method according to claim 26, wherein the basal medium composition is further supplemented with ascorbic acid and transferrin.

28. The method according to claim 26, wherein the basal medium composition comprises F12 or IMDM medium supplemented with ascorbic acid, transferrin, and penicillin-streptomycin.

29. The method according to claim 28, wherein the basal medium composition comprises a 1:1 F12 / IMDM medium and is supplemented with 20 μg / ml ascorbic acid, 10 μg / ml transferrin, and 1% penicillin-streptomycin.

30. The method according to claim 26, wherein the basal medium composition comprises selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

31. The method according to any one of claims 1-30, wherein the PSC is a human CD7+CD75+CD77+CD130+F11R+ naïve pluripotent cell.

32. The method according to any one of claims 1-31, wherein the PSC expresses KLF2 / 4 / 5, ZFP42, ESRRB, DAPP3 / 5, TFCP2L1, FGF4, TBX3, CDH1, PECAM, CD31, NR5A2, and IDID1.

33. A method for generating and maintaining human CD7+CD75+CD77+CD130+F11R+ naïve pluripotent cells in cell culture, comprising: culturing human pluripotent stem cells (PSC) in a culture medium containing an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, an AMPK pathway agonist, a ROCK inhibitor, and a TGF-β1R agonist, such that the culture medium generates and maintains human CD7+CD75+CD77+CD130+F11R+ naïve pluripotent cells in culture.

34. The method according to any one of claims 1-33, wherein the PSC grows in an adherent culture format.

35. The method according to claim 34, wherein the PSC grows on a tissue culture plate.

36. The method according to claim 35, wherein the tissue culture plate is coated with gelatin.

37. The method according to claim 35, wherein the tissue culture plate is coated with vitronectin.

38. The method according to claim 35, wherein the tissue culture plate is coated with 39. The method according to claim 35, wherein the tissue culture plate is coated with 40. The method according to any one of claims 1-33, wherein the PSC grows in the form of cell aggregates in suspension culture.

41. The method according to claim 40, wherein the PSC grows in a bioreactor.

42. A culture medium for maintaining and expanding Oct3 / 4+SOX2+NAN0G+ pluripotent stem cells (PSCs) in cell culture, comprising: an Akt pathway agonist, an FGFR pathway agonist, a JAK7STAT pathway antagonist, a PKC pathway antagonist, and an AMPK pathway agonist.

43. The culture medium according to claim 42, wherein the Akt pathway agonist is selected from the group consisting of: SC79, demethylcoclaurine, LM22B-10, YS-49, YS-49 monohydrate, demethylasindoline B1, relesib, N-oleoyl glycine, NSC45586 sodium, periplocin, CHPG sodium salt, bilobalide, 6-hydroxyflavone, muscone, SEW2871, 8-isoprenyl, lasudafedine, and combinations thereof.

44. The culture medium according to claim 43, wherein the Akt pathway agonist is SC79.

45. The culture medium according to any one of claims 42-44, wherein the FGFR agonist is FGF2 or SUN11602.

46. The culture medium according to claim 45, wherein the FGFR agonist is SUN11602.

47. The culture medium according to any one of claims 42-46, wherein the JAK / STAT signal transduction antagonist is selected from the group consisting of: tofacitinib, ruxolitinib, baricitinib, filgotinib, upadacitinib, peficitinib, orantinib, soctinib, desatinib, digalectinib, deucravacitinib, abrocitinib, lestaurtinib, pacritinib, fedratinib, momelotinib, gandotinib, cedazuridine, GS-829845, GSK2586184, AZD1480, R348, VX-509, GLPG0634, JSI-124, TG101348, AC-430, NS-018, CHZ868, SHR0302, INCB039110, BMS-911543, BMS-986165, PF-04965841, PF-04965842, PF-06263276, PF-06651600, and combinations thereof.

48. The culture medium according to claim 47, wherein the JAK / STAT antagonist is tofacitinib.

49. The culture medium according to any one of claims 42-48, wherein the PKC pathway antagonist is selected from the group consisting of: Go6983, sotrastaurin, enzastaurin, staurosporine, LY31615, Go 6976, GF 109203X, Ro 31-8220 mesylate, and combinations thereof.

50. The culture medium according to claim 49, wherein the PKC pathway antagonist is Go6983.

51. The culture medium according to any one of claims 42-50, wherein the AMPK agonist is selected from the group consisting of metformin, AICAR, broussochalcone B, maritimoside, amygdalin, A769662, PF 06409577, metformin hydrochloride, ZLN 024, ZLN024 hydrochloride, nilotinib, phenformin, nilotinib hydrochloride monohydrate, adenosine 5'-monophosphate monohydrate, homoplantaginin, MK 8722, euphorbia factor L1, ASP4132, GSK621, EX229 (compound 991), trans-ferulic acid, O-304, MK3903, BAM 15, ligustroside, ETC-1002, BC1618, IMM-H007, IM156, japonicoside IVa, pachymic acid, 7-methoxyisoflavone, urolithin B, danthron, demethyleneberberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, malvidin chloride-3-O-arabinoside, RSVA405, etilefrine, COH-SR4, buformin, buformin hydrochloride, PT1, fenbufen, 3a-hydroxy mogrosol, amperozide, and combinations thereof.

52. The culture medium according to claim 51, wherein the AMPK pathway agonist is metformin or AICAR.

53. The culture medium according to any one of claims 42-52, which comprises SC79, SUN11602, tofacitinib, Go6983, and metformin.

54. The culture medium according to any one of claims 42-53, which further comprises a TGF-β1 agonist.

55. The culture medium according to claim 54, wherein the culture medium comprises a TGF-β1 agonist.

56. The culture medium according to claim 54 or 55, wherein the TGF-β1 agonist is selected from the group consisting of TGF-β1, SRI-011381, costunolide, activin A, Nodal, DPS-1, and combinations thereof.

57. The culture medium according to claim 56, wherein the TGF-β1 agonist is TGF-β1 or SRI-011381.

58. The culture medium according to any one of claims 42-57, which further comprises a basal medium composition selected from the group consisting of DMEM, F12, IMDM, CDM2, and combinations thereof.

59. The culture medium according to claim 58, wherein the basal medium composition is further supplemented with ascorbic acid and transferrin.

60. The culture medium according to claim 58, wherein the basal medium composition comprises F12 or IMDM medium supplemented with ascorbic acid, transferrin, and penicillin-streptomycin.

61. The culture medium according to claim 58, wherein the basal medium composition comprises selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

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