Compositions and methods for small molecule-based pluripotent stem cell expansion

Abstract

Methods and compositions for the proliferation and maintenance of Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs) or for the generation and maintenance of CD7+CD75+CD77+CD130+F11R+ naive PSCs in cell culture include the use of small molecule-based culture media containing an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, and an AMPK pathway agonist.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 414,265, filed October 7, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Background of the Invention Pluripotent stem cells have the ability to differentiate into all adult cell types, making them a crucial component of regenerative medicine technologies. Therefore, the mass generation of pluripotent cells with highly consistent phenotypes is of great interest. Several methods have previously been established for the propagation of pluripotent stem cells in culture. Some of the most common methods involve co-culture with mouse embryonic fibroblasts (MEFs) or the use of conditioned medium previously exposed to MEFs. Both methods rely on the use of another cell type to secrete components into the culture medium to sustain the pluripotent phenotype and, by definition, are complex media of unknown composition.

[0003] Well-defined media, such as the commercially available mTeSR and Essential 8 (E8) media, are the most commonly used media developed to maintain pluripotency in culture. Both media rely on the use of proteinaceous components, using high concentrations of FGF2 in the presence of low concentrations of TGF-β to sustain the primed phenotype in human pluripotent cultures. However, proteinaceous components are prone to degradation, resulting in variability during their short shelf life.

[0004] Furthermore, these media maintain human pluripotent cells in a primed phenotype more representative of an ectoderm-biased epiblast population, rather than a naive state of pluripotent cells, which represents an early developmental state consisting of a population resembling the inner cell mass of the preimplantation blastula. Primed pluripotent cells have a lower differentiation potential than naive pluripotent cells.

[0005] Thus, although several approaches are available for the maintenance and expansion of pluripotent stem cells in culture, these approaches have limitations and there remains a need in the art for additional methods and compositions for the maintenance and expansion of pluripotent stem cells in culture, particularly those that maintain the cells in a naive state with high differentiation potential. Summary of the Invention

[0006] The present disclosure provides a well-defined medium composition that can sustain the pluripotent phenotype of stem cells in culture, for example, in a naive state (rather than a primed state) with high differentiation potential. Therefore, the disclosed compositions and methods do not rely on complex medium formulations consisting of unknown components. Because the medium composition is composed of defined components, it is less susceptible to degradation and batch variability. In particular, the present disclosure describes compositions and methods for the continuous maintenance of the pluripotent state of stem cells using a defined culture medium composed of small molecule agonists and antagonists. This has the advantage of sustaining the pluripotent state in the absence of protein components that are subject to variability and degradation. In one aspect, the culture medium of the present invention comprises an Akt agonist, an FGF agonist, a JAK / STAT antagonist, a PKC antagonist, and an AMPK agonist.

[0007] In one aspect, a method for maintaining and expanding Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs) in cell culture includes: culturing 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 the markers Oct3 / 4, SOX2, and NANOG.

[0008] 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 thereof. 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.

[0009] In one embodiment, the Akt pathway agonist is selected from the group consisting of SC79, demethyl-coclaurine, LM22B-10, YS-49, YS-49 monohydrate, demethylasteriquinone B1, resilisib, N-oleoylglycine, NSC45586 sodium, periplosin, CHPG sodium salt, bilobalide, 6-hydroxyflavone, musk ketone, SEW2871, 8-prenylnaringenin, razuprotafib, and combinations thereof. In a more particular embodiment, the Akt pathway agonist is SC79 present in the culture medium at a concentration of 1 ng / ml.

[0010] In one embodiment, the FGFR agonist is FGF2 or SUNl 1602. In a more particular embodiment, the FGFR agonist is SUNl 1602, which is present in the culture medium at a concentration of 5 μM.

[0011] In one embodiment, the JAK / STAT signaling antagonist is selected from the group consisting of tofacitinib, ruxolitinib, baricitinib, filgotinib, upadacitinib, peficitinib, oclacitinib, solcitinib, decernotinib, delgocitinib, deuclavacitinib, abrocitinib, lestaurtinib, pacritinib, fedratinib, momelotinib, gandotinib, cerdulatinib, GS-8298 45, 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 particular embodiment, the JAK / STAT antagonist is tofacitinib, which is present in the culture medium at a concentration of 100 nM.

[0012] In one embodiment, 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. In a more particular embodiment, the PKC pathway antagonist is Go6983 present in the culture medium at a concentration of 5 nM.

[0013] In one embodiment, the AMPK agonist is metformin, AICAR, Cadinol B, malein, amarogentin, A 769662, PF 06409577, metformin hydrochloride, ZLN 024, ZLN 024 hydrochloride, nilotinib, phenformin, nilotinib hydrochloride monohydrate, adenosine 5'-monophosphate monohydrate, hispidulin, MK 8722, euphorbia steroid, ASP4132, GSK621, EX229 (compound 991), trans-ferulic acid, O-304, MK 3903, BAM 15, ligustroflavone, ETC-1002, BC1618, IMM-H007, IM156, chrysanthemum saponin IVa, polycholic acid A, 7-methoxyisoflavone, urolithin B, danthrone, demethylene berberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, malvidin-3-O-arabinoside chloride, RSVA 405, etilefrine, COH-SR4, buformin, buformin hydrochloride, PT1, bempedoic acid, 3a-hydrocymogrol, 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 present in the culture medium at a concentration of 500 μM.

[0014] In some embodiments, the culture medium is used in combination with the protein components FGF2 and / or TGF-β, for example, to increase the rate of proliferation of pluripotent cultures.

[0015] 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, bermosudil, fasudil, hydroxyfasudil, ripasudil, netarsudil, and berosudil. In a more particular embodiment, the ROCK inhibitor is Y27632 present in the culture medium at a concentration of 10 μM.

[0016] 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 particular embodiment, the TGF-β1 agonist is TGF-β1 present in the culture medium at a concentration of 2 ng / ml.

[0017] In one embodiment, the culture medium comprises SC79, SUN11602, tofacitinib, Go6983, and metformin. In another embodiment, the culture medium further comprises selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

[0018] In one embodiment, the culture medium comprises 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 comprises F12 or IMDM medium supplemented with ascorbic acid, transferrin, and penicillin-streptomycin. In a more specific embodiment, the basal medium composition comprises 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 comprises selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

[0019] In one embodiment, the TB5i formulation (described herein) is supplemented in a commonly used basal medium. In another embodiment, the TB5i formulation is supplemented in a developed medium formulation. In another embodiment, the TB5i formulation includes the addition of a ROCK inhibitor and / or a cAMP pathway activator.

[0020] In another aspect, a method for generating and maintaining human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells in cell culture includes culturing 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, 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 the cell culture.

[0021] In some embodiments, PSCs are grown in an adherent culture format, such as on tissue culture plates. In one embodiment, the tissue culture plate is coated with gelatin. In another embodiment, the tissue culture plate is coated with vitronectin. In yet another embodiment, the tissue culture plate is coated with MATRIGEL® or GELTREX®. In one embodiment, the TB5i formulation is used to grow pluripotent stem cells in an adherent culture format.

[0022] In other embodiments, PSCs are grown as cell aggregates in suspension culture. In one embodiment, the TB5i formulation is used to grow pluripotent cells as cell aggregates in suspension culture.

[0023] In other embodiments, PSCs are expanded in a bioreactor. In another embodiment, the TB5i formulation is used to expand pluripotent cells in a bioreactor. [Brief explanation of the drawings]

[0024] [Figure 1]Figure 1 shows a schematic of zygote development to determine pluripotency status. All genes monitored throughout the series of HD-DoE experiments described herein are shown within this schematic. Gene markers for specific lineages are indicated. The optimization strategy throughout this disclosure focused on optimizing expression of naive genes while minimizing primed and lineage-specific genes. [Figure 2] Figures 2A-2C show the results of the HD-DoE experiment for the determination of E8 critical process parameter (CPP) media. Figure 2A shows the reaction conditions for each of the 96 reactions performed during the HD-DoE run. Figure 2B shows the effectors and maximum concentrations used in the experiment. Figure 2C shows the basal media used in all of the experimental reactions. [Figure 3] Figures 3A-3D show the results of establishing CPPs for basal media formulations. Figures 3A-3C show the measured genes representing naive (Figure 3A), pluripotent (Figure 3B), and primed (Figure 3C) states, maximized within MODDE software, and show the overall contribution coefficients to effectors. Figure 3D shows the average contribution coefficients for all effectors used, averaged for each representative state. [Figure 4] Figures 4A-4C show the results of an HD-DoE experiment for pluripotency maintenance. Figure 4A shows the reaction conditions for each of the 96 reactions performed during the HD-DoE. Figure 4B shows the effectors and maximum concentrations used within the experiment. Figure 4C shows the basal medium used in all of the experimental reactions. [Figure 5] Figures 5A-5B show the results of NANOG optimization to identify small molecule pluripotency maintenance formulations. Figure 5A shows the use of MODDE software in optimizing key regulators of the pluripotent state as a function of NANOG gene expression. Figure 5B shows the HD-DoE-informed media additives required for maintenance of the pluripotent state. [Figure 6A]Figures 6A-6B show results demonstrating the combined effect of five additives to sustain the pluripotent state. Figure 6A shows all of the genes characterizing the naive, pluripotent, and primed states, where optimization of the relative contribution coefficients of effectors is shown in the heat map shown. The five components most critical for maintaining the pluripotent state are shown. Figure 6B shows a brightfield image showing pluripotent cultures adapted to the TB5i formulation. [Figure 6B] See legend to Figure 6A. [Figure 7A] Figures 7A-7B show a comparison of TB7i and TB5i media. Figure 7A shows a comparison of day-to-day PSC growth using TB7i and TB5i media formulations. Control cultures were supplemented with FGF2 and TGF-β1. Figure 7B shows individual colonies monitored over consecutive days. [Figure 7B] See legend to Figure 7A. [Figure 8] Figures 8A-8C show that inclusion of FGF2 and TGF-β in TB5i media increases the naive phenotype. Figure 8A shows the effect of including the proteinaceous components FGF2, TGF-β, and insulin in the media when assayed in the presence and absence of TB5i media, where T is TGF-β, F is FGF2, and I is insulin. Figure 8B shows IHC validation of the pluripotent state. Figure 8C shows the underlying rationale behind the use of TB5i media formulations. [Figure 9] Figures 9A-9D show that TB5i medium can sustain pluripotent cells in suspension within a bioreactor. Figure 9A shows a table listing two media formulations used in a 100 ml PBS Vertical Wheel bioreactor. Figure 9B is a graph showing the overall growth of pluripotent cells in the bioreactor compared to STEMSCALE™, a commercially available medium for suspension culture. Figure 9C shows the average aggregate size over a 4-day reactor run. Figure 9D shows aggregates harvested from the bioreactor and plated onto vitronectin-coated plates overnight, then immunofluorescently stained for pluripotency markers the next day. [Figure 10]

[0023] Figure 1 shows the results of a suspension culture validation of TB5i pluripotency maintenance medium. Results show aggregate formation and growth in PBS bioreactors over 4 days in the different medium formulations shown. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description of the Invention Various aspects of the invention are described in further detail in the following subsections.

[0026] I. cell The starting cells for the culture are pluripotent stem cells (PSCs), such as human pluripotent stem cells (hPSCs). Generally, PSCs or hPSCs are defined as stem cells that can differentiate into all cell types of an adult organism, including those characteristic of each germ cell layer (endoderm, mesoderm, and ectoderm). As used herein, the pluripotent state is used in relation to PSCs or hPSCs that express key markers specific for, for example, 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).

[0027] As used herein, the terms "induced pluripotent cells" and "iPSCs" refer to cells taken from a late stage of development that have been induced to have an expression pattern consistent with pluripotency. The source of the cells can be either embryonic or adult. In one embodiment, the iPSCs are the iPSC cell line CR01 (NIH). Further non-limiting examples of induced pluripotent stem cells (iPSCs) include 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 ES03 cells (WiCell Research Institute) and H9 cells (Thomson, JA et al. (1998) Science 282:1145-1147). Human pluripotent stem cells (PSCs) express cell markers that can be used to identify cells as PSCs. 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.

[0028] As used herein, the terms "human embryonic stem cells" and "hESCs" refer to pluripotent cells derived from the inner cell mass of a human blastocyst embryo. The term "inner cell mass" refers to the mass of cells located in the anterior region of the early blastula that gives rise to the entire embryonic body. Important markers for iPSCs or hESCs include, but are not limited to, OCT3 / 4, SOX2, NANOG, and SSEA4. Furthermore, these cells are in a proliferative self-renewal state, accompanied by the expression of TERT and MKi67.

[0029] Both the naive and primed states are considered pluripotent cell states. As used herein, the term "primed state" refers to pluripotent stem cells with an ectodermal bias. This is because the cultures are usually grown in the presence of FGF2 and / or TGF-β. Most commercially available media for maintaining human pluripotency are in this state. The term "naive state" is used in reference to pluripotent cells with greater differentiation potential and characteristics more representative of the inner cell mass, lacking the ectodermal bias characteristic of the primed state in which human pluripotent cells are usually cultured.

[0030] Following initial induction, human embryonic stem cells were previously shown to exhibit distinct genotypic and phenotypic differences from mouse pluripotent cells. Among these differences, mouse embryonic stem cells proliferated faster and produced raised colonies, whereas human embryonic colonies were flatter. These differences were later shown to represent a priming event in hESC cultures, making these cultures more representative of an epiblast population with an ectoderm bias. Induced pluripotent cell cultures followed a similar path, as media conditions used for hESC cultures were adopted for iPSC expansion. Thus, iPSC cultures exhibited a primed phenotype.

[0031] The naive state represents an early developmental state consisting of a population resembling the inner cell mass of the preimplantation blastula, which has greater differentiation potential. The primed state is more representative of the postimplantation epiblast population, which has a preference for differentiation along ectodermal lineages. Advantages of expanding iPSC cells in the naive state include faster proliferation rates, increased differentiation potential, and single-cell clonality. The latter potentially obviates aggregate formation during passaging events and the use of ROCK inhibitors. These factors highlight the benefits of developing growth media capable of expanding the naive state in bioreactor-based platforms.

[0032] Although a growing body of research suggests beneficial attributes of the naive pluripotent state, no commercially available media are currently known to be available for the expansion and maintenance of naive pluripotent cells. Furthermore, most commercially available pluripotency maintenance media rely on incorporating protein components that induce a primed phenotype, significantly increasing the cost of the media. For these reasons, we designed experiments to address the possibility of inducing and maintaining the naive pluripotent state, focusing on identifying small molecules that can mediate this transition. The use of a novel systems biology platform-informed approach, capable of assessing complex interactions in a multidimensional experimental space, enables the discovery of complex combinatorial interactions between several signaling pathways for maintaining pluripotency. Through analysis of a series of experiments, we developed a small-molecule-based culture medium capable of sustaining PSC pluripotency in general, or sustaining the expansion of the naive phenotype in particular.

[0033] II. Culture medium components In one aspect, the disclosed methods relate to the maintenance and expansion of Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs), including primed and / or naive PSCs, in cell culture. The methods involve the use of small molecule-based culture media containing specific agonists and / or antagonists of cell signaling pathways. In some embodiments, the culture media lack serum, lack exogenously added growth factors, lack animal products, are serum-free, xeno-free, and / or do not contain a feeder layer.

[0034] As used herein, an "agonist" of a cell signaling pathway is used in reference to an agent that stimulates (upregulates) a cell signaling pathway. In some embodiments, stimulation of a cell signaling pathway can be initiated extracellularly, for example, by the use of an agonist (e.g., an agonist can be a receptor ligand) that activates a cell surface receptor involved in the signaling pathway. Additionally, or alternatively, stimulation of cell signaling can be initiated intracellularly, for example, by the use of a small molecule agonist that interacts intracellularly with one or more components of the signaling pathway.

[0035] As used herein, an "antagonist" of a cell signaling pathway is used in reference to an agent that inhibits (downregulates) a cell signaling pathway. In some embodiments, inhibition of a cell signaling pathway can be initiated extracellularly, for example, by the use of an antagonist that blocks a cell surface receptor involved in the signaling pathway. Additionally or alternatively, inhibition of cell signaling can be initiated intracellularly, for example, by the use of a small molecule antagonist that interacts intracellularly with one or more components of the signaling pathway.

[0036] The agonist and antagonist used in the method of the present disclosure are known and / or commercially available.They are used in culture medium at effective concentrations to achieve desired results, for example, the generation, expansion and / or maintenance of primed or naive PSCs, which are characterized by the specific corresponding markers described herein.The non-limiting examples of suitable agonist and antagonist agents and effective concentration ranges are further described below.

[0037] In one aspect, a method for maintaining and expanding Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs) in cell culture includes: culturing 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 the markers Oct3 / 4, SOX2, and NANOG.

[0038] 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 thereof. In one embodiment, the PSCs are human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells. In another embodiment, the PSCs express one or more of KLF2 / 4 / 5, ZFP42, ESRRB, DAPP3 / 5, TFCP2L1, FGF4, TBX3, CDH1, PECAM, CD31, NR5A2, and IDID1.

[0039] Agonists of the Akt pathway include agents, molecules, compounds, or substances that can stimulate (upregulate) the signaling pathways of one or more of the serine / threonine kinase Akt family members, 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 proto-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, demethyl-coclaurine, LM22B-10, YS-49, YS-49 monohydrate, demethylasteriquinone B1, resilisib, N-oleoylglycine, NSC45586 sodium, periplosin, CHPG sodium salt, bilobalide, 6-hydroxyflavone, musk ketone, SEW2871, 8-prenylnaringenin, razuprotafib, and combinations thereof.

[0040] In one embodiment, the Akt pathway agonist is present in the culture medium at a concentration within the 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 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 present in the culture medium at a concentration of 1 ng / ml.

[0041] Agonists of the FGFR pathway include agents, molecules, compounds, or substances that can activate (upregulate) signaling through the fibroblast growth factor 2 (FGF2) signaling pathway. In one embodiment, the FGFR pathway agonist is FGF2 or SUN11602.

[0042] In one embodiment, the FGFR pathway agonist is present in the culture medium at a concentration of 100-500 μM, 200-400 μM, or 250-350 μM. In another embodiment, the FGFR pathway agonist is SUN11602 present in the culture medium at a concentration of 1-15 μM, 2-10 μM, or 3-7 μM. In another embodiment, the FGFR pathway antagonist is SUN11602 present in the culture medium at a concentration of 5 μM.

[0043] Antagonists of the JAK / STAT pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) signaling through the JAK / STAT signaling pathway. In one embodiment, the JAK / STAT pathway antagonist is tofacitinib, ruxolitinib, baricitinib, filgotinib, upadacitinib, peficitinib, oclacitinib, solcitinib, decernotinib, delgocitinib, deuclavacitinib, abrocitinib, lestaurtinib, pacritinib, fedratinib, momelotinib, gandotinib, cerduratinib, GS-829845, GSK258 6184, 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.

[0044] In one embodiment, the JAK / STAT pathway antagonist is present in the culture medium at a concentration within the range of 25-250 nM, 50-150 nM, or 75-125 nM. In another embodiment, the JAK / STAT pathway antagonist is tofacitinib present in the culture medium at a concentration of 25-250 nM, 50-150 nM, or 75-125 nM. In another embodiment, the JAK / STAT pathway antagonist is tofacitinib present in the culture medium at a concentration of 100 nM.

[0045] PKC pathway antagonists include agents, molecules, compounds, or substances that can inhibit (downregulate) signaling through the PKC signaling pathway. In one embodiment, 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.

[0046] In one embodiment, the PKC pathway antagonist is present in the culture medium at a concentration ranging from 2 to 10 nM, 2.5 to 7.5 nM, 3 to 6.50 nM, or 4 to 6 nM. In another embodiment, the PKC pathway antagonist is Go6983 present in the culture medium at a concentration of 2 to 10 nM, 2.5 to 7.5 nM, 3 to 6.50 nM, or 4 to 6 nM. In another embodiment, the PKC pathway antagonist is Go6983 present in the culture medium at a concentration of 5 nM.

[0047] Agonists of the AMPK pathway include agents, molecules, compounds, or substances that can activate (upregulate) signaling through the AMPK signaling pathway.In one embodiment, the AMPK pathway agonist is metformin, AICAR, Cadinol B, malein, amarogentin, A 769662, PF 06409577, metformin hydrochloride, ZLN 024, ZLN 024 hydrochloride, nilotinib, phenformin, nilotinib hydrochloride monohydrate, adenosine 5'-monophosphate monohydrate, hispidulin, MK 8722, euphorbia steroid, ASP4132, GSK621, EX229 (compound 991), trans-ferulic acid, O-304, MK 3903, BAM 15, ligstroflavone, ETC-1002, BC1618, IMM-H007, IM156, chrysanthemum saponin IVa, polychoric acid A, 7-methoxyisoflavone, urolithin B, danthron, demethylene berberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, malvidin-3-O-arabinoside chloride, RSVA 405, etilefrine, COH-SR4, buformin, buformin hydrochloride, PT1, bempedoic acid, 3a-hydrocymogrol, ampquinone, and combinations thereof.

[0048] In one embodiment, the AMPK pathway antagonist is present in the culture medium at a concentration of 200-1000 μM, 250-750 μM, 300-650 μM, or 400-600 μM. In another embodiment, the AMPK pathway antagonist is metformin present in the culture medium at a concentration of 200-1000 μM, 250-750 μM, 300-650 μM, or 400-600 μM. In another embodiment, the AMPK pathway antagonist is metformin present in the culture medium at a concentration of 500 μM.

[0049] In one embodiment, the small molecule culture medium comprises a basal medium composition supplemented with SC79, SUN11602, tofacitinib, Go6983, and metformin. In a more particular 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.

[0050] In one embodiment, the small molecule 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 culture medium comprises a basal medium composition supplemented with ascorbic acid and transferrin.

[0051] In one embodiment, the small molecule culture medium comprises a basal medium composition comprising F12 or IMDM medium supplemented with ascorbic acid, transferrin, and penicillin-streptomycin. In a more particular embodiment, the basal medium composition in the culture medium comprises 1:1 F12 / IMDM medium supplemented with 20 μg / ml ascorbic acid, 10 μg / ml transferrin, and 1% penicillin-streptomycin. In another embodiment, the small molecule culture medium comprises a basal medium composition comprising 1:1 F12 / IMDM medium supplemented with 20 μg / ml ascorbic acid, 10 μg / ml transferrin, and 1% penicillin-streptomycin, the basal medium composition further supplemented with either 100 ng / ml FGF2 and 2 ng / ml TGF-β1 (hereinafter "TB5i medium formulation") or 10 μM Y27632 and 1 μM forskolin (hereinafter "TB7i medium formulation").

[0052] In some embodiments, the small molecule-based culture medium comprises a basal medium composition comprising selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

[0053] In some embodiments, small molecule-based culture media are used in combination with the protein components FGF2 and / or TGF-β1 to increase the rate of proliferation of pluripotent stem cell cultures.

[0054] 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.

[0055] ROCK inhibitors include agents, molecules, compounds, or substances that can inhibit (downregulate) signaling through the Rho kinase pathway. In one embodiment, the ROCK inhibitor is selected from the group consisting of Y27632, H1152, GSK429286A, RKI-1447, DJ4, thiazovivin, belmosudil, fasudil, hydroxyfasudil, ripasudil, netarsudil, and velosudil.

[0056] In one embodiment, the ROCK inhibitor is present in the culture medium at a concentration within the range of 2 to 50 μM, 3 to 30 μM, 5 to 20 μM, or 7.5 to 15 μM. In another embodiment, the ROCK inhibitor is Y27632 present in the culture medium at a concentration of 2 to 50 μM, 3 to 30 μM, 5 to 20 μM, or 7.5 to 15 μM. In another embodiment, the ROCK inhibitor is Y27632 present in the culture medium at a concentration of 10 μM.

[0057] TGF-β1 pathway agonists include agents, molecules, compounds, or substances that can activate (upregulate) signaling through the TGF-β1 signaling pathway. In some embodiments, 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.

[0058] In one embodiment, the TGF-β1 pathway agonist is present in the culture medium at a concentration in the range of 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 present in the culture medium at a concentration in the range of 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 present in the culture medium at a concentration of 2 ng / ml.

[0059] In another aspect, a method for generating and maintaining human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells in cell culture includes: culturing pluripotent stem cells (PSCs) in a culture medium according to the present application 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 the cell culture.

[0060] In another aspect, the present application provides small molecule-based culture media for the growth, maintenance, and expansion of Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs), as described herein, and for the generation, growth, maintenance, and expansion of human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells in cell culture.

[0061] 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 medium composition of the present application is supplemented with the aforementioned agonists and antagonists at the concentrations described above.

[0062] In one embodiment, the Akt pathway agonist is SC79 present in the culture medium at a concentration of 1 ng / ml. In another embodiment, the FGFR agonist is SUN11602 present in the culture medium at a concentration of 5 μM. In another embodiment, the JAK / STAT antagonist is tofacitinib present in the culture medium at a concentration of 100 nM. In another embodiment, the PKC pathway antagonist is Go6983 present in the culture medium at a concentration of 5 nM. In another embodiment, the AMPK pathway agonist is metformin present in the culture medium at a concentration of 500 μM. In a preferred embodiment, the small molecule 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.

[0063] In another embodiment, the small molecule culture medium comprises a basal medium composition further supplemented with a ROCK inhibitor, wherein the ROCK inhibitor is Y27632, and wherein the Y27632 is present in the culture medium at a concentration of 10 μM.

[0064] In another embodiment, the small molecule culture medium comprises a basal medium composition further supplemented with a TGF-β1 agonist, wherein the TGF-β1 agonist is TGF-β1, and the TGF-β1 is present in the culture medium at a concentration of 2 ng / ml.

[0065] In a preferred embodiment, the small molecule culture medium comprises a basal medium composition comprising 1:1 F12 / IMDM medium supplemented with 20 μg / ml ascorbic acid, 10 μg / ml transferrin, and 1% penicillin-streptomycin, which is supplemented with 1 ng / ml SC79, 5 μM SUN11602, 100 nM tofacitinib, 5 nM Go6983, and 500 μM metformin, and further supplemented with either 100 ng / ml FGF2 and 2 ng / ml TGF-β1 (i.e., TB5i medium formulation) or 10 μM Y27632 and 1 μM forskolin (i.e., TB7i medium formulation).

[0066] If an agonist or antagonist is used in more than one step of the method, in one embodiment, the same agonist or antagonist is used in each step in which the agent is present in the culture medium, hi another embodiment, different agonists or antagonists that affect the same signaling pathway are used in different steps of the method.

[0067] If an agonist or antagonist is used in more than one step of the method, in one embodiment, the same concentration of agonist or antagonist is used in each step in which the agent is present in the culture medium, hi another embodiment, different concentrations of the same agonist or antagonist are used in different steps of the method.

[0068] III. Culture conditions In combination with the chemically defined and optimized culture medium described in Subsection II above, the methods for maintaining, expanding, and generating PSC cells described above utilize standard culture conditions established in the art for cell culture, such as culturing cells at 37°C and 5% CO2.

[0069] In some embodiments, PSCs are cultured in an adherent culture format using standard culture vessels or plates, such as 6-well, 24-well, or 96-well tissue culture (TC) plates, using the culture medium described herein with daily medium changes. In certain embodiments, PSCs are 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 MATRIGEL®. In another embodiment, the TC plate is coated with GELTREX®.

[0070] The culture medium described herein (e.g., TB5i formulation) has been shown to be effective in expanding and maintaining adherent cultures grown in tissue culture plates. In one exemplary embodiment, PSC cultures, e.g., the CR01 iPSC line, are grown and maintained on vitronectin-coated 6-well TC plates using the culture medium of the present application, e.g., TB5i medium. PSC cultures are generally passaged every 3-4 days and treated with agents to disrupt cell-cell adhesion, such as EDTA or digestive enzymes, e.g., collagenase, accutase, trypsin, or TyrPLE. This can be accomplished by removing the medium and washing each well of the TC plate with 2 ml of PBS. A 3-minute incubation in the presence of 5 mM EDTA can then be performed at 37°C. The wells are then aspirated, and the cells are washed off the plate and seeded in fresh medium. Each passaged well is typically seeded into six wells of a fresh vitronectin-coated TC plate, resulting in an expansion of iPSC lines from one to six.

[0071] In some embodiments, suspension cultures of PSCs can be grown as cell aggregates in a bioreactor, as further described below in Example 3. In one exemplary embodiment, a TB5i media formulation can be used to grow PSCs in suspension culture in a 100 ml PBS VW bioreactor at 60 RPM for 5 consecutive days, with semi-depletion on day 1 and every other day thereafter.

[0072] IV. use The culture media described herein, such as the TB5i medium formulation, can be used to maintain and expand Oct3 / 4+SOX2+NANOG+ PSCs in cell culture, including those in both primed and naive states of differentiation. Furthermore, the culture media can be used to generate and maintain human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells from primed PSCs in cell culture. The ability to maintain and expand pluripotent cells in culture using the disclosed compositions and methods allows for the production of large quantities of these cells, for example, for a variety of regenerative medicine purposes.

[0073] V. composition In other aspects, the present disclosure provides methods and related compositions, including culture media and cell cultures, for the maintenance and expansion of Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs) in cell culture, as well as methods and related compositions, including culture media and cell cultures, for generating and maintaining human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells in cell culture.

[0074] Thus, in one aspect, the present disclosure provides a culture medium for the maintenance and expansion of Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs), comprising an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, and an AMPK pathway agonist.

[0075] In certain embodiments, the culture medium further comprises a ROCK inhibitor, a TGF-β1 agonist, or both.

[0076] In certain embodiments, the culture medium further comprises a basal medium composition. In certain embodiments, the basal medium composition comprises a 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 comprises an F12 or IMDM medium supplemented with ascorbic acid, transferrin, and penicillin-streptomycin. In certain embodiments, the basal medium composition comprises selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

[0077] In another aspect, the present disclosure provides an isolated cell culture comprising Oct3 / 4+SOX2+NANOG+ PSCs cultured in one of the media formulations disclosed herein. Accordingly, in one embodiment, the present disclosure provides an isolated cell culture comprising Oct3 / 4+SOX2+NANOG+ PSCs cultured 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. In certain embodiments, the culture medium further comprises a ROCK inhibitor, a TGF-β1 agonist, or both. In certain embodiments, the culture medium further comprises a basal medium composition. In certain embodiments, the basal medium composition comprises a 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 comprises F12 or IMDM medium supplemented with ascorbic acid, transferrin, and penicillin-streptomycin, hi certain embodiments, the basal medium composition comprises selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

[0078] The present invention is further illustrated by the following examples, which should not be construed as further limiting. All references, patents, and published patent applications cited in the figures, as well as throughout this application, are hereby expressly incorporated by reference. [Example]

[0079] Example 1 Current pluripotency culture media passively maintain pluripotency A network of 54 genes was selected to monitor the differentiation state of pluripotent cultures and track the primed and naive states, as well as any early lineage commitment biases that may arise during the culture process (Figure 1). Other genes monitored throughout this series of experiments include TERT and KI67. Maximum TERT expression is required to sustain the pluripotent state in both naive and primed cells, while KI67, a proliferation marker, is expected to increase in the naive state. Housekeeping genes measured within this QS chip design were used to normalize individual experimental runs. This network of genes was then monitored using HD-DoE methodology (Bukys et al. (2020) Iscience 23:101346), as further described in Example 2, allowing for in silico modeling of the pluripotent state.

[0080] The initial HD-DoE modeling experiment was designed to determine whether any of the components within Essential 8 (E8) medium were important for maintaining pluripotency. The results of this experiment are shown in Figure 2. The components of E8 medium include HEPES, bicarbonate, selenium, ascorbic acid, transferrin, insulin, FGF2, and TGF-β. HEPES, bicarbonate, and selenium were not considered in this HD-DoE design because they are additives that are not specific to maintaining the pluripotent state but are involved in enabling the overall growth of any cell in culture. Furthermore, while these components are present in most common media formulations, the initial HD-DoE experiment herein utilizes a 1:1 F12 / IMDM hybrid medium that already contains HEPES, bicarbonate, and selenium. In addition to E8 components, 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) were evaluated within this design (Figure 2). These components were selected based on previous experiments demonstrating their potential contribution to the maintenance of the pluripotent state.

[0081] Examination of the overall contribution of the aforementioned effectors assayed demonstrated that the only E8 components beneficial to maintaining the pluripotent state were ascorbic acid and transferrin (Figure 3). The only other components within this design determined to potentially aid in maintaining pluripotency were the PKC inhibitor, Go6983, and the ROCK inhibitor, Y27632. The overall pluripotent state was driven by Go6983, while the naive state was supported by Y27632. All other components within this design either exhibited opposing contributions to the pluripotent state or were demonstrated to be clear lineage drivers.

[0082] Example 2 :Defining signaling pathways critical for maintaining pluripotency Using data-driven, high-dimensional design of experiments (HD-DoE)-based variation of pluripotent cultures, several cell signaling pathways known to function within the pluripotent state were assayed. The HD-DOE method was applied to identify conditions for directly inducing a naive state from a pluripotent stem cell state. This example utilizes a method previously described by Bukys et al. (2020) Iscience 23:101346, which uses computational design geometry to simultaneously test multiple process inputs and provide a mathematical model of deep effector / response space. This method enables the discovery of combinatorial signaling inputs that control complex differentiation processes and allows for the rational testing of multiple key process parameters that influence output responses, such as gene expression. Because gene expression provides a hallmark of the phenotype of, for example, human cells, this method can be applied to identify and understand signaling pathways that control cell fate.

[0083] To develop cell culture recipes for expanding and maintaining PSCs in cell culture and for the differentiation of stem cells into naive progenitor cells, we tested and modeled the effects of agonists and antagonists (referred to herein as effectors) of multiple signaling pathways on the expression of preselected genes. The impact of each effector on gene expression levels was defined by a parameter called the factor contribution, which was calculated for each effector during modeling. These effectors are small molecules or proteins commonly used to promote the differentiation of stem cells into specific fates. The selection of effectors was based on current literature on the differentiation of stem cells into naive progenitor cells.

[0084] Both the PKC inhibitor GO6983 and the ROCK inhibitor Y27632 were included in the HD-DoE design (Figure 4A-4B). For this variation matrix (Figure 4C) and all follow-up validation experiments, ascorbic acid and transferrin were added to the basal medium. Focusing on maximizing NANOG as a surrogate for the pluripotent state and a key driver of the naive state, we determined that synergistic pathway driving for pluripotency can be achieved through the combined effects of activating the Akt, FGF, AMPK, and cAMP pathways while antagonizing the Jak-Stat, ROCK, and PKC pathways (Figure 5A).

[0085] Further analysis by sequential optimization of all measured genes representing the naive state, primed state, or general pluripotency markers suggested that neither the cAMP activator forskolin nor the ROCK pathway inhibitor was critical for this process (Figure 6A). Initial validation of TB5i medium demonstrated that colonies rapidly began to cluster tightly, with a raised phenotype characteristic of the naive state (Figure 6B). Direct comparison of TB5i and TB7i confirmed that neither forskolin nor Y27632 were beneficial for the overall expansion of pluripotent cells (Figures 7A-7B).

[0086] The protein additives FGF2, TGF-β1, and insulin were then assayed as additives in the TB5i formulation (Figure 8A). It was determined that insulin had little, if any, beneficial effect on the cultures, thereby confirming the preliminary HD-DoE analysis shown in Figure 3D. While FGF2 increased the proliferation rate of the cultures, TGF-β1 maintained the normal morphology of pluripotent colony outlines, thereby confirming the passive nature of the Essential 8 formulation. Maintenance of pluripotency in TB5i medium was confirmed by immunohistochemistry for the expression of OCT3 / 4 and SOX2. Cultures maintained in both TB5i and TB5i supplemented with TGF-β1 and FGF2 exhibited a higher density phenotype than control cultures (Figure 8B).

[0087] Example 3 TB5i-mediated bioreactor-based pluripotent aggregate expansion To determine whether the TB5i formulation could sustain pluripotency in suspension culture, a PBS VW bioreactor system was used. Three experimental conditions were performed. The first bioreactor served as a control, with cells grown in STEMSCALE™, a commercially available (Gibco) proprietary suspension medium for pluripotent culture in suspension. Cells in the second and third bioreactors were grown in TB5i medium or in TB5i medium supplemented with FGF2 and TGF-β1, respectively (Figure 9A). Consistent with previous observations (Figure 8), both cultures grown in TB5i medium showed proliferation throughout the bioreactor run (Figure 9B), with increased proliferation in TB5i supplemented with FGF2 and TGF-β1. Aggregate proliferation peaked at day 3 (Figures 9C and 10A). Verification of the pluripotent state was achieved by plating the aggregates and performing IHC analysis. Pluripotency markers OCT3 / 4, SOX2, NANOG, and SSEA4 were expressed throughout cultures grown in both conditions (Fig. 9D).

[0088] equivalent 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 invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A method for the maintenance and expansion of Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs) in cell culture, comprising the steps of: 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, and an AMPK pathway agonist, such that the culture medium maintains the PSCs in a primed or naive state containing the markers Oct3 / 4, SOX2, and NANOG.

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

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

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

5. 5. The method of any one of claims 1 to 4, wherein the Akt pathway agonist is selected from the group consisting of SC79, demethyl-coclaurine, LM22B-10, YS-49, YS-49 monohydrate, demethylasteriquinone B1, resilisib, N-oleoylglycine, NSC45586 sodium, periplosin, CHPG sodium salt, bilobalide, 6-hydroxyflavone, musk ketone, SEW2871, 8-prenylnaringenin, razuprotafib, and combinations thereof.

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

7. 7. The method of claim 6, wherein the Akt pathway agonist is SC79 present in the culture medium at a concentration of 1 ng / ml.

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

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

10. 9. The method of claim 8, wherein the FGFR agonist is SUN11602, which is present in the culture medium at a concentration of 5 μM.

11. The JAK / STAT signaling antagonist is selected from the group consisting of tofacitinib, ruxolitinib, baricitinib, filgotinib, upadacitinib, peficitinib, oclacitinib, solcitinib, decernotinib, delgocitinib, deuclavacitinib, abrocitinib, lestaurtinib, pacritinib, fedratinib, momelotinib, gandotinib, cerdulatinib, GS-829845, GSK2586184, and AZD14. 80, 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. 12. The method of claim 11, wherein the JAK / STAT antagonist is tofacitinib.

13. 12. The method of claim 11, wherein the JAK / STAT antagonist is tofacitinib present in the culture medium at a concentration of 100 nM.

14. 14. The method of any one of claims 1 to 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. 15. The method of claim 14, wherein the PKC pathway antagonist is Go6983.

16. 15. The method of claim 14, wherein the PKC pathway antagonist is Go6983 present in the culture medium at a concentration of 5 Nm.

17. The AMPK agonist is selected from the group consisting of metformin, AICAR, Cadinol B, malein, amarogentin, A 769662, PF 06409577, metformin hydrochloride, ZLN 024, ZLN 024 hydrochloride, nilotinib, phenformin, nilotinib hydrochloride monohydrate, adenosine 5'-monophosphate monohydrate, hispidulin, MK 8722, euphorbia steroid, ASP4132, GSK621, EX229 (compound 991), trans-ferulic acid, O-304, MK 3903, BAM 17. The method of any one of claims 1 to 16, wherein the active ingredient is selected from the group consisting of 15, ligstroflavone, ETC-1002, BC1618, IMM-H007, IM156, Chrysanthemum chinense saponin IVa, polychoric acid A, 7-methoxyisoflavone, urolithin B, danthron, demethylene berberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, malvidin-3-O-arabinoside chloride, RSVA 405, etilefrine, COH-SR4, buformin, buformin hydrochloride, PT1, bempedoic acid, 3a-hydrocymogrol, ampkinone, and combinations thereof.

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

19. 18. The method of claim 17, wherein the AMPK pathway agonist is metformin, which is present in the culture medium at a concentration of 500 μM.

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

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

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

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

25. 24. The method of claim 23, wherein the TGF-β1 agonist is TGF-β1 present in the culture medium at a concentration of 2 ng / ml.

26. 26. The method of any one of claims 1 to 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. 27. The method of claim 26, wherein said basal medium composition is further supplemented with ascorbic acid and transferrin.

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

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

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

31. The method of any one of claims 1 to 30, wherein the PSCs are human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells.

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

33. 1. A method for generating and maintaining human CD7+CD75+CD77+CD130+F11R+ naive pluripotent cells in cell culture, comprising the steps of: Culturing human 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, 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 culture.

34. 34. The method of any one of claims 1 to 33, wherein the PSCs are grown in an adherent culture format.

35. 35. The method of claim 34, wherein the PSCs are grown on tissue culture plates.

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

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

38. 36. The method of claim 35, wherein the tissue culture plate is coated with MATRIGEL®.

39. 36. The method of claim 35, wherein the tissue culture plate is coated with GELTREX®.

40. 34. The method of any one of claims 1 to 33, wherein the PSCs are grown as cell aggregates in suspension culture.

41. 41. The method of any one of claims 40, wherein the PSCs are expanded in a bioreactor.

42. A culture medium for the maintenance and expansion of Oct3 / 4+SOX2+NANOG+ pluripotent stem cells (PSCs) in cell culture, comprising an Akt pathway agonist, an FGFR pathway agonist, a JAK / STAT pathway antagonist, a PKC pathway antagonist, and an AMPK pathway agonist.

43. 43. The culture medium of claim 42, wherein the Akt pathway agonist is selected from the group consisting of SC79, demethyl-coclaurine, LM22B-10, YS-49, YS-49 monohydrate, demethylasteriquinone B1, resilisib, N-oleoylglycine, NSC45586 sodium, periplosin, CHPG sodium salt, bilobalide, 6-hydroxyflavone, musk ketone, SEW2871, 8-prenylnaringenin, razuprotafib, and combinations thereof.

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

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

46. 46. ​​The culture medium of claim 45, wherein the FGFR agonist is SUN11602.

47. The JAK / STAT signaling antagonist is selected from the group consisting of tofacitinib, ruxolitinib, baricitinib, filgotinib, upadacitinib, peficitinib, oclacitinib, solcitinib, decernotinib, delgocitinib, deuclavacitinib, abrocitinib, lestaurtinib, pacritinib, fedratinib, momelotinib, gandotinib, cerdulatinib, GS-829845, GSK2586184, and 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. 48. The culture medium of claim 47, wherein the JAK / STAT antagonist is tofacitinib.

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

51. The AMPK agonist is selected from the group consisting of metformin, AICAR, Cadinol B, malein, amarogentin, A 769662, PF 06409577, metformin hydrochloride, ZLN 024, ZLN 024 hydrochloride, nilotinib, phenformin, nilotinib hydrochloride monohydrate, adenosine 5'-monophosphate monohydrate, hispidulin, MK 8722, euphorbia steroid, ASP4132, GSK621, EX229 (compound 991), trans-ferulic acid, O-304, MK 3903, BAM 51. The culture medium of any one of claims 42 to 50, wherein the active ingredient is selected from the group consisting of 15, ligstroflavone, ETC-1002, BC1618, IMM-H007, IM156, Chrysanthemum japonin IVa, polychoric acid A, 7-methoxyisoflavone, urolithin B, danthron, demethylene berberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, malvidin-3-O-arabinoside chloride, RSVA 405, etilefrine, COH-SR4, buformin, buformin hydrochloride, PT1, bempedoic acid, 3a-hydrocymogrol, ampquinone, and combinations thereof.

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

53. 53. The culture medium of any one of claims 42 to 52, comprising SC79, SUN11602, tofacitinib, Go6983, and metformin.

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

55. 55. The culture medium of claim 54, comprising a TGF-β1 agonist.

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

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

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

59. 59. The culture medium of claim 58, wherein said basal medium composition is further supplemented with ascorbic acid and transferrin.

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

61. 59. The culture medium of claim 58, wherein said basal medium composition comprises selenium, ascorbic acid, transferrin, FGF2, and TGF-β1.

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