Serum-free human pluripotent stem cell culture medium

Abstract

A cell culture medium is provided, comprising an enriched basal medium supplemented with ascorbic acid, a member of the fibroblast growth factor (FGF) superfamily, a transforming growth factor-β (TGF-β) superfamily ligand, and a stable glutamine source. Methods for long-term culturing of stem cells in such culture medium, stem cell populations, and kits are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Patent Application No. 16 / 679,932, filed November 11, 2019, the disclosure of which is incorporated herein by reference. [Technical field]

[0002] The present invention relates to a cell culture medium for the expansion and / or long-term maintenance of pluripotent stem cells. [Background technology]

[0003] Stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great promise in disease modeling, tissue engineering and cell-based therapy due to their unique combination of two properties: pluripotency and high proliferative capacity. However, to date, the therapeutic potential of these cells remains largely unrealized.

[0004] The use of stem cells in translational biomedical research has many major technical hurdles to overcome. Particular challenges are the development of efficient methods for stem cell growth under chemically defined and cost-effective conditions, and the production costs of clinical grade stem cells, which are several orders of magnitude higher than other types of human cells. A significant cost is associated with the special coating matrices and growth media involved in stem cell growth, with the specialized growth media typically costing anywhere between 400-1000% of the cost of the matrix.

[0005] The first human embryonic stem cell (hESC) line was derived from a normal human embryo in 1998 by James Thomson (Thomson et al., Science 282, 1145-7 (1998)). Culture conditions include an irradiated mouse embryonic fibroblast (MEF) feeder layer and 20% fetal bovine serum medium. This culture method is labor intensive, difficult to scale up, and it is difficult to consistently maintain undifferentiated hESCs due to undefined components from the fetal bovine serum and MEFs.

[0006] Over the past decade, advances have been made in the production of synthetic (chemically defined) hPSC media to enable the maintenance and growth of hESCs and induced pluripotent stem cells (iPSCs) under feeder-free conditions (Takahashi et al., Cell 131, 861-72 (2007); Yu et al., Science 318, 1917-20 (2007)). The first synthetic feeder-free stem cell medium for hPSC culture, mTeSR1 medium (Ludwig et al., Nat. Biotechnol. 24, 185-7 (2006)), contained several additional components over basal media. Subsequently, a simplified version of mTeSR1, Essential 8 (E8) medium (Chen et al., Nat. Methods 8, 424-9 (2011)), was developed to reduce the number of additional components. However, the proliferation rate of stem cells in chemically defined media and the material cost of the media are still limiting steps in the generation of large quantities of cells required for clinical research. Although efforts have been made to make synthetic media simpler and less expensive to produce, the complex combinatorial effects of various components have so far made it difficult to design a proliferation medium that is completely chemically defined and can support rapid proliferation of stem cells without adversely affecting pluripotency. Summary of the Invention

[0007] In this disclosure, we have identified a unique combination of chemically defined components sufficient for the culture of human embryonic stem cells and human induced pluripotent stem cells (collectively referred to herein as human pluripotent stem cells (hPSCs)). The inventive combination of chemically defined components is sufficient for self-renewal and pluripotency-maintaining hPSCs in long-term culture. The medium may be referred to herein as pluripotent stem cell medium (PSC medium or PSCM). The inventive medium is both effective in supporting the growth of human embryonic stem cells and induced pluripotent stem cells. hPSCs cultured in the inventive PSCM medium exhibited at least two-fold faster proliferation rates when compared to other commercially available media (e.g., mTeSR1 or E8). hPSCs can be maintained in PSCM for long-term culture (e.g., more than six months in culture) and still express high levels of pluripotency markers (such as Oct4, Sox2, and / or Nanog).

[0008] The robust ability to culture both human embryonic stem cells and induced pluripotent stem cells in PSCM should facilitate translational applications of human stem cell research.

[0009] The present disclosure provides a cell culture medium suitable for long-term culturing and maintaining human pluripotent stem cells in an undifferentiated state under serum-free and feeder cell-free conditions. The medium comprises a basal medium such as DMEM or other similar medium, enriched with components from F12, and further supplemented with ascorbic acid, proteins (lipid-enriched albumin, transferrin, and insulin), glutathione, ammonium metavanadate, manganous chloride, sodium selenite, and ethanolamine, a stable source of glutamine, FGF, and TGF-β.

[0010] The present disclosure also provides methods for culturing and expanding human pluripotent stem cells for long periods of time under serum-free and feeder cell-free conditions. The present disclosure also provides methods for rapidly generating large populations of human pluripotent stem cells under chemically defined culture conditions. [Brief description of the drawings]

[0011] Figure 1A shows the proliferation of human pluripotent stem cells (hPSCs) in this medium (PSCM) compared to mTeSR1 medium. The human embryonic stem cell line H9 was passaged five times in PSCM and mTeSR1 at the same seeding density and conditions, with cells cultured in PSCM showing double the cell number after the first day of post-seeding acclimation, when cells show low proliferation in both media.

[0012] Figure IB shows hPSC morphology in PSCM. Morphology of cells cultured in PSCM and mTeSR1 is shown. Cells exhibited similar morphology in both media, suggesting pluripotency.

[0013] FIG. 1C shows hPSC stability in PSCM with respect to expression of pluripotency markers. After 30 passages in PSC medium, hPSCs continue to display pluripotency markers such as Nanog and Oct4 via flow cytometry analysis. Compared to H9 cells cultured in mTeSR1, cells cultured in PSCM express approximately 10-fold higher Nanog levels even after 30 passages. Oct4 is shown on the Y-axis and Nanog is shown on the X-axis.

[0014] Figure ID shows hPSC stability in PSCM with respect to karyotype analysis. H9 cells cultured in PSCM and passaged 30 times show no chromosomal abnormalities as confirmed by karyotype analysis.

[0015] Figures 1E and 1F show hPSC stability in PSCM as observed by immunostaining. Immunostaining for Oct4, Nanog, and Sox2 expression in H9 cells showed that H9 cells cultured in PSCM were positive for all three pluripotency markers (Figure 1E). Immunostaining for pluripotency markers in human iPSC 19-9-11 also showed that human iPSCs were positive for all three pluripotency markers (Figure 1F).

[0016] Figure 2 shows H1Oct4-GFP cells that have been passaged 10 times in PSCM. Bright field (left) and GFP (right) microscopy of the cells shows expression of GFP and therefore Oct4 in the human embryonic stem cell line H1 cells after 10 passages in PSCM.

[0017] Figure 3 shows hPSCs cultured in PSC medium and differentiated into cardiomyocytes. The left side shows highly purified cardiac troponin T (cTnT) positive cells (cardiomyocytes) using flow cytometry, and the right side shows staining for cTnT.

[0018] Figure 4 shows hPSCs cultured in PSC medium and differentiated into endothelial progenitor cells. Left shows a plot of CD31 vs. CD34, and right shows staining for VE-cadherin, a pan-marker for endothelial progenitor cells.

[0019] Figure 5. Nutristem on laminin-521 (登録商標) Figure 1 shows the cell proliferation rate of human embryonic stem cell line HS181 cultured in PSCM compared to XF. Population doubling levels are plotted as a function of days in culture, showing that cells cultured in PSCM exhibit more than double the cell number even after 30 days, after the first acclimation passage by day 5 showing similar population doublings in both media. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure provides an improved cell culture medium for expanding and / or maintaining human pluripotent stem cells. The medium can support feeder cell-independent serum-free culture of human pluripotent stem cells over multiple passages. The medium is a synthetic medium and includes amino acids, inorganic salts, vitamins, proteins, reducing agents, trace elements, energy sources such as glucose and sodium pyruvate, lipids, ethanolamine, hypoxanthine, phenol red, putrescine and thymidine, members of the FGF superfamily, members of the TGF-β superfamily, and a stable source of glutamine. The concentration of ascorbic acid in the medium is at least 0.1 mM. In one embodiment, the medium does not contain one or more of gamma aminobutyric acid (GABA), pipecolic acid (PA), and lithium (Li). Human pluripotent stem cells can be cultured and grown in this medium for long periods under serum-free conditions in an undifferentiated state that maintains their pluripotency even without exposure to any conditioned medium (conditioned medium) derived from or on feeder cells.

[0021] Some (but not all) of the components of the medium of the present invention (i.e., amino acids, inorganic salts, vitamins, proteins, reducing agents, trace elements, energy sources (e.g., glucose and sodium pyruvate), lipids, other components (ethanolamine, hypoxanthine, phenol red, putrescine, and thymidine, etc.)) are present in certain commercially available media. For example, Advanced DMEM / F12 (available, for example, from Thermo Fisher Scientific) provides several components. Advanced DMEM / F12 (ADF) typically requires at least some serum to support the culture of many somatic cells and has not been used as a basal medium for hPSC culture. However, in the present disclosure, it has been surprisingly observed that by modifying ADF by increasing the amount of ascorbic acid and adding certain other components such as members of the FGF superfamily, members of the TGF-β superfamily, and a stable glutamine source, the modified ADF can support the long-term culture and maintenance of pluripotent stem cells under fully defined, serum-free, and feeder cell-free conditions.

[0022] An example of a medium currently used for stem cell culture is mTeSR1. However, mTeSR1 requires gamma-aminobutyric acid (GABA), pipecolic acid (PA), and lithium (Li). In the present disclosure, it has been observed that such additional components are not required for multiple and long-term passage of pluripotent stem cells in the present medium. On the contrary, it has been observed that by reducing the number of components, unexpectedly, a higher proliferation rate can be achieved. Furthermore, human pluripotent stem cells cultured in the present medium express higher levels of pluripotency markers and exhibit long-term pluripotency and karyotype stability compared to cells cultured in mTeSR1 under the same conditions. For example, human pluripotent cells have a shorter doubling time compared to cells cultured in mTeSR1 and can be maintained and cultured in this culture medium for extended periods of time (e.g., at least 6 months or 12 months or more), and the resulting pluripotent stem cells still retain high expression of pluripotency markers (e.g., Oct4, Sox2 and Nanog) and exhibit karyotypic stability (as measured by G-bank karyotyping assay).

[0023] It has been observed that the cell culture medium of the present application causes cells to grow faster, e.g., at least twice as fast (i.e., have a two-fold faster growth rate), compared to other commercially available media, such as mTeSR1. Thus, the cell culture medium of the present invention can enable twice the yield of pluripotent stem cells (or the same yield in half the time), effectively reducing the cost per cell by 50% (not considering the associated labor savings). In clinical-scale situations, where the fixed costs for GMP-grade cell culture equipment are high, even greater savings can be expected, as larger quantities of cells can be produced in a shorter time. Thus, the cell culture medium of the present invention can dramatically reduce cell manufacturing costs, producing more results in a shorter time at the same cost. The stem cell field can utilize resources in a more effective way, lowering the threshold for developing clinical stem cell therapies.

[0024] As discussed herein, the cell culture medium of the present disclosure has fewer components than previously reported stem cell culture media. It will be clear to those skilled in the art that fewer components in stem cell culture medium provide significant advantages in terms of ease of manufacture and cost. Furthermore, by including only the essential components identified herein to maintain cell proliferation and intact pluripotency, the risk of undesirable and / or unknown effects from unnecessary components is also significantly reduced. In general, it is important to minimize or eliminate such effects (especially when the resulting cells are ultimately used in a clinical setting).

[0025] The term "stem cell" generally refers to a cell that, upon division, faces two developmental options: the daughter cells may be identical to the original cell (self-renewal); or they may be progenitors of a specialized cell type (differentiation). Thus, stem cells are capable of adopting one or the other pathway (there are additional pathways by which one of each cell type may be formed). Stem cells are cells that are not terminally differentiated and are capable of producing other types of cells.

[0026] Embryonic stem cells are cells isolated from the inner cell mass of the blastocyst, which is the stage of embryonic development at the time of implantation. However, of course, embryonic stem cells can also be isolated without destroying the embryo, for example, using the method described in Chung et al. (Cell Stem Cell 2: 1-5, 2008). Chung et al. reported the derivation of five human embryonic stem cell (hESC) lines without the destruction of the embryo, and demonstrated that they share the same properties as other hESC lines, including the same expression of pluripotency markers, self-renewal capacity, karyotypic stability, and ability to differentiate into derivatives of all three germ layers both in vitro and in teratomas.

[0027] Pluripotent stem cells are stem cells that have the potential to give rise to any differentiated cell in the body. Induced pluripotent stem cells are a type of pluripotent stem cell that is artificially derived from a non-pluripotent cell (typically an adult somatic cell) by inserting certain genes or by other methods (see, for example, Takahashi & Yamanaka (2006, Cell 126(4) 663)).

[0028] It will be understood that pluripotent stem cells can be derived from any eukaryotic source. For example, they can be derived from a vertebrate source, such as a mammal. For example, pluripotent stem cells can be derived from rats, mice, rabbits, or dogs, or non-human primates or humans. In one embodiment, the pluripotent stem cells are human pluripotent stem cells, and in an embodiment, the PSCs or ESCs or iPSCs are human PSCs or human ESCs or human iPSCs. iPSCs can be derived from any suitable cell type, including lung, fibroblasts (e.g., skin fibroblasts), keratinocytes, blood progenitor cells, bone marrow cells, liver cells, gastric epithelial cells, pancreatic cells, neural stem cells, B lymphocytes, ES derivative cells, and embryonic fibroblasts.

[0029] The term "member of the FGF superfamily" as used herein means any fibroblast growth factor (FGF). FGFs are heparin-binding proteins, and interaction with cell surface-associated heparin sulfate proteoglycans has been shown to be essential for FGF signaling. In humans, 23 members of the FGF superfamily have been identified, all of which are structurally related signaling molecules (Finklestein and Plomaritoglou, 2001, Head Trauma: Basic, Preclinical and Clinical Directions. New York: Wiley pp.165-187; Blaber et al, 1996 Biochemistry 35(7): 2086-94; Ornitz and Itoh, 2001 Genome Biology 2(3): reviews 3005.1-reviews 3005.12). Thus, in one embodiment, the member of the FGF superfamily is one of the 23 members of the FGF superfamily identified in humans (FGF1-FGF23), or their orthologues from other species, e.g., mammalian species: members FGF1-FGF10 all bind to the fibroblast growth factor receptor (FGFR), i.e., the member of the FGF superfamily is preferably one that binds to an FGFR (e.g., any of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, or FGF10). In one embodiment, the member of the FGF superfamily is FGF2 or FGF1. In a preferred embodiment, the member of the FGF superfamily is FGF2. In one embodiment, the FGF is one that is thermostabilized compared to the wild-type protein (such as the thermostable FGF1 described by Chen et al.).

[0030] The amino acid sequences of many members of the FGF superfamily (and the nucleotide sequences of the cDNAs encoding them) are readily available, for example, by reference to GenBank. Also, since the sequence of the human genome is substantially complete, it is possible to deduce the amino acid sequence of human FGF therefrom. FGF may be derived from any source, but is preferably derived from a eukaryotic source. It is preferably derived from a vertebrate source, such as a mammal. For example, FGF may be derived from human, rat, mouse, rabbit or dog, or non-human. The term "derived from" includes situations where a cDNA or gene was originally obtained from a source using genetic material, but the protein is subsequently expressed in any host cell. Thus, it should be understood that human FGF may be expressed in a non-human host cell, such as an insect cell or E. coli, but is considered to be of human origin (derived from human). Members of the FGF superfamily may be expressed and purified using well-known molecular biology and protein expression techniques. Alternatively, members of the FGF superfamily may be commercially supplied, for example, from PeproTech (Rocky Hill, New Jersey) or Sigma.

[0031] The term TGF-β as used herein refers to any TGF-beta, including TGFβ1, TGFβ2, and TGFβ3. In one embodiment, the TGF-β is TGF-β-1. It will be understood that TGF-β can be expressed and purified using well-known molecular biology and protein expression techniques. Alternatively, many are commercially available and may be directly supplied (e.g., from PeproTech). In one embodiment, the TGFβ superfamily ligand includes Activin and Nodal. Examples of Activins include Activin A, Activin B, and Activin AB. Nodal binds to Activin A receptor type IIB ACVR2B. It can then form a receptor complex with Activin A receptor type IB (ACVR1B) or with Activin A receptor type IC (ACVR1C). In one embodiment, the TGF-β ligand is not a bone morphogenetic protein (BMP).

[0032] As used herein, the term "stable glutamine source" refers to any glutamine source that is available to cells when present in the growth medium. Generally, dipeptides containing glutamine are preferred, since free glutamine can be unstable. For example, the glutamine source may be L-alanyl-L-glutamine, which is commercially available (e.g., GlutaMAX from Thermo Fisher Scientific). (登録商標) as).

[0033] The buffer system in this culture medium is bicarbonate-based. In other serum-free media developed for culture, HEPES is considered essential to compensate for the lack of serum. However, surprisingly, in this medium, there is no need to add HEPES, and the bicarbonate in the medium provides adequate CO2 absorption. 2 It has been observed that HEPES provides an adequate buffer system under environmental conditions (typically 5-10%). Thus, in one embodiment, the culture medium does not contain HEPES.

[0034] It will be appreciated that the components of the cell culture media of the present invention can be present at any suitable concentration to support the culture and passaging of cells for up to at least six months, or for at least 50 passages under serum-free and feeder cell-free conditions. Some examples of suitable concentration ranges are provided below.

[0035] In one aspect, the present disclosure provides a synthetic medium for in vitro cell culture comprising amino acids, inorganic salts, vitamins, energy sources such as glucose and sodium pyruvate, proteins, lipids, reducing agents, ethanolamine, hypoxanthine, putrescine, thymidine, a member of the FGF superfamily, a member of the TGF-β superfamily, and a stable source of glutamine, wherein the concentration of ascorbic acid in the medium is at least 0.1 mM. In one embodiment, the culture medium does not contain one or more of gamma-aminobutyric acid (GABA), pipecolic acid (PA), and lithium (Li). In one embodiment, the medium also does not require, and in one embodiment does not contain, 2-mercaptoethanol, pluronic F-68, Tween (80), or any other pluronic or non-ionic detergents. The medium can be used to culture pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The PSCs may be of human or non-human origin. Because PSCs can be cultured in this medium for at least 50 or at least 100 passages, the use of this culture medium is expected to have a dramatic impact on increasing the yield of PSCs, thereby decreasing the production costs of PSCs (e.g., hPSCs and hPSC-derived cells) and should facilitate further translational applications of stem cell research.

[0036] In one embodiment, the present invention provides a cell culture medium comprising, consisting essentially of, or consisting of Advanced DMEM / F12 (ADF), supplemented ascorbic acid, a member of the fibroblast growth factor (FGF) superfamily, a transforming growth factor-β (TGF-β) superfamily ligand, and a glutamine source.

[0037] The cell culture medium of the invention is further described below and in the accompanying Examples. The medium is also referred to herein as PSC medium(s), or PSCM.

[0038] In one embodiment, the present disclosure provides: DMEM (or other similar base medium), Group I components (alanine, asparagine, aspartic acid, glutamic acid, proline, biotin, vitamin B12, copper sulfate, ferrous sulfate, magnesium chloride, dibasic sodium phosphate, zinc sulfate, hypoxanthine, linoleic acid, lipoic acid, putrescine hydrochloride, sodium pyruvate, and thymidine), Group II ingredients (ascorbic acid phosphate, lipid-enriched albumin (AlbuMAX TM etc.), transferrin (human transferrin, holo, etc.), insulin (recombinant, full chain, etc.), monosodium glutathione, ammonium metavanadate, manganese chloride, sodium selenite, and ethanolamine), and Group III components (L-alanyl-L-glutamine, ascorbic acid, FGF, TGF-β) providing a stem cell culture medium comprising, or consisting essentially of, Here, the total concentration of ascorbic acid phosphate in the medium is at least 0.1 mM, and in one embodiment, is 0.1 to 0.8 mM. In one embodiment, the medium does not contain one or more of HEPES, gamma-aminobutyric acid (GABA), pipecolic acid (PA), and lithium (Li), 2-mercaptoethanol, Pluronic F-68, Tween 80, or any other pluronic or non-ionic detergent.

[0039] The Group I components referred to in this specification are present in DMEM / F12 but not in DMEM. The Group II components referred to in this specification are present in Advanced DMEM / F12 (ADF) but not in DMEM / F12. The Group III components referred to in this specification are present in this medium but not in ADF.

[0040] In one embodiment, the present disclosure DMEM / F12 (or other similar enriched media), Group II components (ascorbic acid phosphate, albumin (AlbuMAX TM ), human transferrin (holo), insulin (e.g., recombinant, full chain), glutathione monosodium, ammonium metavanadate, manganese chloride, sodium selenite, and ethanolamine), and Group III components (L-alanyl-L-glutamine, ascorbic acid, FGF, TGF-β) to provide a human pluripotent stem cell culture medium comprising or consisting essentially of only them, wherein the total concentration of ascorbic acid in the medium is at least 0.1 mM, and in one embodiment, is 0.1 - 0.8 mM. In one embodiment, the medium does not contain gamma-aminobutyric acid (GABA), pipecolic acid (PA), and lithium (Li), 2-mercaptoethanol, pluronic F-68, Tween 80, or any other pluronic or non-ionic detergent. In one embodiment, the medium does not contain any of the following: gamma-aminobutyric acid (GABA), pipecolic acid (PA), and lithium (Li), 2-mercaptoethanol, pluronic F-68, Tween 80, or any other pluronic or non-ionic detergent. The medium may or may not contain HEPES.

[0041] In one embodiment, the present disclosure Advanced DMEM / F12 (or other similarly modified medium), and Group III components (L-alanyl-L-glutamine, ascorbic acid, FGF, TGF-β) A human pluripotent stem cell culture medium comprising, or consisting essentially of, Here, the total concentration of ascorbic acid in the medium is at least 0.1 mM (equivalent to about 25 mg / L of ascorbic acid phosphate), and in one embodiment, is 0.1 to 0.8 mM (equivalent to about 25 mg / L to 200 mg / L of ascorbic acid phosphate). In one embodiment, the medium does not contain one or more of HEPES, gamma-aminobutyric acid (GABA), pipecolic acid (PA), and lithium (Li), 2-mercaptoethanol, Pluronic F-68, Tween 80, or any other pluronic or non-ionic detergent. In one embodiment, the medium is free of all of the following: HEPES, gamma-aminobutyric acid (GABA), pipecolic acid (PA), and lithium (Li), 2-mercaptoethanol, Pluronic F-68, Tween 80, or any other pluronic or non-ionic detergent.

[0042] In one embodiment, the cell culture medium of the present disclosure contains 0.1 to 0.8 mM ascorbic acid or a salt thereof or an ester thereof, as described above. For example, the cell culture medium may contain 0.08 to 0.6 mM, or 0.1 to 0.5 mM, or 0.1 to 0.4 mM, or 0.2 to 0.5 mM, or 0.2 to 0.4 mM, or 0.15 to 35 mM, or 0.2 to 0.3 mM, or 0.2 to 0.25 mM ascorbic acid or a salt thereof or an ester thereof. In one embodiment, the cell culture medium contains 0.2 to 0.25 mM (e.g., about 0.24 mM) ascorbic acid or a salt thereof or an ester thereof.

[0043] In one embodiment, the cell culture medium of the present invention contains 25 to 75 μg / L of a member of the FGF superfamily (including any of those mentioned above, such as FGF1 and FGF2). For example, the cell culture medium may contain 30 to 70 μg / L or 35 to 65 μg / L of a member of the FGF superfamily (e.g., FGF2 and FGF1). In one embodiment, the cell culture medium contains 40 to 60 μg / L (e.g., about 50 μg / L) of a member of the FGF superfamily (e.g., FGF2 and FGF1).

[0044] In one embodiment, the cell culture medium of the present invention comprises 0.75 to 2.25 μg / L of a TGF-β superfamily ligand (including any of those mentioned above, such as TGF-β1). For example, the cell culture medium may comprise 0.8 to 2.2 μg / L, or 0.9 to 2.1 μg / L, or 1.0 to 2.0 μg / L, or 1.1 to 1.9 μg / L, or 1.2 to 1.8 μg / L, or 1.3 to 1.7 μg / L of a TGF-β superfamily ligand (e.g., TGF-β1). In one embodiment, the cell culture medium comprises 1.4 to 1.6 μg / L (e.g., about 1.5 μg / L) of a member of the TGF-β superfamily (e.g., TGF-β1).

[0045] In one embodiment, the cell culture medium of the present invention comprises 1 to 5 mM of a glutamine source. For example, the cell culture medium may comprise 1 to 4 mM or 2 to 5 mM or 1 to 3 mM or 3 to 5 mM of a glutamine source (e.g., L-alanyl-L-glutamine). In one embodiment, the cell culture medium comprises 2 to 4 mM of a glutamine source (e.g., L-alanyl-L-glutamine), for example 2 to 3 mM or about 2.5 mM of a glutamine source (e.g., L-alanyl-L-glutamine).

[0046] In one embodiment, the cell culture medium of the present invention can contain 0.1-0.6 mM ascorbic acid, 25-75 μg / L of a member of the FGF superfamily (e.g., FGF2 or FGF1), 0.75-2.25 μg / L of a TGF-β superfamily ligand (e.g., TGF-β1), and 1-5 mM of a glutamine source (e.g., L-alanyl-L-glutamine). For example, a cell culture medium can include about 0.25 mM ascorbic acid, about 50 μg / L of a member of the FGF superfamily (e.g., FGF2 or FGF1), about 1.5 μg / L of a TGF-β superfamily ligand (e.g., TGF-β1, and about 2.5 mM of a glutamine source. In one embodiment, a cell culture medium of the present invention includes about 0.25 mM ascorbic acid, about 50 μg / L of FGF-2, about 1.5 μg / L of TGF-β-1, and about 2.5 mM of L-alanyl-L-glutamine.

[0047] In one embodiment, the cell culture medium of the invention comprises or consists essentially of all of the components listed in Table 1 (PSCM) below, and in some embodiments, comprises all of the components listed in Table 1 at the corresponding concentrations provided for each component in the table. In one embodiment, the components are present in the amounts shown in Table 1 ±10%. For example, glycine may be present in the range of about 16.8 mg / L to 20.6 mg / L, insulin may be present in the range of about 9 to 11 mg / L, and so on. In some embodiments, the medium may contain the components at the listed concentrations shown in Table 1 ±1 to 20%. For example, the medium may contain the components at the listed concentrations shown in Table 1 ±5, 10, 15, or 20%. In one embodiment, the medium does not contain one or more of HEPES, gamma-aminobutyric acid (GABA), pipecolic acid (PA), and lithium (Li), 2-mercaptoethanol, Pluronic F-68, Tween 80, or any other pluronic or non-ionic detergent. In one embodiment, the medium does not contain all of the following: HEPES, gamma-aminobutyric acid (GABA), pipecolic acid (PA), and lithium (Li), 2-mercaptoethanol, Pluronic F-68, Tween 80, or any other pluronic or non-ionic detergent. [Table 1] TIFF0007681037000002.tif168169

[0048] In one embodiment, the cell culture medium can include one or more antibiotics, such as penicillin, streptomycin, or a mixture thereof. The antibiotics penicillin and streptomycin are used to prevent bacterial contamination in cell cultures due to their effective combined action against gram-positive and gram-negative bacteria. A penicillin-streptomycin mixture is commercially available as Pen-Strep by Thermo Fisher Scientific.

[0049] In one embodiment, the cell culture medium of the present invention may further comprise an apoptosis inhibitor, for example a ROCK inhibitor, such as Y-27632 [(R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, Y-27632 dihydrochloride] (Watanabe et al, Nature Biotechnology 25, 681-686 (2007)), which is commercially available, for example, from Sigma Aldrich.

[0050] The cell culture medium of the present disclosure may be xeno-free, meaning that none of its components are derived from non-human animals, but one or more of its components may be derived from humans. As an example, a xeno-free version of PSCM can be produced by replacing the AlbuMAX present in ADF with human recombinant albumin (HRA) or human serum albumin (HSA) from rice. The HRA or HSA concentration in the PSCM can be optimized to support hPSC self-renewal. In the present disclosure, similar data was obtained using Albumax and albumin from human sources such as human serum albumin or HRA.

[0051] The medium can be prepared by conventional methods. If prepared in liquid form, the ingredients can be added to water. The ingredients may also be provided as a powder mixture or in lyophilized form.

[0052] The cell culture medium can be used for the growth, passaging and / or maintenance of pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The cells may be the following hESCs: H1, H7, H9, H13, H14; iPSC lines: 19-9-11, 6-9-9, 19-9-7 (available from WiCell (wicell.org)).

[0053] Human pluripotent stem cells can be cultured and maintained in the present medium. Generally, cells are passaged when they are about 60-70% confluent, but they may be passaged at any desired confluence. For passaging, cells are generally dissociated by exposure to EDTA, trypsin or chymotrypsin or a similar enzyme cocktail, separated from the substrate, separated from the medium (generally by sedimentation, low speed centrifugation or filtration), re-seeded on a suitable substrate, and grown in the presence of the present medium.

[0054] Incubation conditions for cell culture are known in the art. For example, such conditions typically include culturing at a temperature of 32-40° C., such as at about 37° C. 2 The concentration is generally about 1-10%, such as about 2-7%, or about 5%, or any range or value between 1-10%. Oxygen tension is generally adjusted to provide normoxic conditions, preferably about 20%.

[0055] The cells may be cultured on a suitable substrate. For example, suitable substrates include Matrigel, collagen IV, fibronectin, laminin, collagen, vitronectin, polylysine, iMatrix-511, etc. These materials are commercially available and routinely used for cell culture. In the examples, iMatrix-511 can be used at about 5 μg / ml, vitronectin can be used at about 10 μg / ml, and Matrigel can be used at 80 μg / ml.

[0056] In one embodiment, cells may be cultured as suspension cultures without the need for a substrate. In suspension cultures, cells are suspended in culture medium and grown under the same conditions as substrate-attached cultures. Cells are generally grown in shaker or spinner flasks or bottles. For subculture, enzyme treatment is not required to detach cells from the substrate. EDTA may be used to treat cell clusters to generate smaller cell clusters. A portion of the cell suspension may be removed and diluted with a fresh batch of medium and grown.

[0057] In the present disclosure, it has been observed that human pluripotent stem cells grow more rapidly under similar conditions than in other known commonly used culture media (such as mTeSR1). Thus, in one embodiment, the cell culture medium of the present invention is generally a medium in which hPSCs grow at least 1.5 times faster, e.g., at least 2 times faster, 2.5 times faster, 3 times faster, 4 times faster, or 5 times faster than they grow in mTeSR1 medium under similar culture conditions. Culturing under similar conditions generally means that, apart from the medium, parameters such as seeding density, growth matrix, incubation conditions, temperature, agitation, (all of which may affect the growth rate) are the same. In one embodiment, at least one of the pluripotency markers is expressed at at least twice the expression level in cells cultured in mTeSR1 under similar culture conditions. In one embodiment, at least one of the pluripotency markers is expressed at at least 10 times the expression level in cells cultured in mTeSR1 under similar culture conditions.

[0058] Measurement of the proliferation rate of hPSCs can be performed by any suitable method in the art, and generally involves evaluating cell number over time at regular time points. Cell number can be determined directly, for example, by microscopic or electronic counting, or indirectly, for example, by using chromogenic dyes, incorporating radioactive precursors (e.g., by incorporation of labeled nucleosides into DNA, e.g., quantification with thymidine), or measuring metabolic activity of cellular enzymes. Any convenient method can be used. A convenient measure of proliferation rate is doubling time. This is the time it takes to double the number of cells.

[0059] Human pluripotent stem cells can be maintained in long-term culture in this medium under serum-free, feeder cell layer-free, conditioned medium-free conditions in an undifferentiated state for several months. For example, an undifferentiated phenotype can be observed for at least 6 months. In one embodiment, an undifferentiated phenotype is observed for at least 7, 8, 9, 10, 11, or 12 months or longer.

[0060] Subculture or passage is necessary to culture cells for a long period of time. hPSCs can be maintained in culture in the medium by passage for several passages. For example, hPSCs can be passaged from 1 passage to at least 100 passages or more (e.g., 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more). Even after passage of cells for several passages, it has been observed that the cells still maintain pluripotency and / or karyotypic stability. The expression of pluripotency of PSCs may be via displaying any one or more characteristics of cells with pluripotency. The characteristics may include morphological characteristics, immunohistochemical characteristics, molecular biological characteristics, or may include biological activity. All or a substantial portion of the cells may retain one or more characteristics. This may be 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or substantially 100% of the treated cells.

[0061] hPSCs cultured in this culture medium are expected to retain the ability to differentiate into all three cell lineages, i.e., endoderm, ectoderm, and mesoderm. For example, hPSCs may differentiate into cardiomyocytes, neural progenitor cells, and other cells. Pluripotency can be assessed by any suitable method in the art. For example, methods for inducing hPSCs to differentiate into each of these lineages are known in the art and may be used to analyze the ability of cells to differentiate. Alternatively, pluripotency can be determined by assessing the expression of one or more pluripotency markers (e.g., Oct4, Sox2, Nanog, and / or TRA-1-61). Expression of one or more pluripotency markers can be assessed at one or more time points during the culture of the cells in the medium. Expression can be assessed using standard techniques in the art for measuring expression levels, including assessment of nucleic acid (e.g., RNA) and / or protein levels. If the marker is a protein, the technique is typically based on immunoassays. The antibody selective for the pluripotency marker of interest may itself be labeled, for example, with a radioactive label or with a fluorescent label or with an enzyme. Alternatively, and preferably, it is detected by a labeled secondary antibody that binds to the antibody selective for the pluripotency marker. Antibodies against pluripotency markers available from commercial sources may be used. Examples of other suitable methods include flow cytometry (FACS), histochemical staining, Western blotting, enzyme-linked immunosorbent assay (ELISA). Flow immunocytochemistry may be used to detect cell surface markers, and immunohistochemistry (e.g., of fixed cells or tissue sections) may be used for intracellular or cell surface markers. Western blot analysis may be performed on cell extracts, and ELISA may be used for cell extracts or products secreted into the medium.

[0062] hPSCs cultured in culture medium may retain a normal karyotype during or after expansion. A "normal" karyotype includes a karyotype that is identical, similar, or substantially similar to the karyotype of the parent hPSCs. For example, there are no macroscopic abnormalities such as translocations, chromosomal losses, deletions, etc. Karyotypes can be assessed in a number of ways, for example, visually under a light microscope. Karyotypes can be prepared and analyzed as described in McWhir et al (Methods Mol Biol, 331: 77-90, 2006) and Hewitt et al (Stem Cells, 25(1): 10-8, 2007). Cells can also be karyotyped using standard G-banding techniques (available at many clinical diagnostic laboratories that provide routine karyotyping services, such as Cytogenetics Lab in Oakland Calif) and compared to published hPSC karyotypes.

[0063] In one embodiment, the present disclosure provides a cell culture medium comprising, or consisting essentially of, amino acids, inorganic salts, vitamins, trace elements, an energy source (an energy source selected from the group consisting of glucose, sodium pyruvate or combinations thereof), proteins, lipids, ethanolamine, hypoxanthine, putrescine, thymidine, a member of the FGF superfamily, a ligand of the TGF-β superfamily, and a stable glutamine source, at concentrations sufficient to enable in vitro passaging of undifferentiated hPSCs under serum-free, feeder cell-free conditions for at least 50 passages, wherein the concentration of ascorbic acid phosphate in the medium is in the range of 0.1-0.8 mM.

[0064] In one embodiment, the disclosure provides a cell culture medium comprising or consisting essentially of DMEM, alanine, asparagine, aspartic acid, glutamic acid, proline, biotin, vitamin B12, copper sulfate, ferrous sulfate, magnesium chloride, dibasic sodium phosphate, zinc sulfate, hypoxanthine, linoleic acid, lipoic acid, putrescine hydrochloride, sodium pyruvate, and thymidine, an ascorbic acid source, lipid-enriched albumin, transferrin, insulin, monosodium glutathione, ammonium metavanadate, manganese chloride, sodium selenite, and ethanolamine, L-alanyl-L-glutamine, a member of the FGF superfamily, a member of the TGF-β superfamily ligand, in concentrations sufficient to enable in vitro passaging of undifferentiated hPSCs for at least 50 passages under serum-free, feeder cell-free conditions, wherein the concentration of ascorbic acid in the medium is 0.1-0.8 mM.

[0065] In one embodiment, the disclosure provides a cell culture medium comprising or consisting essentially of DMEM / F12, an ascorbic acid source, lipid-enriched albumin, transferrin, insulin, monosodium glutathione, ammonium metavanadate, manganese chloride, sodium selenite, and ethanolamine, L-alanyl-L-glutamine, a member of the FGF superfamily, a member of the TGF-β superfamily ligand, in concentrations sufficient to allow in vitro passaging of undifferentiated hPSCs for at least 50 passages under serum-free, feeder cell-free conditions, wherein the concentration of ascorbic acid in the medium is 0.1-0.8 mM.

[0066] In one aspect, the present disclosure provides a method for culturing and maintaining human pluripotent stem cells, comprising growing / maintaining cells in serum-free culture medium as described herein, wherein the cells are not and have not been exposed to feeder cells or conditioned medium derived therefrom. The cells may be maintained for at least 30 passages and may express pluripotency markers (e.g., one or more of Nanog, Oct4, and Sox2). In one embodiment, the pluripotency markers may be expressed at least twice the expression level in cells cultured in mTeSR1 under similar conditions. In one embodiment, the pluripotency markers may be expressed at least 5 or 10 times the expression level in cells cultured in mTeSR1 under similar conditions.

[0067] In one aspect, the present disclosure provides a kit comprising Advanced DMEM / F12 (ADF), a source of ascorbic acid, a member of the fibroblast growth factor (FGF) superfamily, a transforming growth factor-β (TGF-β) superfamily ligand, and a source of glutamine. In one embodiment, the FGF in the kit is FGF2 or FGF1. In one embodiment, the TGF-β is TGF-β1. In one embodiment, the glutamine source is L-alanyl-L-glutamine. In one embodiment, all components of the medium may be premixed and provided in powder form, which is accompanied by instructions related to the preparation of the culture medium. The components may be provided in a sterile form. The kit may further comprise an antibiotic, such as penicillin and / or streptomycin, and gentamicin. The kit may also comprise an apoptosis inhibitor, such as a ROCK inhibitor (e.g., Y-27632).

[0068] In one embodiment, the present disclosure provides: i) DMEM, ii) combinations of alanine, asparagine, aspartic acid, glutamic acid, proline, biotin, vitamin B12, copper sulfate, ferrous sulfate, magnesium chloride, dibasic sodium phosphate, zinc sulfate, hypoxanthine, linoleic acid, lipoic acid, putrescine hydrochloride, sodium pyruvate, and thymidine, a source of ascorbic acid, lipid-enriched albumin, transferrin, insulin, monosodium glutathione, ammonium metavanadate, manganese chloride, sodium selenite, and ethanolamine, L-alanyl-L-glutamine, a member of the FGF superfamily, a member of the TGF-β superfamily ligand, and iii) optionally, instructions for preparing cell culture medium. wherein the components are provided in sufficient concentrations to allow in vitro subculture of undifferentiated hPSCs for at least several passages, e.g., up to 50 passages, under serum-free and feeder cell-free conditions, and wherein after preparation of the cell culture medium, the concentration of ascorbic acid in the medium is 0.1-0.8 mM.

[0069] In one embodiment, the present disclosure provides: i) DMEM / F12, ii) sources of ascorbic acid (e.g., ascorbic acid phosphate), lipid-enriched albumin, transferrin, insulin, monosodium glutathione, ammonium metavanadate, manganese chloride, sodium selenite, and ethanolamine, L-alanyl-L-glutamine, members of the FGF superfamily, members of the TGF-β superfamily ligands, and iii) optionally, instructions for preparing cell culture medium. wherein the components are provided in concentrations sufficient to allow in vitro passaging of undifferentiated hPSCs for at least 50 passages under serum-free and feeder cell-free conditions, and wherein after preparation of the cell culture medium, the concentration of ascorbic acid in the medium is 0.1-0.8 mM, 0.15-0.7 mM, or 0.2-0.4 mM, or 0.15-0.3 mM.

[0070] In one embodiment, the present disclosure provides: i) Advanced DMEM / F12, ii) a source of ascorbic acid (e.g., ascorbic acid phosphate), L-alanyl-L-glutamine, a member of the FGF superfamily, a member of the TGF-β superfamily ligand, and iii) optionally, instructions for preparing cell culture medium. wherein the components are provided in sufficient concentrations to allow in vitro subculture of undifferentiated hPSCs for at least several passages, e.g., up to 50 passages, under serum-free and feeder cell-free conditions, and wherein after preparation of the cell culture medium, the concentration of ascorbic acid in the medium is 0.1-0.8 mM.

[0071] In one aspect, the present disclosure provides a method of maintaining undifferentiated hPSCs in culture for extended periods of time under fully defined chemical conditions, which includes culturing and passaging the cells in the present medium under serum-free, feeder cell-free, feeder cell-derived conditioned medium-free conditions for multiple passages. The cells can be frozen at any passage and then thawed and continue to be passaged, or the cells may be used to generate differentiated cells of any desired lineage.

[0072] In one aspect, the disclosure provides a population of pluripotent stem cells cultured in the medium under serum-free and feeder cell layer-free conditions, hi one embodiment, the cells are human pluripotent stem cells.

[0073] The following examples further illustrate the invention and are not intended to be limiting in any way. EXAMPLES

[0074] This example describes the preparation of one embodiment of a PSC medium and the use of the medium to culture human pluripotent stem cells.

[0075] method

[0076] Preparation of PSC medium

[0077] 6.25 ml of GlutaMAX (Thermo Fisher Scientific), 305 μl of ascorbic acid phosphate solution (100 mg / ml) (Sigma), 500 μl of FGF2 solution (50 μg / ml) (Peprotech), and 500 μl of TGFβ1 solution (1.5 μg / ml) (Peprotech) were added to 500 ml of Advanced DMEM / F12 (Thermo Fisher Scientific).

[0078] Passaged hPSC colonies cultured in PSC medium containing EDTA (0.5 mM)

[0079] Matrigel-coated dishes equilibrated to room temperature were used. PSC medium was also equilibrated to room temperature. hPSCs growing on the culture sites (6-well plates) were detached by exposure to EDTA for about 3 minutes. The EDTA solution was removed and 3 ml of PSCM + 5 μM Y27632 (ROCK inhibitor) was added to each plate. Once the cells had detached and rounded, they were removed from the wells. The contents of the wells were pooled in a sterile conical tube containing 9 ml of PSCM + 5 μM Y27632 and mixed gently to obtain a cell suspension. 2 ml of the cell suspension was added to each well of a new Matrigel-coated 6-well plate (1:6 split ratio). The cells were then continued to be cultured. The medium was replenished the next day and then refreshed every 3-3 days. When the cells reached about 80% confluency, they were split again.

[0080] Flow cytometry analysis

[0081] The cells were then incubated with Accutase (登録商標)Cells were dissociated into single cells using PBS (10 min), then fixed with 1% paraformaldehyde for 20 min at room temperature and stained with primary and secondary antibodies in PBS + 0.1% Triton X-100 and 0.5% BSA. Data were collected on a FACSCaliber flow cytometer (Beckton Dickinson) and analyzed using FlowJo. FACS gating was based on corresponding isotype antibody controls. Oct4 and Nanog antibodies were used.

[0082] immunostaining

[0083] Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then stained with primary and secondary antibodies in PBS + 0.4% Triton X-100 and 5% nonfat dry milk. Nuclei were stained with Gold Anti-fade Reagent with DAPI. A Nikon epifluorescence microscope was used for imaging analysis. Antibodies used were Oct4 antibody (Mouse IgG2b clone: ​​C-10 sc-5279, available from Santa Cruz), Nanog antibody (Nanog(D73G4) XP from Cell Signaling Technologies), and IgG2b clone (Cell Signaling Technologies). 登録商標 rabbit mAb #4903), and Sox2 antibody (Sox2(D6D9) XP from Cell Signaling Technologies 登録商標 The antibody was rabbit mAb #3579.

[0084] result

[0085] Increased growth rate

[0086] H9 cells cultured in PSCM compared to mTeSR1 (at the same seeding density and under the same conditions) for five passages show double the cell number after the first day of post-seeding acclimation (cells show slower growth in both media) (Figure 1A). The morphology of cells cultured in PSCM and mTeSR1 is shown in Figure 1B.

[0087] long term stability

[0088] After 30 passages in PSC medium, hPSCs continue to display pluripotency markers such as Nanog and Oct3 / 4 by flow cytometry analysis (Figure 1C). Compared with H9 cells cultured in mTeSR1, cells cultured in PSCM express approximately 10-fold higher Nanog levels even after 30 passages (Figure 1C). H9 cells cultured in PSCM for 30 passages do not show chromosomal abnormalities as confirmed by karyotype analysis (Figure 1D). Immunostaining of Oct4, Nanog, and Sox2 expression in H9 cells was performed, and H9 cells cultured in PSCM were positive for all three pluripotency markers (Figure 1E). Immunostaining of pluripotency markers in human iPSC 19-9-11 was performed, and human iPSCs were also positive for all three pluripotency markers (Figure 1F). EXAMPLES

[0089] Human embryonic stem cell line H1-Oct4-GFP cells were cultured for 10 passages in PSC medium containing L-alanyl-L-glutamine. As is evident from Figure 2, even after 10 passages, GFP expression can still be observed. The expression of GFP is driven by a gene called Oct4, which is a stem cell pluripotency marker. Thus, if the stem cells express Oct4, they are pluripotent. Because these cells express GFP driven by Oct4, they remain pluripotent. EXAMPLES

[0090] In this example, we generated differentiated cells from hPSCs. We generated cardiomyocytes and endothelial progenitor cells.

[0091] Cardiac Differentiation: When hPSCs maintained on Matrigel plates reached confluence, the cells were treated with CHIR99021 in RPMI / B27-insulin for 24 hours (days 0-1). The medium was changed to RPMI / B27-insulin. On day 3, 5 μM of Wnt Production inhibitor (IWP) 2 or IWP 4 was added and removed during the medium change on day 5. The cells were maintained in RPMI / B27 (starting on day 7) and the medium was changed every 3 days. The results are shown in Figure 3. hPSCs were differentiated into cardiomyocytes, followed by the GiWi protocol. On day 15 of differentiation, the differentiated cells were immunostained with cTnT.

[0092] Differentiation of endothelial progenitor cells: On day 0, hPSCs were treated with 6-10 μM CHIR99021 for 2 days in Advanced DMEM / F12, 2.5 mM GlutaMAX, and 60 μg / ml ascorbic acid phosphate. After 2 days, the CHIR99021-containing medium was aspirated and cells were maintained in basal medium without CHIR99021 for an additional 3-4 days. Results are shown in Figure 4. hPSCs were treated with CHIR99021 for 2 days, followed by an additional 3 days in defined medium. On day 5 of differentiation, cells were immunostained for VE-cadherin. Cells were analyzed for CD31 and CD34 expression by flow cytometry. EXAMPLES

[0093] In this example, the human embryonic stem cell line HS181 was plated on laminin-coated Laminin 521 (Ln 521) (used at 5 μg / ml) and the cells were cultured in this medium containing human serum albumin (hence the xeno-free version) and Nutristem hESC XF. No ROCK inhibitor was used. The seeding density was 30,000 cells / cm for HS181. 2 and 50,000 cells / cm for iPSC3. 2The results were consistent across the 10-year follow-up period. Cells were fed daily and passaged every 3 days. Cells were passaged 10 times. Cells were observed to proliferate faster with PSCM compared to Nutristem, especially at later passages (Figure 5). Immunostaining for pluripotency factors Oct4, DAPI, and SSEA4 was similar for cells in both media. This example demonstrates that the medium can be used to passage and maintain stem cells plated on different substrates.

Claims

1. 1. A cell culture medium comprising: Advanced DMEM / F12, L-alanyl-L-glutamine at a concentration of 1-5 mM, with additional ascorbic acid phosphate as shown in the table below; wherein the concentration of ascorbic acid in the culture medium is 0.1-0.8 mM, the concentration of the member of the FGF superfamily is 25-75 μg / L, and the concentration of the member of the TGF-β superfamily ligand selected from the group consisting of TGF-β1, TGF-β2, and TGF-β3 is 0.75-2.25 μg / L; The medium allows for in vitro subculture of undifferentiated human pluripotent stem cells for at least 50 passages under serum-free and feeder cell-free conditions, and the undifferentiated cells express Oct4, Sox2, and Nanog and maintain karyotypic stability for at least 50 passages. Cell culture medium.

2. 2. The cell culture medium of claim 1, wherein the culture medium does not contain one or more of gamma-aminobutyric acid (GABA), pipecolic acid (PA), and lithium (Li).

3. 2. The cell culture medium of claim 1, wherein the culture medium does not contain one or more of the following: 2-mercaptoethanol, Pluronic F-68, Tween 80.

4. 2. The cell culture medium of claim 1, wherein the concentration of ascorbic acid in the medium is 0.1 mM to 0.6 mM.

5. 5. The cell culture medium of claim 4, wherein the concentration of ascorbic acid is 0.15 to 0.30 mM.

6. The cell culture medium of claim 1 , wherein the member of the FGF superfamily is FGF1 or FGF2.

7. The cell culture medium of claim 1 , wherein the TGF-β superfamily ligand is TGF-β1.

8. 2. The cell culture medium of claim 1, wherein ascorbic acid is 0.1 to 0.8 mM, a member of the FGF superfamily is 25 to 65 μg / L, a TGF-β superfamily ligand is 0.8 to 2.2 μg / L, and L-alanyl-L-glutamine is 1 to 5 mM.

9. 9. The cell culture medium of claim 8, wherein ascorbic acid is about 0.25 mM, FGF-2 is about 50 μg / L, TGF-β superfamily ligand is about 1.5 μg / L, and L-alanyl-L-glutamine is about 2.5 mM.

10. 2. The cell culture medium of claim 1, wherein the albumin in the Advanced DMEM / F12 is bovine serum albumin, human serum albumin, or recombinant human or bovine albumin.

11. A cell culture medium containing the following components present at ±10% of the concentrations shown in the table:

12. 12. The cell culture medium of claim 11, wherein the components are present at the concentrations indicated in the table.

13. 13. A method for culturing and maintaining human pluripotent stem cells comprising growing cells in the culture medium of claim 1, wherein the cell culture medium is serum-free.

14. 14. The method of claim 13, wherein the cells are not exposed to feeder cells or conditioned medium derived therefrom.

15. 14. The method of claim 13, wherein the cells are maintained in culture for at least 30 passages, and optionally express pluripotency markers after at least 30 passages.

16. The method of claim 13 , wherein the cells are grown on a substrate.

17. 17. The method of claim 16, wherein the substrate is vitronectin, laminin or matrigel.

18. The method of claim 15, wherein the pluripotency markers are Nanog, Oct4, and / or Sox2.

19. The method of claim 13, wherein the cells are human embryonic stem cells or human induced pluripotent stem cells.

20. A kit for preparing the cell culture medium of claim 1, comprising: i) Advanced DMEM / F12; ii) a mixture comprising ascorbic acid phosphate, L-alanyl-L-glutamine, a member of the FGF superfamily, and a member of the TGF-β superfamily ligand selected from the group consisting of TGFβ1, TGFβ2, and TGFβ3; wherein the components are provided such that the TGF-β superfamily ligand has a concentration of 0.75-2.25 μg / L, the ascorbic acid has a concentration of 0.1-0.8 mM, the FGF superfamily has a concentration of 25-75 μg / L, and the L-alanyl-L-glutamine has a concentration of 1-5 mM; and iii) Instructions for preparing cell culture medium Including the kit.

21. The kit of claim 20, wherein the albumin is xeno-free.

22. the member of the FGF superfamily is FGF1 or FGF2; The kit according to claim 20 , wherein the member of the TGF-β superfamily ligand is TGF-β1.

23. 2. The cell culture medium of claim 1, wherein the proliferation rate of human pluripotent stem cells (hPSCs) is at least 1.5 times that of hPSCs cultured in mTeSR1 under similar culture conditions.

24. 24. The cell culture medium of claim 23, wherein the proliferation rate of the hPSCs and the expression of the pluripotency marker Nanog are at least twice as high as hPSCs cultured in mTeSR1 under similar culture conditions.

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