Method for inhibiting differentiation of pluripotent stem cells

Abstract

In the present invention, pluripotent stem cells are suspension cultured while being maintained in an undifferentiated state. During suspension culturing of the pluripotent stem cells, an undifferentiated state is maintained by adding a tankyrase inhibitor (TNKS inhibitor) and a PKCβ inhibitor from among PKC inhibitors.

Description

[Technical Field]

[0001] The present invention relates to a method for producing pluripotent stem cells by suspension culture, and also to a method for producing a pluripotent stem cell population by suspension culture. [Background technology]

[0002] Pluripotent stem cells, such as ES cells and iPS cells, have the ability to proliferate indefinitely and differentiate into various somatic cells. The practical application of therapies involving the transplantation of somatic cells induced to differentiate from pluripotent stem cells has the potential to fundamentally revolutionize the treatment of intractable diseases and lifestyle-related diseases. For example, technology has already been developed to induce differentiation of pluripotent stem cells in vitro into a wide variety of somatic cells, including nerve cells, cardiomyocytes, blood cells, and retinal cells.

[0003] On the other hand, regenerative medicine using pluripotent stem cells has many challenges to overcome before it can be put to practical use. One of these challenges is the productivity of pluripotent stem cells. For example, liver regeneration requires approximately 2 × 10 11 It is said that 10 cells are needed to culture pluripotent stem cells. The culture methods for pluripotent stem cells are roughly divided into adhesion culture, in which cells are cultured by adhering them to a flat substrate, and suspension culture, in which cells are cultured by suspending them in a liquid medium. To culture the above number of cells by adhesion culture, 10 6 cm 2 This requires at least 100 substrates, which is equivalent to approximately 20,000 standard 10-cm dishes. Thus, because the number of cells obtained in adherent culture on a substrate surface depends on the culture area, scaling up requires a huge area, making it difficult to supply the amount of cells required for regenerative medicine. In suspension culture, cells are cultured in a liquid medium while suspended in the medium, so the number of cells obtained depends on the medium volume. Therefore, suspension culture is easy to scale up and is suitable for mass cell production. For example, Non-Patent Document 1 discloses a method of suspension culture of pluripotent stem cells using a spinner flask as a cell culture vessel for suspension culture while stirring the liquid medium.

[0004] Another challenge facing the practical application of regenerative medicine is the productivity of target somatic cells. Efforts to efficiently produce target somatic cells include efforts to improve differentiation induction efficiency, and various methods for doing so have been reported. For example, Non-Patent Document 2 discloses a method for suppressing spontaneous differentiation of pluripotent stem cells in adherent culture by adding a protein kinase C (PKC) inhibitor to the medium. Furthermore, Non-Patent Document 3 discloses a method for suppressing spontaneous differentiation of pluripotent stem cells in adherent culture by adding a tankyrase (TNKS) inhibitor to the medium.

[0005] As mentioned above, various methods have been developed for efficiently producing target somatic cells from pluripotent stem cells. However, the methods in Non-Patent Documents 2 and 3 only describe adhesion culture, and do not describe the effect of inhibiting spontaneous differentiation in suspension culture, nor do they describe or suggest the effect of combining a PKC inhibitor with a TNKS inhibitor. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Olmer R.et al.,Tissue Engineering:Part C,Volume 18(10):772-784(2012) [Non-patent document 2] M.Kinehara et al.,PLoS One.2013;8(1):e54122 [Non-patent document 3] T.Sumi et al.,PLoS One.2013;8(5):e63378 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a technique for suppressing spontaneous differentiation into all three germ layers, i.e., endoderm, mesoderm, and ectoderm, and maintaining the undifferentiated state of pluripotent stem cells. [Means for solving the problem]

[0008] As a result of extensive research to solve the above problems, the present inventors have succeeded in suppressing spontaneous differentiation into all three germ layers and maintaining the undifferentiated state of pluripotent stem cells by coexisting a PKCβ inhibitor and a TNKS inhibitor in suspension culture of pluripotent stem cells. The present invention was completed based on these research results and includes the following:

[0009] (1) A method for producing a pluripotent stem cell population, comprising a step of suspension culturing pluripotent stem cells in a liquid medium containing a PKCβ inhibitor and a TNKS inhibitor. (2) The method according to (1), wherein the concentration of the PKCβ inhibitor in the liquid medium is 25 nM or more and 15 μM or less. (3) The method according to (1), wherein the concentration of the TNKS inhibitor in the liquid medium is 90 nM or more and 40 μM or less. (4) The method according to (1), wherein the ratio of the concentrations of the PKCβ inhibitor and the TNKS inhibitor contained in the liquid medium is in the range of 167:1 or more and 1:1600 or less. (5) The method according to (1), wherein the liquid medium contains at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate. (6) The method according to (1), wherein the liquid medium contains FGF2 and / or TGF-β1. (7) The method according to (1), wherein the liquid medium contains a ROCK inhibitor. (8) The method according to (7), wherein the ROCK inhibitor is Y-27632. (9) The method according to (1), wherein the step of suspension culture includes a step of forming cell aggregates. (10) The method according to (1), wherein the step of suspension culture includes a step of recovering cell aggregates. (11) The method according to (1), wherein the pluripotent stem cell population has a ratio of OCT4-positive cells of 90% or more, a ratio of SOX2-positive cells of 90% or more, and a ratio of Nanog-positive cells of 90% or more. (12) The method according to (1), wherein the pluripotent stem cells are ES cells and / or induced pluripotent stem cells. (13) The method according to (1), wherein the suspension culture is carried out until the lactic acid concentration in the liquid medium reaches 5 to 15 mM. (14) The method according to (1), wherein the suspension culture is an agitation culture with an impeller tip speed of 0.05 m / s or more and 1.37 m / s or less. (15) A pluripotent stem cell population produced by the method according to any one of (1) to (14) above. (16) Pluripotent stem cell differentiation inhibitors, including PKCβ inhibitors and TNKS inhibitors. (17) The pluripotent stem cell differentiation inhibitor according to (16), wherein the PKCβ inhibitor is contained at a concentration of 50 nM or more and 200 mM or less. (18) The pluripotent stem cell differentiation inhibitor according to (16), wherein the TNKS inhibitor is contained at a concentration of 180 nM or more and 113 mM or less. (19) The pluripotent stem cell differentiation inhibitor according to (16), wherein the ratio of the concentrations of the PKCβ inhibitor and the TNKS inhibitor contained in the differentiation inhibitor is in the range of 167:1 or more and 1:1600 or less. (20) A kit for inhibiting pluripotent stem cell differentiation, comprising the pluripotent stem cell differentiation inhibitor according to any one of (16) to (19) above.

[0010] (21) The method according to (1) and the pluripotent stem cell differentiation inhibitor according to (16), wherein the PKCβ inhibitor is at least one selected from the group consisting of Go6983, GF109203X, LY-333531, Enzastaurin, Sotrastaurin, Ro-31-8220-mesylate, Ro-32-0432-hydrochloride, Go6976, Rottlerin, Midostaurin, Daphnetin, Dequalinium Chloride, Baicalein, Quercetin, Luteolin, Bisindolylmaleimide II, Calphostin C, Chelerythrine chloride, L-threo Dihydrosphingosine, and Melittin. (22) The method described in (1) and the pluripotent stem cell differentiation inhibitor described in (16), wherein the TNKS inhibitor is at least one selected from the group consisting of IWR-1-endo, XAV939, G007-LK, G244-LM and WIKI4. (23) The method according to (1) and the pluripotent stem cell differentiation inhibitor according to (16), wherein the pluripotent stem cells are primed pluripotent stem cells. (24) The method according to (1), wherein the liquid medium does not contain LIF. (25) The kit for inhibiting pluripotent stem cell differentiation according to (20), which comprises a liquid medium composition that does not contain LIF. (26) The method according to (1), wherein the liquid medium does not contain a GSK3 inhibitor. (27) The kit for inhibiting pluripotent stem cell differentiation according to (20), comprising a liquid medium composition that does not contain a GSK3 inhibitor. (28) The method according to (1), wherein the liquid medium does not contain a GSK3 inhibitor or a MEK / ERK inhibitor. (29) The kit for inhibiting pluripotent stem cell differentiation according to (20), comprising a liquid medium composition that does not contain a GSK3 inhibitor or a MEK / ERK inhibitor. (30) The method according to (1) and the pluripotent stem cell differentiation inhibitor according to (16), wherein the PKCβ inhibitor is a compound having the following structural formula [Formula I]:

[0011] [ka] A compound represented by the formula (I) or a salt thereof. In Formula I, R1 is a hydrogen atom or an alkoxy group having 1 to 3 carbon atoms (preferably a methoxy group), R2 is a hydrogen atom, an alkyl group having 1 to 3 carbon atoms (preferably a methyl group), or —N(R A an alkyl group having 1 to 3 carbon atoms substituted with —(CH)—N(CH), R A are independently an ethyl group or a methyl group (preferably a methyl group), R3 is [ka] , [ka] , or [ka] is a group represented by R B represents a hydrogen atom, an alkyl group having 2 to 4 carbon atoms substituted with -SC(=NH)(-NH2) (preferably -(CH2)3-SC(=NH)(-NH2)), or [ka]

[0012] is a group represented by Or, R2 and R B and combine to form the following divalent group: [ka] where # indicates the bond to the attachment point of R and ## indicates the bond to R B refers to the bond to the bonding position of The configuration of the asymmetric carbon atom contained in the divalent group is not particularly limited, but preferably, [ka] and R C is -N(R D an alkyl group having 1 to 3 carbon atoms (preferably —(CH)—N(CH)), substituted with R D are independently an ethyl group or a methyl group (preferably a methyl group). Examples of salts of the compound represented by formula I include hydrochlorides and sulfates. This specification includes the disclosure of Japanese Patent Application No. 2020-021843, from which this application claims priority. [Effects of the Invention]

[0013] According to the method for producing a pluripotent stem cell population of the present invention, a pluripotent stem cell population that maintains an undifferentiated state can be produced by suspension culture. The pluripotent stem cell differentiation inhibitor of the present invention can maintain the undifferentiated state of pluripotent stem cells in suspension culture. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows phase contrast images of cell aggregates photographed in Comparative Examples 1 to 3 and Example 1. [Figure 2] FIG. 1 is a characteristic diagram showing the results of quantitative real-time PCR analysis performed in Example 2. [Figure 3] 10 shows phase contrast images of cell aggregates photographed in Comparative Examples 4, 5-1 to 5-4 and Examples 3-1 to 3-3. [Figure 4] FIG. 1 is a characteristic diagram showing the relative gene expression level of PAX6 in pluripotent stem cells cultured in Reference Example 2, Comparative Examples 4, 5-1 to 5-4, and Examples 3-1 to 3-3. [Figure 5] FIG. 1 is a characteristic diagram showing the positive rates of undifferentiation markers OCT4, SOX2, and NANOG in pluripotent stem cells cultured in Reference Example 3, Comparative Examples 6, 7-1 to 7-4, and Examples 5-1 to 5-2. [Figure 6]FIG. 1 is a characteristic diagram showing the relative gene expression level of T in pluripotent stem cells cultured in Reference Example 4, Comparative Examples 8 and 9, and Example 7. [Figure 7] FIG. 1 is a characteristic diagram showing the relative gene expression level of SOX17 in pluripotent stem cells cultured in Reference Example 4, Comparative Examples 8 and 9, and Example 7. [Figure 8] 10 shows phase contrast images of cell aggregates photographed in Comparative Example 10 and Example 9. [Figure 9] FIG. 10 is a characteristic diagram showing the relative gene expression level of T in pluripotent stem cells cultured in Reference Example 5, Comparative Example 10, and Example 9. [Figure 10] FIG. 10 is a characteristic diagram showing the relative gene expression level of SOX17 in pluripotent stem cells cultured in Reference Example 5, Comparative Example 10, and Example 9. [Figure 11] 1 is a characteristic diagram showing the relative gene expression level of PAX6 in pluripotent stem cells cultured in Reference Example 5, Comparative Example 10, and Example 9. FIG. [Figure 12] 10 shows phase contrast images of pluripotent stem cells cultured in adhesion in Comparative Example 11 (with and without the addition of a TNKS inhibitor and a PKCβ inhibitor). [Figure 13] 10 shows phase contrast images of pluripotent stem cells cultured in suspension in Example 11 (with and without the addition of a TNKS inhibitor and a PKCβ inhibitor). [Figure 14] FIG. 10 is a characteristic diagram showing the expansion rates of pluripotent stem cells cultured in Comparative Example 11 and Example 11. [Figure 15] 10 is a phase-contrast image of pluripotent stem cells cultured in suspension in Comparative Example 12. 11 is a phase-contrast image of a captured cell aggregate. [Figure 16] 10 is a phase-contrast image of pluripotent stem cells cultured in suspension in Example 12. 11 is a phase-contrast image of a captured cell aggregate. [Figure 17] FIG. 10 is a characteristic diagram showing the relative gene expression levels of OCT4 and SOX2 in pluripotent stem cells cultured in Comparative Example 12 and Example 12. [Figure 18] FIG. 10 is a characteristic diagram showing the OCT4 positivity rate in pluripotent stem cells cultured in Comparative Example 12 and Example 12. [Figure 19] FIG. 10 is a characteristic diagram showing the viable cell densities achieved in pluripotent stem cells cultured in Comparative Example 12 and Example 12. [Figure 20] FIG. 10 is a characteristic diagram showing the concentration of lactic acid contained in the medium for pluripotent stem cells cultured in Example 12. [Figure 21] FIG. 1 is a characteristic diagram showing the relative gene expression levels of OCT4, SOX2, NANOG, T, SOX17, and PAX6 in pluripotent stem cells cultured in Example 13 and Reference Example 6. [Figure 22] FIG. 11 is a characteristic diagram showing the results of G-Band analysis of the pluripotent stem cells cultured in Example 13. [Figure 23] This is a characteristic diagram showing the results of measuring the relative gene expression levels of CDX2, PDGFRa, SOX17, and PAX6 in pluripotent stem cells cultured in Example 13 and Reference Example 6 (maintenance of undifferentiation) and Example 14 and Reference Example 7 (induction of differentiation). DETAILED DESCRIPTION OF THE INVENTION

[0015] 1. Pluripotent stem cell differentiation inhibitor Overview The pluripotent stem cell differentiation inhibitor of the present invention is characterized by containing a protein kinase C β (PKCβ) inhibitor and a tankyrase (TNKS) inhibitor. Furthermore, the pluripotent stem cell differentiation inhibitor of the present invention can be a composition comprising a PKCβ inhibitor and a TNKS inhibitor. The pluripotent stem cell differentiation inhibitor of the present invention maintains the undifferentiated state of cells in suspension culture, making it possible to easily produce a pluripotent stem cell population by suspension culture.

[0016] 1-2.Definition of Terms As used herein, the following terms are defined. ≪Cell≫ As used herein, the term "pluripotent stem cells" refers to cells that possess the multipotency (multiple-potency) to differentiate into all cell types that constitute a living organism and can continue to proliferate indefinitely while maintaining pluripotency when cultured in vitro under appropriate conditions. More specifically, pluripotency refers to the ability to differentiate into the germ layers that constitute an individual (the three germ layers in vertebrates: ectoderm, mesoderm, and endoderm). Examples of such cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), and induced pluripotent stem cells (iPS cells). "ES cells" refer to pluripotent stem cells prepared from early embryos. "EG cells" are pluripotent stem cells prepared from fetal primordial germ cells (Shamblott MJ et al., 1998, Proc. Natl. Acad. Sci. USA., 95:13726-13731). "GS cells" are pluripotent stem cells prepared from testis (Conrad S., 2008, Nature, 456:344-349). "iPS cells" are pluripotent stem cells that can be reprogrammed to an undifferentiated state by introducing genes encoding a small number of reprogramming factors into differentiated somatic cells.

[0017] The pluripotent stem cells referred to herein may be cells derived from a multicellular organism. They are preferably animal-derived cells, more preferably mammalian-derived cells. Examples include rodents such as mice, rats, hamsters, and guinea pigs; livestock or pets such as dogs, cats, rabbits, cows, horses, sheep, and goats; and primates such as humans, rhesus monkeys, gorillas, and chimpanzees. Human-derived cells are particularly preferred.

[0018] As used herein, pluripotent stem cells include naive pluripotent stem cells and primed pluripotent stem cells. Naive pluripotent stem cells are defined as a state similar to the pluripotency observed in the inner cell mass before implantation, while primed pluripotent stem cells are defined as a state similar to the pluripotency observed in the epiblast after implantation. Compared to naive pluripotent stem cells, primed pluripotent stem cells are characterized by a lower frequency of contribution to ontogeny, a single transcriptional activity of the X chromosome, and a high level of transcriptionally repressive histone modifications. Furthermore, the marker gene for primed pluripotent stem cells is OTX2, while the marker genes for primed pluripotent stem cells are REX1 and the KLF family. Furthermore, primed pluripotent stem cells form flat colonies, while naive pluripotent stem cells form dome-shaped colonies. It is particularly preferred to use primed pluripotent stem cells as the pluripotent stem cells used herein.

[0019] The pluripotent stem cells used herein may be commercially available cells, cells provided by a donor, or newly prepared cells. Although not limited thereto, when used in each invention herein, the pluripotent stem cells are preferably iPS cells or ES cells.

[0020] When the iPS cells used herein are commercially available, examples include, but are not limited to, the 253G1 strain, 253G4 strain, 201B6 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, TkDN4-M strain, and TkDA strain. Strains that can be used include the hiPSC3-1 strain, TkDA3-2 strain, TkDA3-4 strain, TkDA3-5 strain, TkDA3-9 strain, TkDA3-20 strain, hiPSC38-2 strain, MSC-iPSC1 strain, BJ-iPSC1 strain, RPChiPS771-2, WTC-11 strain, 1231A3 strain, 1383D2 strain, 1383D6 strain, 1210B2 strain, 1201C1 strain, and 1205B2 strain.

[0021] Furthermore, when the iPS cells used herein are newly generated cells, the combination of genes for the reprogramming factors to be introduced is not limited, but examples include the combination of OCT3 / 4, KLF4, SOX2, and c-Myc genes (Yu J, et al. 2007, Science, 318:1917-20), and the combination of OCT3 / 4, SOX2, LIN28, and Nanog genes (Takahashi K, et al. 2007, Cell, 131:861-72). The manner in which these genes are introduced into cells is not particularly limited, but may be, for example, gene transfer using a plasmid, synthetic RNA, or protein. iPS cells generated by methods using microRNA, RNA, small molecules, or the like may also be used. Furthermore, newly generated clinical-grade iPS cells may also be used.

[0022] When the ES cells used herein are commercially available, for example, but not limited to, the KhES-1 strain, KhEs-2 strain, KhEs-3 strain, KhEs-4 strain, KhEs-5 strain, SEES1 strain, SEES2 strain, SEES3 strain, SEES-4 strain, SEEs-5 strain, SEEs-6 strain, SEEs-7 strain, HUES8 strain, CyT49 strain, H1 strain, H9 strain, HS-181 strain, etc. can be used.

[0023] ≪Cell aggregate≫ As used herein, a "cell aggregate" refers to a mass-like cell group formed by cell aggregation in suspension culture, and is also called a spheroid. Cell aggregates usually have a roughly spherical shape. The cells that make up the cell aggregate are not particularly limited, as long as they are one or more types of cells. For example, a cell aggregate made up of pluripotent stem cells such as human pluripotent stem cells or human embryonic stem cells contains cells that express a pluripotent stem cell marker and / or are positive for the pluripotent stem cell marker.

[0024] Pluripotent stem cell markers are gene markers that are specifically or excessively expressed in pluripotent stem cells, and examples include alkaline phosphatase, Nanog, OCT4, SOX2, TRA-1-60, c-Myc, KLF4, LIN28, SSEA-4, and SSEA-1.

[0025] Pluripotent stem cell markers can be detected by any detection method known in the art. Methods for detecting cell markers include, but are not limited to, flow cytometry. When a fluorescently labeled antibody is used as a detection reagent in flow cytometry, cells that emit stronger fluorescence than a negative control (isotype control) are determined to be "positive" for the marker. The proportion of cells that test positive for a fluorescently labeled antibody analyzed by flow cytometry is sometimes referred to as the "positive rate." Furthermore, any fluorescently labeled antibody known in the art can be used, including, but not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc.

[0026] When the cells constituting the cell aggregate are pluripotent stem cells, the positivity rate for the pluripotent stem cell marker can be preferably 80% or more, more preferably 90% or more, more preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and more preferably 100% or less. A cell aggregate in which the proportion of cells expressing and / or positive for a pluripotent stem cell marker is within the above range is a highly undifferentiated and more homogeneous cell population.

[0027] <Culture and medium> "Suspension culture" is a cell culture method in which cells are grown in a suspended state in a medium. As used herein, "suspension state" refers to a state in which cells are not adherent to an external matrix such as a culture vessel. "Suspension culture" is a method of culturing cells in suspension, in which cells exist as aggregated cell masses in the culture medium. An alternative culture method to suspension culture is adhesion culture. "Adhesion culture" is a method of culturing cells in an adherent manner. "Adhesion culture" refers to adhering cells to an external matrix such as a culture vessel and growing them, in principle, in a monolayer. Note that the aforementioned adherent cells can usually be cultured not only in adhesion culture but also in suspension culture.

[0028] As used herein, the term "medium" refers to a liquid or solid substance prepared for culturing cells. In principle, it contains the minimum amount of components essential for cell growth and / or maintenance. Unless otherwise specified, the medium referred to in this specification refers to a liquid medium for animal cells used to culture animal-derived cells.

[0029] As used herein, the term "basal medium" refers to a medium that serves as the basis for various animal cell culture media. Culture can be performed using the medium alone, or various culture additives can be added to prepare media specific to various cells depending on the purpose. Examples of basal media used herein include, but are not limited to, BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof (e.g., DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)). As the DMEM / F12 medium, a medium in which DMEM medium and Ham's F12 medium are mixed at a weight ratio of preferably 60 / 40 to 40 / 60, for example, 58 / 42, 55 / 45, 52 / 48, 50 / 50, 48 / 52, 45 / 55, or 42 / 58, is particularly used. In addition, media used for culturing human iPS cells and human ES cells can also be suitably used. The medium used in the present invention is preferably a medium that does not contain serum, ie, a serum-free medium.

[0030] As used herein, the term "culture additive" refers to a substance other than serum that is added to a culture medium for the purpose of culture. Specific examples of culture additives include, but are not limited to, L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, growth factors, fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and antibiotics. Insulin, transferrin, and cytokines may be naturally derived substances isolated from tissues or serum of animals (preferably humans, mice, rats, cattle, horses, goats, etc.), or may be recombinant proteins produced by genetic engineering. Growth factors that can be used include, but are not limited to, basic fibroblast growth factor-2 (FGF2), transforming growth factor-β1 (TGF-β1), activin A, IGF-1, MCP-1, IL-6, PAI, PEDF, IGFBP-2, LIF, and IGFBP-7. Antibiotics that can be used include, but are not limited to, penicillin, streptomycin, and amphotericin B. Particularly preferred growth factors as culture additives for the medium used in the present invention are FGF2 and / or TGF-β1.

[0031] The medium preferably contains a ROCK inhibitor, such as Y-27632. By including a ROCK inhibitor in the medium, cell death in suspension culture of pluripotent stem cells can be significantly suppressed.

[0032] Furthermore, when culturing primed pluripotent stem cells, the medium preferably does not contain LIF. Furthermore, when culturing primed pluripotent stem cells, the medium preferably does not contain either a GSK3 inhibitor or a MEK / ERK inhibitor, preferably both. A medium that does not contain LIF, a GSK3 inhibitor, or a MEK / ERK inhibitor allows primed pluripotent stem cells to be cultured without naivetization and while maintaining an undifferentiated state.

[0033] The medium used in the present invention may contain one or more of the above culture additives. The medium to which the above culture additives are added is generally, but not limited to, the above basal medium.

[0034] Culture additives can be added to the medium in the form of a solution, derivative, salt, mixed reagent, or the like. For example, L-ascorbic acid may be added to the medium in the form of a derivative such as magnesium 2-ascorbate phosphate, and selenium may be added to the medium in the form of a selenite (sodium selenite, etc.). Insulin, transferrin, and selenium can also be added to the medium in the form of an ITS reagent (insulin-transferrin-selenium). Commercially available media supplemented with at least one selected from L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate can also be used. Commercially available media supplemented with insulin and transferrin include CHO-S-SFM II (Life Technologies Japan, Inc.), Hybridoma-SFM (Life Technologies Japan, Inc.), eRDF Dry Powdered Media (Life Technologies Japan, Inc.), and UltraCULTURE TM (BioWhittaker), UltraDOMA TM (BioWhittaker), UltraCHO TM (BioWhittaker), UltraMDCK TM (BioWhittaker), STEMPRO® hESC SFM (Life Technologies Japan), Essential8 TM (Life Technologies Japan, Inc.), StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.), mTeSR1 (Veritas), and TeSR2 (Veritas).

[0035] The most preferred medium for use in the present invention is a serum-free medium containing L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, and at least one growth factor, and particularly preferred is serum-free DMEM / F12 medium containing L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, and at least one growth factor (preferably FGF2 and TGF-β1).

[0036] <PKCβ inhibitor> As used herein, the term "protein kinase C β (PKC β) inhibitor" refers to a substance that inhibits or suppresses the activity of PKC β. Protein kinases have a C-terminal catalytic domain and an N-terminal regulatory domain. The catalytic domain contains a sequence that recognizes phosphorylated residues on substrate proteins and a sequence that recognizes ATP / Mg 2+ The regulatory region consists of the C1 and C2 domains.

[0037] PKC includes conventional isozymes PKCα, PKCβI, PKCβII, and PKCγ, novel isozymes PKCδ, PKCε, PKCθ, and PKCη, and atypical isozymes PKCζ, PKCλ, and PKCμ.

[0038] As used herein, the term "PKCβ" refers to both PKCβI and PKCβII or to either PKCβI or PKCβII. Furthermore, as used herein, the term "PKCβ inhibitor" refers to a substance that inhibits at least PKCβI and / or PKCβII among the conventional, novel, and atypical isozymes. In other words, the term "PKCβ inhibitor" includes substances that inhibit or suppress only the activity of PKCβI, substances that inhibit or suppress only the activity of PKCβII, and substances that inhibit or suppress the activities of PKCβI and PKCβII.

[0039] The PKCβ inhibitor may be a substance that specifically inhibits or suppresses only the activity of PKCβ, or may be a substance that inhibits or suppresses the activity of isozymes other than PKCβI or PKCβII. For example, the PKCβ inhibitor may be a substance that inhibits or suppresses the activity of all of the conventional, novel, and atypical isozymes described above, including PKCβI and PKCβII. The PKCβ inhibitor may also be a substance that inhibits or suppresses the activity of the conventional isozymes PKCα and PKCγ in addition to PKCβI and PKCβII. Furthermore, the PKCβ inhibitor may be a substance that inhibits or suppresses the activity of the novel isozymes PKCδ, PKCε, PKCθ, and PKCη in addition to PKCβI and PKCβII.

[0040] PKCβ inhibitors include compounds that act directly or indirectly on PKCβ, antisense nucleic acids against the gene encoding PKCβ, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof.

[0041] An example of a PKCβ inhibitor is a compound having the following structural formula [Formula I]:

[0042] [ka] A compound represented by the formula (I) or a salt thereof. In Formula I, R1 is a hydrogen atom or an alkoxy group having 1 to 3 carbon atoms (preferably a methoxy group), and R2 is a hydrogen atom, an alkyl group having 1 to 3 carbon atoms (preferably a methyl group), or —N(R A an alkyl group having 1 to 3 carbon atoms substituted with —(CH)—N(CH), R A are independently an ethyl group or a methyl group (preferably a methyl group), R3 is [ka] , [ka] , or [ka] is a group represented by R B represents a hydrogen atom, an alkyl group having 2 to 4 carbon atoms substituted with -SC(=NH)(-NH2) (preferably -(CH2)3-SC(=NH)(-NH2)), or [ka] is a group represented by Or, R2 and R B and combine to form the following divalent group: [ka] where # indicates the bond to the attachment point of R and ## indicates the bond to R B refers to the bond to the bonding position of The configuration of the asymmetric carbon atom contained in the divalent group is not particularly limited, but preferably, [ka] and R C is -N(R D an alkyl group having 1 to 3 carbon atoms (preferably —(CH)—N(CH)), substituted with R D are independently an ethyl group or a methyl group (preferably a methyl group).

[0043] Examples of salts of the compound represented by formula I include hydrochlorides and sulfates. Specific examples of PKCβ inhibitors having the above structural formula [Formula I] include compounds selected from the group consisting of Go6983, GF109203X, LY-333531, enzastaurin, sotrastaurin, Ro-31-8220-mesylate, Ro-32-0432-hydrochloride, Go6976, rottlerin, midostaurin, daphnetin, dequalinium chloride, baicalein, quercetin, luteolin, bisindolylmaleimide II, calphostin C, chelerythrine chloride, L-threodihydrosphingosine, and melittin. Among PKCβ inhibitors having the above structural formula, it is particularly preferred to use a compound selected from the group consisting of Go6983, GF109203X, and LY-333531.

[0044] The structural formula of Go6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione) is shown below.

[0045] [ka]

[0046] The structural formula of GF109203X (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide) is shown below.

[0047] [ka]

[0048] The structural formula of LY-333531 ((9S)-[(dimethylamino)methyl]-6,7,10,11-tetrahydro-9H,18H-5,21:12,17-dimethenodibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecine-18,20(19H)-dione, monohydroxychloride) is shown below.

[0049] [ka]

[0050] The structural formula of Enzastaurin (3-(1-methylindol-3-yl)-4-[1-[1-(pyridin-2-ylmethyl)piperidin-4-yl]indol-3-yl]pyrrole-2,5-dione) is shown below.

[0051] [ka]

[0052] The structural formula of sotrastaurin (3-(1H-indol-3-yl)-4-(2-(4-methylpiperazin-1-yl)quinazolin-4-yl)-1H-pyrrole-2,5-dione) is shown below.

[0053] [ka]

[0054] The structural formula of Ro-31-8220-mesylate (3-[3-[2,5-dihydro-4-(1-methyl-1H-indol-3-yl)-2,5-dioxo-1H-pyrrol-3-yl]-1H-indol-1-yl]propylcarbamimidothioic acid ester mesylate) is shown below.

[0055] [ka]

[0056] The structural formula of Ro-32-0432-hydrochloride (3-[(8S)-8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl]-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione hydrochloride) is shown below.

[0057] [ka]

[0058] <TNKS inhibitors> As used herein, the term "tankyrase (TNKS) inhibitor" refers to a substance that inhibits or suppresses the activity of tankyrase. Tankyrase (sometimes referred to as tankyrase) belongs to the poly(ADP-ribosyl)transferase (PARP) family, which poly(ADP-ribosyl)ates target proteins, and tankyrase-1 (tankyrase-1 / PARP-5a) and tankyrase-2 (tankyrase-2 / PARP-5b) are known. Tankyrase is known to promote telomere elongation by telomerase by poly(ADP-ribosyl)ating the telomeric protein TRF1 and releasing it from telomeres.

[0059] As used herein, the term "TNKS" refers to both tankyrase 1 and tankyrase 2, or to either tankyrase 1 or tankyrase 2. Furthermore, as used herein, the term "TNKS inhibitor" refers to a substance that inhibits tankyrase 1 and / or tankyrase 2. That is, the term "TNKS inhibitor" includes substances that inhibit or suppress only the activity of tankyrase 1, substances that inhibit or suppress only the activity of tankyrase 2, and substances that inhibit or suppress the activities of tankyrase 1 and tankyrase 2.

[0060] TNKS inhibitors include compounds that act directly or indirectly on TNKS, antisense nucleic acids against the gene encoding TNKS, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof.

[0061] Examples of TNKS inhibitors include compounds selected from the group consisting of IWR-1-endo, XAV939, G007-LK, G244-LM, MSC2504877, and WIKI4. Among TNKS inhibitors, it is particularly preferable to use IWR-1-endo and / or XAV939.

[0062] The structural formula of IWR-1-endo (4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide) is shown below.

[0063] [ka]

[0064] The structural formula of XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one) is shown below.

[0065] [ka]

[0066] The structural formula of G007-LK (4-[5-[(1E)-2-[4-(2-chlorophenyl)-5-[5-(methylsulfonyl)-2-pyridinyl]-4H-1,2,4-thiazol-3-yl]ethenyl]-1,3,4-oxadiazol-2-yl]-benzonitrile) is shown below.

[0067] [ka]

[0068] The structural formula of G244-LM (3,5,7,8-tetrahydro-2-[4-[2-(methylsulfonyl)phenyl]-1-piperazinyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one) is shown below.

[0069] [ka]

[0070] The structural formula of WIKI4 (2-[3-[[4-(4-methoxyphenyl)-5-(4-pyridinyl)-4H-1,2,4-thiazol-3-yl]thio]propyl]-1H-benzo[de]isoquinoline-1,3(2H)-dione) is shown below.

[0071] [ka]

[0072] 1-3.Configuration The pluripotent stem cell differentiation inhibitor according to the present invention contains, as active ingredients, the PKCβ inhibitor and the TNKS inhibitor defined in 1-2 above.

[0073] Here, the PKCβ inhibitor in the pluripotent stem cell differentiation inhibitor may be one type, or a combination of two or more different types.

[0074] The lower limit of the concentration of the PKCβ inhibitor is not particularly limited, and can be determined depending on the range in which it has an inhibitory effect on differentiation of pluripotent stem cells.

[0075] For example, the final concentration of the PKCβ inhibitor in the liquid medium can be 25 nM or more, 30 nM or more, 50 nM or more, 80 nM or more, 100 nM or more, 150 nM or more, 200 nM or more, 500 nM or more, 700 nM or more, 1 μM or more, 3 μM or more, 5 μM or more, or 10 μM or more.

[0076] The upper limit of the concentration of the PKCβ inhibitor is not particularly limited, and can be determined depending on the range in which the inhibitor has an inhibitory effect on differentiation of pluripotent stem cells, the solubility of the PKCβ inhibitor, and the like.

[0077] For example, the final concentration of the PKCβ inhibitor in the liquid medium can be 15 μM or less, 10 μM or less, 5 μM or less, 3 μM or less, 1 μM or less, 700 nM or less, 500 nM or less, 200 nM or less, 150 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, or 30 nM or less.

[0078] In addition, when the pluripotent stem cell differentiation inhibitor is a composition, the concentration of the PKCβ inhibitor in the composition can be determined so that it is within the above range when added to a culture medium. For example, when the composition is used after being diluted 2-fold, the lower limit of the PKCβ inhibitor in the composition can be 50 nM or more, 60 nM or more, 100 nM or more, 160 nM or more, 200 nM or more, 300 nM or more, 400 nM or more, 1 μM or more, 1.4 μM or more, 2 μM or more, 6 μM or more, 10 μM or more, or 20 μM or more.

[0079] The upper limit of the PKCβ inhibitor in the composition is not particularly limited and can be determined by the solubility of the PKCβ inhibitor. Specifically, the upper limit of the PKCβ inhibitor in the composition can be set to 200 mM.

[0080] On the other hand, the TNKS inhibitor in the pluripotent stem cell differentiation inhibitor may be one type, or a combination of two or more different types.

[0081] The lower limit of the concentration of the TNKS inhibitor is not particularly limited, and can be determined depending on the range in which it has an inhibitory effect on differentiation of pluripotent stem cells.

[0082] For example, the final concentration of the TNKS inhibitor in the liquid medium can be 90 nM or more, 100 nM or more, 150 nM or more, 200 nM or more, 300 nM or more, 400 nM or more, 500 nM or more, 600 nM or more, 700 nM or more, 800 nM or more, 900 nM or more, 1 μM or more, 1.5 μM or more, 3 μM or more, 5 μM or more, 10 μM or more, 15 μM or more, 30 μM or more, or 35 μM or more.

[0083] The upper limit of the concentration of the TNKS inhibitor is not particularly limited, and can be determined depending on the range in which the inhibitor has an inhibitory effect on differentiation of pluripotent stem cells, the solubility of the TNKS inhibitor, and the like.

[0084] For example, the final concentration of the TNKS inhibitor in the liquid medium can be 40 μM or less, 35 μM or less, 30 μM or less, 15 μM or less, 10 μM or less, 5 μM or less, 3 μM or less, 1.5 μM or less, 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 150 nM or less, or 100 nM or less.

[0085] In addition, when the pluripotent stem cell differentiation inhibitor is a composition, the concentration of the TNKS inhibitor in the composition can be determined so that it is within the above range when added to a culture medium.For example, when the composition is diluted 2 times and used, the lower limit of the TNKS inhibitor in the composition can be 180nM or more, 200nM or more, 300nM or more, 400nM or more, 600nM or more, 800nM or more, 1μM or more, 1.2μM or more, 1.4μM or more, 1.6μM or more, 1.8μM or more, 2μM or more, 3μM or more, 6μM or more, 10μM or more, 20μM or more, 30μM or more, 60μM or more, or 70μM or more.

[0086] The upper limit of the TNKS inhibitor in the composition is not particularly limited and can be the solubility of the TNKS inhibitor. Specifically, the upper limit of the TNKS inhibitor in the composition can be 113 mM.

[0087] Furthermore, the lower limit of the ratio of the concentrations of the PKCβ inhibitor to the TNKS inhibitor in the liquid medium is not particularly limited, but can be, for example, 167:1 or more, 111:1 or more, 56:1 or more, 33:1 or more, 11:1 or more, 7.8:1 or more, 5.6:1 or more, 2.2:1 or more, 1.7:1 or more, or 1.1:1 or more. The upper limit of the ratio of the concentrations of the PKCβ inhibitor to the TNKS inhibitor in the liquid medium is not particularly limited, but can be, for example, 1:1600 or less, 1:1400 or less, 1:1200 or less, 1:600 ​​or less, 1:400 or less, 1:200 or less, 1:120 or less, 1:60 or less, 1:40 or less, 1:36 or less, 1:32 or less, 1:28 or less, 1:24 or less, 1:20 or less, 1:16 or less, 1:12 or less, 1:8 or less, 1:6 or less, or 1:4 or less. The ratio of the concentrations of the PKCβ inhibitor to the TNKS inhibitor in the liquid medium is not particularly limited, but can be, for example, in the range of 167:1 to 1:1600. In addition, the ratio of the concentrations of the PKCβ inhibitor and the TNKS inhibitor in the liquid culture medium can be in the range of 111:1 or more and 1:1600 or less, 56:1 or more and 1:1600 or less, 33:1 or more and 1:1600 or less, 11:1 or more and 1:1600 or less, 7.8:1 or more and 1:1600 or less, 5.6:1 or more and 1:1600 or less, 2.2:1 or more and 1:1600 or less, 1.7:1 or more and 1:1600 or less, or 1.1:1 or more and 1:1600 or less.Furthermore, the ratio of the concentrations of the PKCβ inhibitor and the TNKS inhibitor in the liquid medium can be in the range of 167:1 or more and 1:1400 or less, 167:1 or more and 1:1200 or less, 167:1 or more and 1:600 ​​or less, 167:1 or more and 1:400 or less, 167:1 or more and 1:200 or less, 167:1 or more and 1:120 or less, 167:1 or more and 1:60 or less, 167:1 or more and 1:40 or less, The ratio may be in the range of 167:1 to 1:36, 167:1 to 1:32, 167:1 to 1:28, 167:1 to 1:24, 167:1 to 1:20, 167:1 to 1:16, 167:1 to 1:12, 167:1 to 1:8, 167:1 to 1:6, or 167:1 to 1:4.

[0088] On the other hand, when the pluripotent stem cell differentiation inhibitor is a composition, other ingredients to be combined with the active ingredients, PKCβ inhibitor and TNKS inhibitor, include carriers, which include solvents and / or excipients.

[0089] Examples of solvents include water, buffers (including PBS), saline, organic solvents (DMSO, DMF, xylene, lower alcohols), and the like.

[0090] Examples of excipients include antibiotics, buffers, thickeners, colorants, stabilizers, surfactants, emulsifiers, antiseptics, preservatives, and antioxidants. Antibiotics are not particularly limited, but examples include penicillin, streptomycin, and amphotericin B. Examples of buffers include phosphate buffer, Tris-HCl buffer, and glycine buffer. Examples of thickeners include gelatin and polysaccharides. Examples of colorants include phenol red. Examples of stabilizers include albumin, dextran, methylcellulose, and gelatin. Examples of surfactants include cholesterol, alkyl glycosides, alkyl polyglucosides, alkyl monoglyceryl ethers, glucosides, maltosides, neopentyl glycols, polyoxyethylene glycols, thioglucosides, thiomaltosides, peptides, saponins, phospholipids, fatty acid sorbitan esters, and fatty acid diethanolamides. Examples of emulsifiers include glycerin fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, and sucrose fatty acid esters. Examples of preservatives include aminoethylsulfonic acid, benzoic acid, sodium benzoate, ethanol, sodium edetate, agar, dl-camphor, citric acid, sodium citrate, salicylic acid, sodium salicylate, phenyl salicylate, dibutylhydroxytoluene, sorbic acid, potassium sorbate, nitrogen, dehydroacetic acid, sodium dehydroacetate, 2-naphthol, sucrose, honey, isobutyl parahydroxybenzoate, isopropyl parahydroxybenzoate, ethyl parahydroxybenzoate, butyl parahydroxybenzoate, propyl parahydroxybenzoate, methyl parahydroxybenzoate, l-menthol, and eucalyptus oil. Preservatives include benzoic acid, sodium benzoate, ethanol, sodium edetate, dried sodium sulfite, citric acid, glycerin, salicylic acid, sodium salicylate, dibutylhydroxytoluene, D-sorbitol, sorbic acid, potassium sorbate, sodium dehydroacetate, isobutyl parahydroxybenzoate, isopropyl parahydroxybenzoate, ethyl parahydroxybenzoate, butyl parahydroxybenzoate, propyl parahydroxybenzoate, methyl parahydroxybenzoate, propylene glycol, and phosphoric acid.Antioxidants include citric acid, citric acid derivatives, vitamin C and its derivatives, lycopene, vitamin A, carotenoids, vitamin B and its derivatives, flavonoids, polyphenols, glutathione, selenium, sodium thiosulfate, vitamin E and its derivatives, alpha-lipoic acid and its derivatives, pycnogenol, flavangenol, superoxide dismutase (SOD), glutathione peroxidase, glutathione-S-transferase, glutathione reductase, catalase, ascorbate peroxidase, and mixtures thereof.

[0091] Furthermore, the pluripotent stem cell differentiation inhibitor may contain one or more growth factors, such as FGF2 and TGF-β1.

[0092] The pluripotent stem cell differentiation inhibitor may be in the form of a liquid or solid (including granules, powders, and dust). The application form of the pluripotent stem cell differentiation inhibitor is not particularly limited. For example, it may be in the form of a medium used for suspension culture, or it may be in the form of an additive to be added when preparing a medium for suspension culture.

[0093] 1-4.Effects The pluripotent stem cell differentiation inhibitor of the present invention can maintain the undifferentiated state of pluripotent stem cells in a suspension culture system and form cell aggregates, thereby enabling the efficient production of large amounts of pluripotent stem cells. 2. Method for producing pluripotent stem cell populations

[0094] 2-1. Overview The method for producing a pluripotent stem cell population according to the present invention comprises the step of suspension culturing pluripotent stem cells in the presence of a protein kinase C β (PKCβ) inhibitor and a tankyrase (TNKS) inhibitor. According to the method for producing a pluripotent stem cell population according to the present invention, the presence of the PKCβ inhibitor and the TNKS inhibitor maintains the undifferentiated state of the pluripotent stem cells, making it possible to easily produce a pluripotent stem cell population by suspension culture.

[0095] 2-2. Method The method of this embodiment includes a suspension culture step as an essential step, and a maintenance culture step and a recovery step as optional steps. Each step will be explained below.

[0096] 2-2-1. Maintenance culture process The "maintenance culture step" is a step of culturing a cell population before the suspension culture step, or a cell aggregate obtained after the suspension culture step or the subsequent recovery step, in order to proliferate the cells while maintaining their undifferentiated state. The maintenance culture can utilize animal cell culture methods known in the art. For example, the cells may be cultured by adhesion to a culture substrate such as a container or carrier, or by suspension culture.

[0097] Hereinafter, although not limited thereto, examples of animal cell culture methods used in this step and in the suspension culture step described below will be given and explained.

[0098] (cell) The cells used in this step are cells capable of cell aggregation in suspension culture. As described above in the section "Culture and Medium" in "1-2. Definition of Terms," ​​animal cells are preferred, and human cells are more preferred. Furthermore, the type of cells used is pluripotent stem cells, with pluripotent stem cells such as iPS cells and ES cells being particularly preferred.

[0099] (Culture container) The culture vessel used for the culture is preferably one with low cell adhesion to the vessel inner surface. Examples of vessels with low cell adhesion to the vessel inner surface include plates with hydrophilic surface treatment using a biocompatible substance. For example, Nunclon TM Sphera (Thermo Fisher Scientific Inc.) can be used as a culture vessel.

[0100] The shape of the culture vessel is not particularly limited, and examples thereof include culture vessels in the shape of a dish, flask, well, bag, spinner flask, etc.

[0101] The capacity of the culture vessel to be used can be selected appropriately and is not particularly limited, but the lower limit of the area of ​​the bottom surface of the part containing the culture medium when viewed in plan is 0.32 cm 2 Over 0.65cm 2 Over 1.9cm 2 3.0cm or more 2 Over 3.5cm 2 Over 9.0cm 2 or more, or 9.6 cm 2 Above, the upper limit is 1000 cm 2 Below, 500cm 2 Below, 300cm 2 Below, 150cm 2 Below, 75cm 2 Below, 55cm 2 Below, 25cm 2 Below, 21cm 2 Below, 9.6cm 2 Less than or equal to 3.5cm 2 It is preferable that:

[0102] (Culture medium) The volume of the medium or culture solution can be adjusted depending on the culture vessel used. For example, a 12-well plate (with a well bottom area of ​​3.5 cm per well in plan view) 2 When using a 6-well plate (where the area of ​​the well bottom in a plan view is 9.6 cm per well), the amount per well can be 0.5 mL or more and 1.5 mL or less, preferably about 1.3 mL. 2When using a 125 mL Erlenmeyer flask (a 125 mL Erlenmeyer flask), the lower limit of the amount per well can be 1.5 mL or more, 2 mL or more, or 3 mL or more, and the upper limit can be 6.0 mL or less, 5 mL or less, or 4 mL or less. Furthermore, when using a 125 mL Erlenmeyer flask (a 125 mL Erlenmeyer flask), the lower limit of the amount per container can be 10 mL or more, 15 mL or more, 20 mL or more, 25 mL or more, or 30 mL or more, and the upper limit can be 50 mL or less, 45 mL or less, or 40 mL or less. Furthermore, when using a 500 mL Erlenmeyer flask, the lower limit of the amount per container can be 100 mL or more, 105 mL or more, 110 mL or more, 115 mL or more, or 120 mL or more, and the upper limit can be 150 mL or less, 145 mL or less, 140 mL or less, 135 mL or less, 130 mL or less, or 125 mL or less. Furthermore, when using a 1000 mL Erlenmeyer flask, the lower limit of the amount per container can be 250 mL or more, 260 mL or more, 270 mL or more, 280 mL or more, or 290 mL or more, and the upper limit can be 350 mL or less, 340 mL or less, 330 mL or less, 320 mL or less, or 310 mL or less. Furthermore, for example, when a disposable culture bag with a capacity of 2 L is used, the amount per bag can be at least 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or 1000 mL, and at most 2000 mL, 1900 mL, 1800 mL, 1700 mL, 1600 mL, 1500 mL, 1400 mL, 1300 mL, 1200 mL, or 1100 mL. When a disposable culture bag with a capacity of 10 L is used, the amount per bag can be at least 500 mL, 1 L, 2 L, 3 L, 4 L, or 5 L, and at most 10 L, 9 L, 8 L, 7 L, or 6 L.

[0103] (seeding density) In suspension culture, the density of cells seeded in new medium (seeding density) can be adjusted appropriately taking into account the culture time, cell state after culture, and the number of cells required after culture. Although not limited, the lower limit is usually 0.01 × 10 5 cells / mL or more, 0.1×10 5 cells / mL or more, 1×10 5 cells / mL or more, or 2×10 5 cells / mL or more, with an upper limit of 20 × 10 5 cells / mL or less, or 10 x 10 5 It is sufficient if the range is below cells / mL.

[0104] (Culture conditions) Culture conditions, such as culture temperature, time, and CO2 concentration, are not particularly limited. They may be within the range of conventional methods in the art. For example, the culture temperature may be 20°C or higher or 35°C or higher, and 45°C or lower or 40°C or lower, preferably 37°C. The culture time may be 0.5 hours or more or 6 hours or more, and 7 days or less, 120 hours or less, 96 hours or less, 72 hours or less, or 48 hours or less. The CO2 concentration during culture may be 4% or more or 4.5% or more, and 10% or less or 5.5% or less, preferably 5%. Furthermore, medium changes can be performed at appropriate frequencies. The frequency of medium changes varies depending on the cell type being cultured, but may be, for example, at least once every 5 days, at least once every 4 days, at least once every 3 days, at least once every 2 days, at least once a day, or at least twice a day, but is not limited to these. For medium replacement, the cells are recovered in the same manner as in the recovery step, and then fresh medium is added, the cell aggregates are gently dispersed, and the cells are cultured again. Note that the frequency and method of medium replacement are not limited to those described above, and an optimal method may be adopted as appropriate.

[0105] The timing of terminating the culture and the timing of changing the medium can also be determined based on, for example, the concentration of lactic acid in the medium. Lactic acid is produced by the cells during culture and accumulates in the medium. Lactic acid produced by the cells or lactic acid originally contained in the medium is known to damage the cells, thereby inhibiting the maintenance of the undifferentiated state of pluripotent stem cells and adversely affecting proliferation, particularly the cell proliferation ability after passage. Therefore, by determining the timing of terminating the culture and / or the timing of changing the medium based on the concentration of lactic acid in the medium, it is possible to avoid the inhibitory effect of lactic acid on the maintenance of the undifferentiated state and the decrease in cell proliferation ability caused by lactic acid.

[0106] In particular, when pluripotent stem cells are cultured in suspension in the presence of a protein kinase C β (PKCβ) inhibitor and a tankyrase (TNKS) inhibitor, the above-mentioned effects of lactic acid can be reduced. In other words, when pluripotent stem cells are cultured in suspension in the presence of a protein kinase C β (PKCβ) inhibitor and a tankyrase (TNKS) inhibitor, the undifferentiated state can be maintained or the cell proliferation ability can be maintained even at a higher concentration of lactic acid than in conventional cell culture. Specifically, culture can be performed suitably even when the lactic acid concentration in the medium reaches 5 mM or higher. Furthermore, culture can be performed suitably even when the lactic acid concentration in the medium reaches 7 mM or higher, 9 mM or higher, 10 mM or higher, 11 mM or higher, or 12 mM or higher.

[0107] On the other hand, the lactic acid concentration in the medium is preferably 15 mM or less. In particular, the lactic acid concentration in the medium is preferably 14 mM or less, 13 mM or less, 12 mM or less, or 10 mM or less. If the lactic acid concentration in the medium is within this range, a significant decrease in the medium pH can be suppressed, and the above-mentioned effects on cells can be avoided.

[0108] As described above, when pluripotent stem cells are cultured in suspension in the presence of a protein kinase C β (PKCβ) inhibitor and a tankyrase (TNKS) inhibitor, the timing for terminating the culture and changing the medium can be set to when the lactate concentration in the medium reaches, for example, 15 mM, 14 mM or less, 13 mM or less, 12 mM or less, or 10 mM or less. In another embodiment, the timing for terminating the culture and changing the medium can also be set to when the lactate concentration in the medium is lower than this range.

[0109] (Culture method) The state of flow of the medium during culture is not important, and either static culture or flow culture may be used, with flow culture being preferred.

[0110] "Static culture" refers to culturing in a culture vessel in a stationary state. This static culture is usually used in adherent culture.

[0111] "Fluid culture" refers to culturing under conditions in which the medium is flowing. In the case of fluid culture, a method in which the medium is flowed so as to promote cell aggregation is preferred. Examples of such culture methods include rotation culture, rocking culture, agitation culture, and combinations thereof.

[0112] "Rotation culture" (including shaking culture) refers to a culture method in which the medium flows so that cells gather at one point due to stress from the swirling flow (centrifugal force, centripetal force). Specifically, this is done by rotating a culture vessel containing a medium containing cells along a generally horizontal plane, tracing a closed orbit such as a circle, ellipse, flattened circle, or flattened ellipse.

[0113] The rotation speed is not particularly limited, but the lower limit can be 1 rpm or more, 10 rpm or more, 50 rpm or more, 60 rpm or more, 70 rpm or more, 80 rpm or more, 83 rpm or more, 85 rpm or more, or 90 rpm or more. Meanwhile, the upper limit can be 200 rpm or less, 150 rpm or less, 120 rpm or less, 115 rpm or less, 110 rpm or less, 105 rpm or less, 100 rpm or less, 95 rpm or less, or 90 rpm or less. The amplitude of the shaker used for rotation culture is not particularly limited, but the lower limit can be, for example, 1 mm or more, 10 mm or more, 20 mm or more, or 25 mm or more. Meanwhile, the upper limit can be, for example, 200 mm or less, 100 mm or less, 50 mm or less, 30 mm or less, or 25 mm or less. The rotation radius during rotation culture is also not particularly limited, but is preferably set so that the amplitude is within the above range. The lower limit of the rotation radius is, for example, 5 mm or more or 10 mm or more, and the upper limit can be, for example, 100 mm or less or 50 mm or less. In particular, in the method for producing cell aggregates described below, it is preferable to set the rotation conditions within the above range, as this makes it easier to produce cell aggregates of appropriate sizes.

[0114] "Rock culture" refers to a culture method in which a rocking flow is imparted to the culture medium by linear reciprocating motion, such as rocking agitation. Specifically, rocking is performed by rocking a culture vessel containing a cell-containing culture medium in a plane perpendicular to a generally horizontal plane. The rocking speed is not particularly limited. For example, assuming one reciprocating motion is one cycle, the lower limit is 2 or more, 4 or more, 6 or more, 8 or more, or 10 or more times per minute, while the upper limit is 15 or fewer, 20 or fewer, 25 or fewer, or 50 or fewer times per minute. During rocking, it is preferable to set the culture vessel at a slight angle, i.e., an induction angle, relative to the vertical plane. The rocking angle is not particularly limited. For example, the lower limit can be 0.1° or more, 2° or more, 4° or more, 6° or more, or 8° or more, while the upper limit can be 20° or less, 18° or less, 15° or less, 12° or less, or 10° or less. In the cell aggregate manufacturing method described below, it is preferable to set the rocking conditions within the above range, since this makes it easier to manufacture cell aggregates of an appropriate size.

[0115] Furthermore, the culture can be performed while stirring by a combination of the above-mentioned rotation and rocking movements.

[0116] "Agitation culture" refers to a culture method in which the culture vessel is left stationary while the medium in the vessel is agitated using an agitation means such as a stirrer bar or impeller. For example, agitation culture can be achieved by using a spinner flask-type culture vessel equipped with an agitation impeller. Such culture vessels are commercially available and can also be used. When using a commercially available spinner flask-type culture vessel, the amount of cell culture composition recommended by the manufacturer can be suitably used. The agitation speed of the agitation means is not particularly limited, but the lower limit can be 1 rpm or more, 10 rpm or more, 30 rpm or more, 50 rpm or more, 70 rpm or more, 90 rpm or more, 110 rpm or more, or 130 rpm or more. On the other hand, the upper limit can be 200 rpm or less, or 150 rpm or less.

[0117] Furthermore, in the "agitated culture method," it is preferable to control the shear stress applied to the cells during culture. Animal cells, including pluripotent stem cells, are generally more susceptible to physical stress than other cells. Therefore, if the shear stress applied to the cells during agitated culture is too great, the cells may suffer physical damage, resulting in a decrease in their proliferation ability or, in the case of pluripotent stem cells, in the inability to maintain their undifferentiated state.

[0118] The shear stress imposed on cells in agitated culture depends on, but is not limited to, the impeller tip speed. The impeller tip speed is the peripheral speed of the impeller tip and can be calculated as impeller diameter [m] × pi × rotation speed [rps] = impeller tip speed [m / s]. Note that if multiple impeller diameters can be determined depending on the shape of the impeller tip, the largest distance can be used.

[0119] In particular, when pluripotent stem cells are cultured in suspension in the presence of a protein kinase C β (PKCβ) inhibitor and a tankyrase (TNKS) inhibitor, the above-mentioned effects of the applied shear stress can be reduced. In other words, when pluripotent stem cells are cultured in suspension in the presence of a protein kinase C β (PKCβ) inhibitor and a tankyrase (TNKS) inhibitor, they can maintain their undifferentiated state or their cell proliferation ability even under conditions in which higher shear stress than that of normal cell culture is applied. Specifically, even at extremely high impeller tip speeds of 0.23 m / s or more, agitation culture can be performed while maintaining the undifferentiated state of pluripotent stem cells.

[0120] Furthermore, the blade tip speed is preferably 0.05 m / s or more, preferably 0.08 m / s or more, preferably 0.10 m / s or more, preferably 0.13 m / s or more, preferably 0.17 m / s or more, preferably 0.20 m / s or more, preferably 0.23 m / s or more, preferably 0.25 m / s or more, and preferably 0.30 m / s or more. By maintaining the blade tip speed within this range, it is possible to suppress excessive aggregation of cells while maintaining the undifferentiated state of the pluripotent stem cells.

[0121] Furthermore, the tip speed of the impeller is preferably 1.37 m / s or less, preferably 1.00 m / s or less, preferably 0.84 m / s or less, preferably 0.50 m / s or less, preferably 0.42 m / s or less, preferably 0.34 m / s or less, and preferably 0.30 m / s or less. By keeping the tip speed within this range, the medium flow state in the culture system can be stabilized while maintaining the undifferentiated state of the pluripotent stem cells.

[0122] The extent to which the number of cells should be increased in this step and the state of the cells should be adjusted may be determined appropriately depending on the type of cells to be cultured, the purpose of cell aggregation, the type of medium, and the culture conditions.

[0123] The size of the individual cell aggregates produced by the suspension culture method of this embodiment is not particularly limited, but when observed under a microscope, the lower limit of the maximum width in the observed image may be 30 μm or more, 40 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, or 100 μm or more. On the other hand, the upper limit may be 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 400 μm or less, or 300 μm or less. Cell aggregates within this range are preferred as a cell growth environment because oxygen and nutrients can be easily supplied to the cells inside.

[0124] Of the population of cell aggregates produced by the suspension culture method of this embodiment, it is preferable that the lower limit of 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100% are cell aggregates within the above size range on a weight basis.

[0125] The population of cell aggregates produced by the suspension culture method of this embodiment preferably has a ratio of live cells (viability) among the cells constituting the population of 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. Cell aggregates with a viability within the above ranges are likely to maintain an aggregated state, which is a favorable state for cell proliferation.

[0126] (Post-processing) After this step, the cells are separated from the culture medium by a conventional method and recovered. At this time, it is preferable to recover the cells as single cells by detachment or dispersion treatment. Specific methods will be described in detail in the recovery step below. The recovered cells can be used as is, or, if necessary, washed with a buffer (including PBS buffer), saline, or medium (preferably the medium used in the next step or basal medium), and then subjected to the next step.

[0127] 2-2-2. Suspension culture process The "suspension culture step" is a step of suspension culture of cells in a medium containing the above-mentioned pluripotent stem cell differentiation inhibitor or its active ingredients, a PKCβ inhibitor and a TNKS inhibitor.

[0128] The cell culture method in this step basically conforms to the culture method described above in "2-2-1. Maintenance culture step." Therefore, the explanation common to the method already described in the maintenance culture step will be omitted here, and only the features unique to this step will be described in detail.

[0129] (cell) The cells used in this step are not limited, but are preferably cells prepared after the maintenance culture step. As described in the maintenance culture step, the type of cells is pluripotent stem cells, with pluripotent stem cells such as iPS cells and ES cells being particularly preferred. Furthermore, the cells are preferably in a single-cell state when seeded in a medium. Furthermore, the pluripotent stem cells used in this step may be a single cell or a cell population (pluripotent stem cell population) consisting of multiple cells. When the pluripotent stem cells are a pluripotent stem cell population, the percentage (proportion) of cells in the population that express pluripotent stem cell markers (e.g., OCT4, SOX2, Nanog) and / or are positive for the pluripotent stem cell markers is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or less.

[0130] (Culture medium) This step is characterized by culturing in a medium containing a PKCβ inhibitor and a TNKS inhibitor, which differs from the medium used in the maintenance culture step. The type of medium is not limited as long as it contains a PKCβ inhibitor and a TNKS inhibitor and is capable of growing and / or maintaining the cells.

[0131] The concentrations of the PKCβ inhibitor and the TNKS inhibitor contained in the medium in this step are as described in the above section 1-3. Composition. The method of adding the PKCβ inhibitor and the TNKS inhibitor is not particularly limited, as long as the concentrations of the PKCβ inhibitor and the TNKS inhibitor in the medium at the start of this step are within the above-mentioned ranges. For example, the medium may be prepared by directly adding one or more PKCβ inhibitors and TNKS inhibitors to the medium so that the total amount falls within the above-mentioned concentration range.

[0132] (Culture method) In this step, suspension culture is performed. Therefore, the culture method is preferably flow culture in which the medium is flowed. By culturing pluripotent stem cells in suspension in a medium containing a PKCβ inhibitor and a TNKS inhibitor, the undifferentiated state of the pluripotent stem cells can be maintained.

[0133] In this step, a portion of the pluripotent stem cells can be removed during the culture process to confirm whether they maintain an undifferentiated state. For example, by measuring the expression of pluripotent stem cell markers expressed in the pluripotent stem cells removed during culture, it can be confirmed whether they maintain an undifferentiated state. As described above, examples of pluripotent stem cell markers include alkaline phosphatase, Nanog, OCT4, SOX2, TRA-1-60, c-Myc, KLF4, LIN28, SSEA-4, and SSEA-1. As described above, methods for detecting these pluripotent stem cell markers include, for example, flow cytometry.

[0134] When the positive rate of pluripotent stem cell markers among the pluripotent stem cells extracted during culture is preferably 80% or more, more preferably 90% or more, more preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, or more preferably 100% or less, it can be determined that the stem cells remain undifferentiated.

[0135] Furthermore, in this step, by measuring the expression of three germ layer markers (endodermal cell markers, mesodermal cell markers, and ectodermal cell markers) in pluripotent stem cells removed during the culture process, it can be confirmed whether the undifferentiated state is maintained. That is, when the positive rates of these endodermal cell markers, mesodermal cell markers, and ectodermal cell markers are all preferably 20% or less, more preferably 10% or less, more preferably 9% or less, more preferably 8% or less, more preferably 7% or less, more preferably 6% or less, more preferably 5% or less, more preferably 4% or less, more preferably 3% or less, more preferably 2% or less, more preferably 1% or less, and more preferably below the detection limit, it can be determined that the undifferentiated state is maintained.

[0136] Endodermal cell markers are genes specific to endodermal cells, and examples include SOX17, FOXA2, CXCR4, AFP, GATA4, EOMES, etc. Endodermal cells differentiate into tissues of organs such as the digestive tract, lung, thyroid, pancreas, and liver, cells of secretory glands that open into the digestive tract, peritoneum, pleura, larynx, Eustachian tube, trachea, bronchi, and urinary tract (bladder, most of the urethra, and part of the ureter), etc.

[0137] Mesodermal cell markers are genes specific to mesodermal cells, and examples include T(BRACHYURY), MESP1, MESP2, FOXF1, HAND1, EVX1, IRX3, CDX2, TBX6, MIXL1, ISL1, SNAI2, FOXC1, and PDGFRα. Mesodermal cells differentiate into body cavities and the mesothelium lining them, muscles, skeletons, skin dermis, connective tissue, heart, blood vessels (including vascular endothelium), blood (including blood cells), lymphatic vessels, spleen, kidneys, ureters, and gonads (testes, uterus, and gonadal epithelium).

[0138] Ectodermal cell markers are genes specific to ectodermal cells, such as FGF5, NESTIN, SOX1, and PAX6. Ectodermal cells form the epidermis of the skin, the epithelium of the distal part of the male urethra, hair, nails, skin glands (including mammary glands and sweat glands), sensory organs (including the epithelium of the distal parts of the oral cavity, pharynx, nose, and rectum, and salivary glands), lenses, and the peripheral nervous system. Furthermore, a portion of the ectoderm invaginates into a groove during development to form the neural tube, which also serves as the source of neurons and melanocytes in the central nervous system, such as the brain and spinal cord.

[0139] The expression of these three germ layer markers (endodermal cell markers, mesodermal cell markers, and ectodermal cell markers) can be measured by any detection method known in the art. Methods for measuring the expression of the three germ layer markers (endodermal cell markers, mesodermal cell markers, and ectodermal cell markers) include, but are not limited to, quantitative real-time PCR analysis, RNA-Seq, Northern hybridization, and hybridization using DNA arrays. In quantitative real-time PCR analysis, the expression level of the marker gene to be measured is converted to a relative expression level with respect to the expression level of an internal standard gene, and the expression level of the marker gene can be evaluated based on this relative expression level. Examples of internal standard genes include the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and the β-actin (ACTB) gene.

[0140] 2-2-3. Recovery process The "recovery step" is a step of recovering cultured cells from the culture medium after the maintenance culture step or suspension culture step, and is a selection step in the method of the present invention.

[0141] As used herein, "recovery (of cells)" refers to obtaining cells by separating the cells from the culture medium. The method for recovering cells may be any conventional method used in cell culture methods in the art, and is not particularly limited.

[0142] After the suspension culture process, cells exist in a suspended state in the culture medium. Therefore, cell recovery can be achieved by removing the liquid components of the supernatant by static or centrifugation. Cells can also be recovered using a filtration filter or hollow fiber separation membrane. When removing the liquid components by statically leaving the culture medium in a static state, the container containing the culture medium can be left in a static state for about 5 minutes, and the supernatant can be removed, leaving behind the settled cells and cell aggregates. Centrifugation can be performed at a centrifugal acceleration and for a processing time that does not damage the cells due to centrifugal force. For example, the lower limit of the centrifugal acceleration is not particularly limited as long as it can settle the cells, but it can be, for example, 100 × g or more, 300 × g or more, 800 × g or more, or 1000 × g or more. On the other hand, the upper limit can be a speed at which the cells are not or are least likely to be damaged by centrifugal force, such as 1400 × g or less, 1500 × g or less, or 1600 × g or less. The lower limit of the treatment time is not particularly limited as long as it is a time that allows cells to settle due to the centrifugal acceleration, but it may be, for example, 30 seconds or more, 1 minute or more, 3 minutes or more, or 5 minutes or more. The upper limit may be a time that does not or is unlikely to damage the cells due to the centrifugal acceleration, for example, 10 minutes or less, 8 minutes or less, 6 minutes or less, or 30 seconds or less. When removing liquid components by filtration, for example, the culture medium may be passed through a nonwoven fabric or mesh filter to remove the filtrate, and the remaining cell aggregates may be recovered. When removing liquid components using a hollow fiber separation membrane, for example, the culture medium and cells may be separated and recovered using an apparatus equipped with a hollow fiber separation membrane, such as a cell concentration and washing system (Kaneka Corporation).

[0143] The collected cells can be washed as needed. The washing method is not limited. For example, the washing method may be the same as that described in the "Post-process treatment" section of the maintenance culture process. Buffers (including PBS buffer), saline, or culture media (basal culture media are preferred) may be used as washing solutions.

[0144] (single cell) As used herein, "single cell formation" refers to dispersing a cell aggregate in which multiple cells adhere or aggregate to each other, such as a monolayer cell fragment or a cell aggregate, into a single, free cell state.

[0145] The single cell dissociation is performed using a detachment agent and / or a chelating agent. Examples of detachment agents include, but are not limited to, trypsin, collagenase, pronase, hyaluronidase, elastase, as well as commercially available Accutase (registered trademark), Accumax (registered trademark), and TrypLE. TM Express Enzyme (Life Technologies Japan, Inc.), TrypLE TM Select Enzyme (Life Technologies Japan, Inc.), Dispase (registered trademark), etc. can be used. For example, when trypsin is used for dissociating single cells, the lower limit of the concentration in the solution is not particularly limited as long as it is a concentration that can disperse cell aggregates, but it may be, for example, 0.15 vol% or more, 0.18 vol% or more, 0.20 vol% or more, or 0.24 vol% or more. On the other hand, the upper limit of the concentration in the solution is not particularly limited as long as it is a concentration that does not affect the cells themselves, such as by lysing them, but it may be 0.30 vol% or less, 0.28 vol% or less, or 0.25 vol% or less. Furthermore, although the treatment time depends on the trypsin concentration, the lower limit is not particularly limited as long as it is a time that allows the cell aggregates to be sufficiently dispersed by the action of trypsin, and it may be, for example, 5 minutes or more, 8 minutes or more, 10 minutes or more, 12 minutes or more, or 15 minutes or more. On the other hand, the upper limit of the treatment time is not particularly limited as long as the cells themselves are not affected by the action of trypsin, such as being lysed. For example, it may be 30 minutes or less, 28 minutes or less, 25 minutes or less, 22 minutes or less, 20 minutes or less, or 18 minutes or less. When using a commercially available detachment agent, it should be used at a concentration that can disperse the cells into a single cell state, as described in the attached protocol. The dissociation into single cells can be promoted by gentle physical treatment after treatment with the detachment agent and / or chelating agent. This physical treatment is not limited, but examples include pipetting the cells together with the solution multiple times. Furthermore, if necessary, the cells may be passed through a strainer or mesh.

[0146] The single-cells can be recovered by leaving the cells to stand or centrifuging them to remove the supernatant containing the release agent. The recovered cells may be washed as needed. The centrifugation conditions and washing method may be the same as those described above.

[0147] 3. Cell aggregate production method and cell aggregate obtained by the method Overview By applying the above-mentioned "2. Method for producing a pluripotent stem cell population," a cell aggregate can be obtained as a product. This production method makes it possible to produce a cell aggregate that maintains the undifferentiated state of pluripotent stem cells.

[0148] 3-2. Cell aggregate production method The basic steps in the cell aggregate production method of this embodiment are similar to those described above in "2. Method for producing a pluripotent stem cell population." That is, the method includes a suspension culture step as an essential step, a maintenance culture step as a selection step, and a recovery step. Each step is as described above.

[0149] 3-3.Cell aggregates The cell aggregate production method of this embodiment makes it possible to produce a cell aggregate that maintains an undifferentiated state.

[0150] The size of individual cell aggregates produced by the cell aggregate production method of this embodiment is not particularly limited, but when observed under a microscope, the lower limit of the maximum width in the observed image may be 30 μm or more, 40 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, or 100 μm or more. Meanwhile, the upper limit may be 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, or 200 μm or less. Incidentally, a single human iPS cell has a size of approximately 10 μm. Cell aggregates within this range are preferable as a cell growth environment, as they facilitate the supply of oxygen and nutrients to the cells inside.

[0151] Of the population of cell aggregates produced by the cell aggregate production method of this embodiment, it is preferred that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% by weight are cell aggregates within the above-mentioned size range. In a population of cell aggregates containing 20% ​​or more cell aggregates within the above-mentioned size range, oxygen and nutrients are easily supplied to the cells inside each individual cell aggregate, making it a preferred environment for cell growth.

[0152] The cell aggregate population produced by the cell aggregate production method of this embodiment preferably has a proportion of live cells (viability) among the cells constituting the population of, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. Cell aggregates with a viability within the above ranges are likely to maintain an aggregated state, which is a favorable state for cell proliferation.

[0153] When the cells constituting a cell aggregate are pluripotent stem cells, the undifferentiated state of the pluripotent stem cells constituting the cell aggregate may be determined based on the proportion of cells expressing and / or positive for a pluripotent stem cell marker. For example, the positive rate of the pluripotent stem cell marker may be preferably 80% or more, more preferably 90% or more, more preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and more preferably 100% or less. A cell aggregate in which the positive rate of the pluripotent stem cell marker is within the above range is composed of a population of pluripotent stem cells that maintain an undifferentiated state, and can be said to be a more homogeneous cell population.

[0154] Pluripotent stem cell markers can be detected by any detection method known in the art. Methods for detecting cell markers include, but are not limited to, flow cytometry. When a fluorescently labeled antibody is used as a detection reagent in flow cytometry, cells that emit stronger fluorescence than a negative control (isotype control) are determined to be "positive" for the marker. The proportion of cells that test positive for a fluorescently labeled antibody analyzed by flow cytometry is sometimes referred to as the "positive rate." Any fluorescently labeled antibody known in the art can be used, including, but not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc.

[0155] In the present invention, the cells constituting the cell aggregate may be composed of a single type of cell or two or more types of cells. For example, the cell aggregate may be composed of only iPS cells, or may be composed of iPS cells and ES cells.

[0156] Effects According to the cell aggregate production method of this aspect, it is possible to produce a cell aggregate in which the undifferentiated state of pluripotent stem cells is maintained.

[0157] 4. Method for producing pluripotent stem cell clusters Overview A pluripotent stem cell aggregate can be produced using the above-mentioned "2. Method for producing a pluripotent stem cell population." This production method makes it possible to produce a cell aggregate in which pluripotent stem cells maintain their undifferentiated state.

[0158] 4-2. Method for producing pluripotent stem cell clusters The basic steps in the method for producing a pluripotent stem cell cluster of this embodiment are similar to those described above in "2. Method for producing a pluripotent stem cell population." That is, the essential step includes a step (first step) of performing suspension culture in a medium containing a PKCβ inhibitor and a TNKS inhibitor, and the selection steps include a maintenance culture step and a recovery step. Each step is basically as described above in "2. Method for producing a pluripotent stem cell population."

[0159] The culture time in the first step is preferably 0.5 hours or more, more preferably 12 hours or more, and preferably 7 days or less, 6 days or less, 5 days or less, or 4 days or less, more preferably 72 hours or less, more preferably 48 hours or less, and most preferably 24 hours or less. The culture time in the maintenance culture step is preferably 0.5 hours or more, more preferably 12 hours or more, 24 hours or more, or 48 hours or more, and preferably 7 days or less, 6 days or less, 5 days or less, more preferably 4 days or less, and most preferably 72 hours or less.

[0160] The medium can be changed at an appropriate frequency during the first step, between the first step and the maintenance culture step, and / or during the maintenance culture step. In particular, the medium is changed between the first step and the maintenance culture step. The frequency of medium change varies depending on the cell type being cultured, but may be, for example, at least once every 5 days, at least once every 4 days, at least once every 3 days, at least once every 2 days, at least once a day, or at least twice a day, but is not limited to these. The frequency and method of medium change are not limited to those described above, and an optimal method may be adopted as appropriate.

[0161] 4-3. Pluripotent stem cell aggregates According to the method for producing a pluripotent stem cell cluster of this embodiment, it is possible to produce an aggregate of pluripotent stem cells of an appropriate size while maintaining the undifferentiated state of the pluripotent stem cells. For example, in the pluripotent stem cell cluster obtained by the method for culturing a pluripotent stem cell cluster of this embodiment, the percentage (proportion) of cells that express pluripotent stem cell markers (e.g., OCT4, SOX2, NANOG) and / or are positive for the pluripotent stem cell markers is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or less.

[0162] Effects According to the cell aggregate production method of this aspect, it is possible to produce a cell aggregate in which the undifferentiated state of pluripotent stem cells is maintained.

[0163] 5. Suspension culture medium Overview This embodiment is a medium for suspension culture of cells. Use of the suspension culture medium of this embodiment makes it possible to maintain the undifferentiated state of pluripotent stem cells in the medium.

[0164] 5-2.Configuration The medium for suspension culture of cells in this embodiment contains at least the differentiation inhibitor described above in "1. Pluripotent stem cell differentiation inhibitor" or its active ingredients, a PKCβ inhibitor and a TNKS inhibitor, and is composed of a cell proliferation medium for maintaining the undifferentiated state of pluripotent stem cells in suspension culture.

[0165] The composition of the suspension culture medium of this embodiment is described in detail in the section "Culture and medium" in "1-2. Definition of terms" of "1. Pluripotent stem cell differentiation inhibitor" above.

[0166] The composition of the differentiation inhibitor, its active ingredients, the PKCβ inhibitor and the TNKS inhibitor, and the concentration of each in the differentiation inhibitor are described in detail in "1-3. Composition" of "1. Pluripotent stem cell differentiation inhibitor" above.

[0167] The medium for suspension culture is used as a liquid medium at least during cell culture, but for storage, it may be in a powdered, prepared solid form, for example. In this case, it may be dissolved in sterilized water or the like and diluted to an appropriate concentration before use as a medium.

[0168] Furthermore, the suspension culture medium can be stored frozen until use and then thawed at the time of use.

[0169] Effects By culturing cells in suspension using the suspension culture medium of this embodiment, the undifferentiated state of pluripotent stem cells can be maintained.

[0170] 6. Pluripotent Stem Cell Differentiation Inhibition Kit Overview The kit containing the pluripotent stem cell differentiation inhibitor of the present invention is a kit used for culturing pluripotent stem cells, which contains a pluripotent stem cell differentiation inhibitor consisting of a PKCβ inhibitor and a TNKS inhibitor. By using this pluripotent stem cell differentiation inhibitor kit, pluripotent stem cells can be proliferated in suspension culture while maintaining their undifferentiated state.

[0171] 6-2.Configuration The pluripotent stem cell differentiation inhibiting kit includes, as essential components, a pluripotent stem cell differentiation inhibiting agent consisting of a PKCβ inhibitor and a TNKS inhibitor, and, as optional components, a medium and / or a protocol.

[0172] The protocol describes how to use the kit for inhibiting pluripotent stem cell differentiation, including the amounts of the PKCβ inhibitor and TNKS inhibitor to be added to the culture medium, the amounts of other components to be added, and a suitable suspension culture method for maintaining undifferentiated cells. [Example]

[0173] The pluripotent stem cell differentiation inhibitor and method for producing a pluripotent stem cell population according to the present invention will be explained in more detail below using examples, but the technical scope of the present invention is not limited to the following examples.

[0174] <Reference Example 1: Adherent culture of human iPS cell line WTC-11> iMatrix-511 Silk (Nippi Co., Ltd.) at 0.25 μg / cm 2 Human iPS cells WTC-11 (Coriell Institute) were seeded on cell culture dishes coated with PEG-400 and cultured at 37°C in a 5% CO atmosphere. StemFit® AK02N (Ajinomoto Co.) medium was used and changed daily. A ROCK inhibitor (Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.)) was added to the medium at a final concentration of 10 μM only when the cells were seeded.

[0175] Comparative Example 1: Suspension culture of human iPS cell line WTC-11 Human iPS cell line WTC-11, which had been cultured in adherent culture as described in Reference Example 1, was washed with Dulbecco's PBS (Nacalai Tesque) and then treated with 0.5 mM EDTA solution in Dulbecco's PBS (Nacalai Tesque) for 5 minutes. After aspirating the EDTA solution, StemFit® AK02N medium was added to the dish. The cells were detached using a cell scraper and dispersed to single cells by pipetting. The cells were suspended in StemFit® AK02N medium containing Y-27632 (Fujifilm Wako Pure Chemical Industries) at a final concentration of 10 μM. A portion of the suspension was stained with trypan blue to measure the viable cell count. The cell suspension was diluted to 1 × 10 cells per mL using StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM. 5The cells were prepared to contain 1000 cells. Four milliliters of cell suspension was seeded per well into a 6-well suspension culture plate (Sumitomo Bakelite Co., Ltd.). The seeded plate was rotated at 90 rpm on a rotary shaker (Optima Co., Ltd.) in a 25-mm diameter circle along the horizontal plane, and suspension culture was performed at 37°C in a 5% CO2 environment. The day the cells were seeded was designated day 0 of culture, and subculture was performed on day 5 of culture. Suspension culture was continued until day 10 of culture. The medium was replaced on days 2, 4, 7, and 9 of culture. For medium replacement, 3 mL of culture supernatant was removed from the well by aspiration, followed by the addition of 3 mL of StemFit® AK02N medium without Y-27632. On day 5 of culture, the cell aggregates and culture supernatant were collected from the well into a centrifuge tube and left to stand for approximately 5 minutes to allow the cell aggregates to settle, after which the culture supernatant was removed. After washing with Dulbecco's PBS, 1 mL of Accutase was added to the cell aggregates, treated at 37°C for 10 minutes, and then dispersed into single cells by pipetting. The cells were suspended in StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM, and a portion of the suspension was stained with trypan blue to measure the number of viable cells. The cell suspension was diluted to 1 × 10 per mL using StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM. 5 The suspension was prepared to contain 100 cells, and the cells were seeded in the same manner as on day 0 of culture, and suspension culture was continued. On day 10 of culture, a phase-contrast image of the cell aggregate was taken using a phase-contrast microscope. The phase-contrast image of the cell aggregate is shown in Figure 1. Figure 1 confirms the formation of cell aggregates.

[0176] Comparative Example 2: Addition of a TNKS inhibitor to suspension culture of human iPS cell line WTC-11 Suspension culture was carried out using the same procedure as in Comparative Example 1, except that the seeding medium and medium exchange medium for suspension culture were prepared to contain the TNKS inhibitor IWR-1-endo (Fujifilm Wako Pure Chemical Industries, Ltd.) at a final concentration of 10 μM.

[0177] Comparative Example 3: Addition of a PKCβ inhibitor to suspension culture of human iPS cell line WTC-11 Suspension culture was carried out using the same procedure as in Comparative Example 1, except that the seeding medium and medium exchange medium for suspension culture were prepared to contain the PKCβ inhibitor Go6983 (Fujifilm Wako Pure Chemical Industries, Ltd.) at a final concentration of 10 μM.

[0178] Example 1: Addition of TNKS inhibitor and PKCβ inhibitor to suspension culture of human iPS cell line WTC-11 Suspension culture was carried out in the same manner as in Comparative Example 1, except that the seeding medium and medium exchange medium for suspension culture were prepared to contain IWR-1-endo and Go6983 at final concentrations of 10 μM, respectively.

[0179] <Example 2: Quantitative real-time PCR analysis> Quantitative real-time PCR analysis was performed according to the following procedure. Cells from Reference Example 1 on day 5 of culture, and cells from Comparative Examples 1 and 2 and Example 1 on day 10 of culture, were washed with Dulbecco's PBS, and total RNA was purified using the Monarch Total RNA Miniprep Kit (New England BioLabs Japan). The purification method followed the instructions provided with the kit. 1000 ng of total RNA was separated using a microspectrophotometer. The isolated RNA was mixed with 1 μL of ReverTra Ace® (Toyobo Co., Ltd.), 4 μL of 5x RT buffer (Toyobo Co., Ltd.), 2 μL of 10 mM dNTPs Mixture (Toyobo Co., Ltd.), 1 μL of RNase inhibitor (10 units / μL) (Toyobo Co., Ltd.), 1 μL of random primer (25 pmol / μL) (Toyobo Co., Ltd.), and RNase-free dH2O to a total volume of 20 μL, and cDNA synthesis was carried out using a SimpliAmp Thermal Cycler (Thermo Fisher Scientific). The reaction conditions for cDNA synthesis were 10 minutes at 30°C, followed by 60 minutes at 42°C and 5 minutes at 99°C, followed by cooling to 4°C. The synthesized cDNA solution was diluted 5-fold with sterile water. The diluted cDNA solution was added to a reaction plate at 0.5 μL / well, sterile water at 3.98 μL / well, THUNDERBIRD® Probe qPCR Mix (Toyobo Co., Ltd.) at 5 μL / well, Taqman® probe at 0.5 μL / well, and 50X ROX reference dye (Toyobo Co., Ltd.) at 0.02 μL / well, and mixed. The Taqman probes used were GAPDH, OCT4, T, SOX17, and PAX6. Quantitative real-time PCR analysis was performed using a TaqMan® QuantStudio3 Real-Time PCR System (Thermo Fisher Scientific). The reaction conditions are shown in Table 1.

[0180] [Table 1]

[0181] The Assay IDs of the Taqman (registered trademark) probes used in quantitative real-time PCR analysis are shown below. GAPDH:Hs02786624_g1 OCT4:Hs01088114_m1 T:Hs00610080_m1 SOX17:Hs00751752_s1 PAX6:Hs04260367_gH The results of measuring gene expression are shown in Table 2 and FIG.

[0182] [Table 2]

[0183] As shown in Table 2 and Figure 2, in Reference Example 1, in which adhesion culture was performed, the expression levels of T (mesodermal marker gene), SOX17 (endodermal marker gene), and PAX6 (ectodermal marker gene) were low, and the undifferentiated state of the pluripotent stem cells was maintained.

[0184] On the other hand, in Comparative Example 1, where suspension culture was performed, the expression levels of T, SOX17, and PAX6 were increased compared to Reference Example 1. This indicates that spontaneous differentiation into mesoderm, endoderm, and ectoderm occurred in suspension culture, and the undifferentiated state of pluripotent stem cells could not be maintained. Furthermore, in Comparative Example 2, where suspension culture was performed with the addition of a TNKS inhibitor, the expression levels of T and SOX17 were suppressed to the same level as in Reference Example 1, but the expression level of PAX6 was higher than in Reference Example 1. Furthermore, in Comparative Example 3, where suspension culture was performed with the addition of a PKCβ inhibitor, the expression level of PAX6 was suppressed to the same level as in Reference Example 1, but the expression levels of T and SOX17 were higher than in Comparative Example 1. In Example 1, where both a TNKS inhibitor and a PKCβ inhibitor were added, the expression levels of T, SOX17, and PAX6 were all similar to those in Reference Example 1.

[0185] These results indicate that TNKS inhibitors effectively suppress spontaneous differentiation into mesoderm and endoderm, but have a weak inhibitory effect on ectoderm differentiation. Furthermore, PKCβ inhibitors suppress spontaneous differentiation into ectoderm, but promote mesoderm and endodermal differentiation. On the other hand, the addition of both TNKS inhibitors and PKCβ inhibitors in suspension cultures suppressed spontaneous differentiation into all three germ layers: mesoderm, endoderm, and ectoderm.

[0186] In other words, it was revealed that adding only one of a TNKS inhibitor and a PKCβ inhibitor is not sufficient to effectively suppress the spontaneous differentiation of pluripotent stem cells into mesoderm, endoderm, and ectoderm and maintain the undifferentiated state of pluripotent stem cells; only by adding both a TNKS inhibitor and a PKCβ inhibitor can spontaneous differentiation into all of the mesoderm, endoderm, and ectoderm be suppressed and the undifferentiated state of pluripotent stem cells be maintained.

[0187] <Reference Example 2: Adherent culture of human iPS cell line 201B7> Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Thermo Fisher Scientific) at 0.5 μg / cm 2 Human iPS cells 201B7 (Centre for iPS Cell Research and Application, Kyoto University) were seeded onto cell culture dishes coated with α-methylbutyric acid (ABA), and adherent culture was performed at 37°C in a 5% CO atmosphere. StemFit® AK02N (Ajinomoto Co.) medium was used, and the medium was changed daily. Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium to a final concentration of 10 μM only when the cells were seeded.

[0188] Comparative Example 4: Suspension culture of human iPS cell line 201B7 Human iPS cell line 201B7, which had been cultured in adherent culture using the same procedure as in Reference Example 2, was treated with Accutase (Innovative Cell Technology, Inc.) for 5 minutes to detach it from the culture surface and dispersed into single cells by pipetting. The cells were suspended in StemFit (registered trademark) AK02N medium containing Y-27632 at a final concentration of 10 μM, and a portion of the suspension was stained with trypan blue to measure the number of viable cells. The cell suspension was diluted to 2 × 10 per mL using StemFit (registered trademark) AK02N medium containing Y-27632 at a final concentration of 10 μM. 5 The cells were prepared to contain 1000 cells. 4 mL of cell suspension was seeded per well in a 6-well suspension culture plate. The seeded plate was rotated on a rotary shaker at 83 rpm, drawing a circle with a diameter of 25 mm along the horizontal plane, and suspension culture was performed at 37°C and 5% CO2. The day the cells were seeded was designated culture day 0. Subculture was performed on culture day 4, and suspension culture was continued until culture day 8. Medium changes were performed daily. To change the medium, the entire volume of the medium containing the cell aggregates was collected in a centrifuge tube and left to stand for approximately 5 minutes to allow the cell aggregates to settle. The culture supernatant was then removed, gently resuspended in StemFit® AK02N medium, and returned to the original well. During medium changes, Y-27632 was added to the medium to a final concentration of 5 μM on culture days 1 and 5, and to a final concentration of 2 μM on culture days 2 and 6. On the fourth day of culture, the cell aggregates and culture supernatant were collected from the wells into centrifuge tubes and left to stand for approximately 5 minutes to allow the cell aggregates to settle, after which the culture supernatant was removed. 1 mL of Accutase was added to the cell aggregates, treated at 37°C for 10 minutes, and then dispersed into single cells by pipetting. The cells were suspended in StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM, and a portion of the suspension was stained with trypan blue to measure the number of viable cells. The cell suspension was diluted to 2 × 10 per mL using StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM. 5The suspension was prepared to contain 100 cells, and the cells were seeded in the same manner as on day 0 of culture, and suspension culture was continued. On day 8 of culture, a phase-contrast image of the cell aggregate was taken using a phase-contrast microscope. The phase-contrast image of the cell aggregate is shown in Figure 3. Figure 3 confirms the formation of cell aggregates.

[0189] Comparative Example 5: Addition of TNKS inhibitor and PKC inhibitor to suspension culture of human iPS cell line 201B7 To confirm whether PKC inhibitors that inhibit PKC isozymes other than PKCβ have a differentiation-suppressing effect, suspension culture was performed using the following method. Suspension culture was carried out using the same procedure as in Comparative Example 4, except that a TNKS inhibitor and a PKC inhibitor (which inhibits the activity of PKC isozymes other than PKCβ) were added to the seeding medium and medium exchange medium for suspension culture in the combinations shown in Table 3.

[0190] [Table 3]

[0191] Example 3: Addition of TNKS inhibitor and PKCβ inhibitor to suspension culture of human iPS cell line 201B7 In order to confirm whether or not a PKCβ inhibitor other than Go6983 used in Example 1 has a differentiation-suppressing effect, suspension culture was performed by the following method. Suspension culture was carried out using the same procedure as in Comparative Example 4, except that a TNKS inhibitor and a PKCβ inhibitor were added to the seeding medium and medium replacement medium for suspension culture in the combinations shown in Table 4.

[0192] [Table 4]

[0193] <Example 4: Quantitative real-time PCR analysis> Quantitative real-time PCR analysis was carried out according to the following procedure. The cells on the fourth day of culture in Reference Example 2 and the cells on the eighth day of culture in Comparative Examples 4 and 5 and Example 3 were treated with TRIzol TMThe DNA was lysed using TRIzol Reagent (Thermo Fisher Scientific). The RNA was then lysed using the PureLink® RNA Mini Kit (Thermo Fisher Scientific). TM Total RNA was isolated and purified from the solution dissolved in reagent. The concentration of the purified RNA was measured using a BioSpec-nano (Shimadzu Corporation), and 500 ng of the purified RNA was collected. 2 μL of ReverTra Ace® qPCR RT Master Mix (Toyobo Co., Ltd.) and RNase-free dH2O were added to the collected RNA to make a 10 μL solution, and cDNA synthesis was performed using a SimpliAmp Thermal Cycler (Thermo Fisher Scientific). The cDNA synthesis reaction conditions were 15 minutes at 37°C, followed by 5 minutes at 50°C and 5 minutes at 98°C, followed by cooling to 4°C. The synthesized cDNA solution was diluted 100-fold with 10 mM Tris-HCl pH 8.0 (Nacalai Tesque) and added to a 384-well PCR plate (Thermo Fisher Scientific) at 5 μL / well. KOD SYBR® qPCR Mix (Toyobo Co., Ltd.), a 50 μM forward primer, a 50 μM reverse primer, and DEPC-treated water (Nacalai Tesque) were mixed in a ratio of 100:1:1:48, and this mixture was added to the 384-well PCR plate at 15 μL / well. Primers used were GAPDH, OCT4, T, SOX17, and PAX6. The 384-well PCR plate was centrifuged to remove air bubbles from the wells, and quantitative real-time PCR analysis was performed using a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific). The reaction conditions are shown in Table 5.

[0194] [Table 5]

[0195] The base sequences of the primers used in quantitative real-time PCR analysis are shown below. ACTB (Forward): 5'-CCTCATGAAGATCCTCACCGA-3' (SEQ ID NO: 1) ACTB (Reverse): 5'-TTGCCAATGGTGATGACCTGG-3' (SEQ ID NO: 2) OCT4 (Forward): 5'-AGTGGGTGGAGGAAGCTGACAAC-3' (SEQ ID NO: 3) OCT4 (Reverse): 5'-TCGTTGTGCATAGTCGCTGCTTGA-3' (SEQ ID NO: 4) SOX2 (Forward): 5'-CACCAATCCCATCCACACTCAC-3' (SEQ ID NO: 5) SOX2 (Reverse): 5'-GCAAAGCTCCTACCGTACCAC-3' (SEQ ID NO: 6) NANOG (Forward): 5'-AGCCTCCAGCAGATGCAAGAACTC-3' (SEQ ID NO: 7) NANOG (Reverse): 5'-TTGCTCCACATTGGAAGGTTCCCA-3' (SEQ ID NO: 8) T (Forward): 5'-TCACAAAGAGATGATGGAGGAAC-3' (SEQ ID NO: 9) T (Reverse): 5'-ACATGCAGGTGAGTTGTCAG-3' (SEQ ID NO: 10) SOX17 (Forward): 5'-ATCTGCACTTCGTGTGCAAG-3' (SEQ ID NO: 11) SOX17 (Reverse): 5'-GAGTCTGAGGATTTCCTTAGCTC-3' (SEQ ID NO: 12) PAX6 (Forward): 5'-AGGAATGGACTTGAAACAAGG-3' (SEQ ID NO: 13) PAX6 (Reverse): 5'-GCAAAGCTTGTTGATCATGG-3' (SEQ ID NO: 14) The results of measuring gene expression are shown in Table 6 and FIG.

[0196] [Table 6]

[0197] As shown in Table 6 and Figure 4, in Comparative Example 4, in which suspension culture was performed, the expression level of PAX6 was increased compared to adhesion culture (Reference Example 2), indicating the occurrence of spontaneous differentiation into ectoderm. In Comparative Examples 5-1 to 5-4, in which a TNKS inhibitor and a PKC inhibitor (which inhibits the activity of PKC isozymes other than PKCβ) were added, the expression level of PAX6 was similar to that of Comparative Example 4. In Examples 3-1 to 3-3, in which a TNKS inhibitor and a PKCβ inhibitor were added, the expression level of PAX6 was lower than that of Comparative Example 4 and similar to that of Reference Example 2.

[0198] None of the four PKC inhibitors added in Comparative Examples 5-1 to 5-4 have an inhibitory effect on PKCβ. On the other hand, LY-333531 added in Example 3 is a selective inhibitor of PKCβ, and Go6983 and GF109203X are broad-spectrum inhibitors that inhibit the activity of multiple PKC isozymes, including PKCβ. These findings demonstrate that inhibition of PKCβ is effective in suppressing spontaneous differentiation into ectoderm, and that PKCβ inhibitors are useful as inhibitors of pluripotent stem cell differentiation into ectoderm.

[0199] <Reference Example 3: Preparation of cells for flow cytometry analysis> To analyze the undifferentiated state of the adherent cultured cell population by flow cytometry, the cells were cultured for 4 days using the same procedure as in Reference Example 2, and then the cells were prepared.

[0200] Comparative Example 6: Preparation of cells for flow cytometry analysis To analyze the undifferentiated state of the suspension-cultured cell population by flow cytometry, the cells were cultured for 8 days using the same procedure as in Comparative Example 4 to prepare cells.

[0201] <Comparative Example 7: Preparation of cells for flow cytometry analysis> To perform flow cytometry analysis of the undifferentiated state of a cell population cultured in suspension with the addition of a TNKS inhibitor and a PKC inhibitor (which inhibits the activity of PKC isozymes other than PKCβ), cells were prepared by culturing for 8 days using the same procedure as in Comparative Example 5. The combinations of TNKS inhibitors and PKC inhibitors (which inhibit the activity of PKC isozymes other than PKCβ) used are shown in Table 7.

[0202] [Table 7]

[0203] <Example 5: Preparation of cells for flow cytometry analysis> To analyze the undifferentiated state of the cell populations cultured in suspension with the addition of a TNKS inhibitor and a PKCβ inhibitor by flow cytometry, the cells were prepared by culturing for 8 days using the same procedures as in Examples 3-2 and 3-3. The combinations of TNKS inhibitors and PKCβ inhibitors used are shown in Table 8.

[0204] [Table 8]

[0205] <Example 6: Flow cytometry analysis> Flow cytometry analysis was performed according to the following procedure. The cells obtained in Reference Example 3, Comparative Example 6, Comparative Example 7, and Example 5 were treated with Accutase and dispersed to single cells by pipetting. The cells were washed with phosphate-buffered saline (PBS). They were then fixed with 4% PFA (paraformaldehyde) at room temperature for 20 minutes, washed three times with PBS, and permeabilized with cold methanol at -20°C overnight. After washing three times with PBS, the cells were blocked with 3% FBS (fetal bovine serum) / PBS at room temperature for 1 hour. The cell sample was then divided into two aliquots and resuspended in 50 μL each. Fluorescently labeled anti-OCT4, anti-SOX2, and anti-NANOG antibodies were added to one aliquot and mixed, while a fluorescently labeled isotype control antibody was added to the other aliquot and mixed. The aliquots were stained at 4°C for 1 hour in the dark. The antibodies used and their amounts are shown in Table 9.

[0206] [Table 9]

[0207] After washing once with 3% FBS (fetal bovine serum) / PBS, the cells were passed through a cell strainer and analyzed using a Guava easyCyte 8HT (Lumix). For samples treated with isotype control antibodies, all regions in the cell population extracted by the FSC / SSC dot plot where the percentage of cells with higher fluorescence intensity was 1.0% or less were selected. For samples treated with anti-OCT4, anti-SOX2, and anti-NANOG antibodies, the percentage of cells within each region in the cell population extracted by the FSC / SSC dot plot was calculated, and this was used as the percentage of cells positive for OCT4, SOX2, and NANOG. The results are shown in Table 10 and Figure 5.

[0208] [Table 10]

[0209] As shown in Table 10 and Figure 5, in Reference Example 3, in which adhesion culture was performed, the proportion of cells positive for the undifferentiation markers OCT4, SOX2, and NANOG was 90% or more, and a pluripotent stem cell population that maintained an undifferentiated state was obtained. On the other hand, in Comparative Example 6, in which suspension culture was performed, and Comparative Example 7, in which suspension culture was performed with the addition of a TNKS inhibitor and a PKC inhibitor, the proportion of cells positive for OCT4 was reduced, indicating the emergence of spontaneously differentiated cell populations and the failure to maintain the undifferentiated state of pluripotent stem cells. In Example 5, in which suspension culture was performed with the addition of a TNKS inhibitor and a PKCβ inhibitor, the proportion of cells positive for OCT4, SOX2, and NANOG was 90% or more, suggesting that spontaneous differentiation was suppressed and a cell population that maintained an undifferentiated state was obtained. These results also demonstrate that suspension culture using a TNKS inhibitor and a PKCβ inhibitor can suppress spontaneous differentiation and obtain a pluripotent stem cell population that maintains an undifferentiated state.

[0210] <Reference Example 4: Adherent culture of human iPS cell line 201B7> Adherent culture was carried out in the same manner as in Reference Example 2.

[0211] Comparative Example 8: Suspension culture of human iPS cell line 201B7 Human iPS cell line 201B7, which had been cultured in an adherent culture using the same procedure as in Reference Example 2, was treated with Accutase for 5 minutes to detach it from the culture surface and dispersed into single cells by pipetting. The cells were suspended in StemFit (registered trademark) AK02N medium containing Y-27632 at a final concentration of 10 μM, and a portion of the suspension was stained with trypan blue to measure the number of viable cells. The cell suspension was diluted to 2 × 10 cells per mL using StemFit (registered trademark) AK02N medium containing Y-27632 at a final concentration of 10 μM. 5The cells were prepared to contain 1000 cells. 1 mL of cell suspension was seeded per well into a 12-well suspension culture plate (Sumitomo Bakelite Co., Ltd.). The seeded plate was rotated on a rotary shaker at 98 rpm, drawing a circle with a diameter of 25 mm along the horizontal plane, and suspension culture was performed at 37°C and 5% CO2. The day the cells were seeded was designated as day 0 of culture, and subculture was performed on day 5 of culture. Suspension culture was continued until day 10 of culture. Medium changes were performed daily. To change the medium, the entire volume of the medium containing the cell aggregates was collected into a centrifuge tube and left to stand for approximately 5 minutes to allow the cell aggregates to settle. The culture supernatant was then removed, and the cells were gently resuspended in StemFit® AK02N medium and returned to the original well. During medium changes, Y-27632 was added to the medium at a final concentration of 5 μM on days 1 and 6 of culture, and at a final concentration of 2 μM on days 2 and 7 of culture. On day 5 of culture, the cell aggregates and culture supernatant were collected from the wells into centrifuge tubes and left to stand for approximately 5 minutes to allow the cell aggregates to settle, after which the culture supernatant was removed. 1 mL of Accutase was added to the cell aggregates, which were then treated at 37°C for 10 minutes and dispersed to single cells by pipetting. The cells were suspended in StemFit® AK02N medium containing a final concentration of 10 μM Y-27632, and a portion of the suspension was stained with trypan blue to measure the viable cell count. The cell suspension was diluted to 2 × 10 per mL using StemFit® AK02N medium containing a final concentration of 10 μM Y-27632. 5 The suspension was prepared so as to contain 100 cells, and the cells were seeded in the same manner as on day 0 of culture, and suspension culture was continued.

[0212] Comparative Example 9: Addition of PORCN inhibitor and PKCβ inhibitor to suspension culture of human iPS cell line 201B7 The TNKS inhibitor used in Example 1 is known to have an inhibitory effect on WNT signaling. Therefore, to confirm whether a porcupine (PORCN) inhibitor, which has an inhibitory effect on WNT signaling in addition to the TNKS inhibitor, also has a differentiation-suppressing effect, suspension culture was performed using the following method. Suspension culture was performed using the same procedure as in Comparative Example 8, except that IWP-2 (Fujifilm Wako Pure Chemical Industries, Ltd.) or WNT-C59 (Cayman Chemical Co., Ltd.) was added as a PORCN inhibitor to the seeding medium and medium replacement medium for suspension culture, and GF109203X or LY-333531 was added as a PKCβ inhibitor.

[0213] Example 7: Addition of TNKS inhibitor and PKCβ inhibitor to suspension culture of human iPS cell line 201B7 In order to confirm whether a TNKS inhibitor other than IWR-1-endo used in Example 1 has a differentiation-suppressing effect, suspension culture was performed using the following method. Suspension culture was performed using the same procedure as in Comparative Example 8, except that IWR-1-endo or XAV939 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added as a TNKS inhibitor and GF109203X or LY-333531 was added as a PKCβ inhibitor to the seeding medium and medium exchange medium for suspension culture.

[0214] <Example 8: Quantitative real-time PCR analysis> Quantitative real-time PCR analysis was performed on the cells on day 4 of culture in Reference Example 4 and on day 10 of culture in Comparative Example 8, Comparative Example 9, and Example 7 using the same procedure as in Example 4. The results of measuring gene expression are shown in Tables 11 and 12 and Figures 6 and 7.

[0215] [Table 11]

[0216] [Table 12]

[0217] As shown in Tables 11 and 12 and Figures 6 and 7, in Reference Example 4, in which adhesion culture was performed, the expression levels of T, SOX17, and PAX6 were low, and the undifferentiated state of pluripotent stem cells was maintained. On the other hand, in Comparative Example 8, in which suspension culture was performed, the expression levels of T and SOX17 increased, indicating that spontaneous differentiation into mesoderm and endoderm occurred and the undifferentiated state of pluripotent stem cells was not maintained. In Comparative Example 9, in which suspension culture was performed with the addition of a PORCN inhibitor and a PKCβ inhibitor, the expression levels of T and SOX17 were lower than in Comparative Example 8, indicating that PORCN inhibitors suppress spontaneous differentiation into mesoderm and endoderm. Furthermore, in Example 7, in which suspension culture was performed with the addition of a TNKS inhibitor and a PKCβ inhibitor, the expression levels of T and SOX17 were even lower than in Comparative Example 9, indicating that TNKS inhibitors can more potently suppress spontaneous differentiation into mesoderm and endoderm than PORCN inhibitors. Although both PORCN inhibitors and TNKS inhibitors have inhibitory effects on WNT signaling, it was revealed that TNKS inhibitors are more effective in suppressing spontaneous differentiation into mesoderm and endoderm when combined with PKCβ inhibitors.

[0218] <Reference Example 5: Adherent culture of human iPS cell line 201B7> Adherent culture was carried out in the same manner as in Reference Example 2.

[0219] Comparative Example 10: Suspension culture of human iPS cell line 201B7 Suspension culture was carried out using the same procedure as in Comparative Example 8. On the eighth day of culture, a phase-contrast image of the cell aggregate was obtained using a phase-contrast microscope. The phase-contrast image of the cell aggregate is shown in Figure 8. From Figure 8, it was confirmed that a cell aggregate had been formed.

[0220] <Example 9: Examination of the concentrations of TNKS inhibitors and PKCβ inhibitors in suspension culture of human iPS cell line 201B7> Suspension culture was performed using the same procedure as in Comparative Example 8, except that IWR-1-endo was added as a TNKS inhibitor to the seeding medium and medium replacement medium at a final concentration of 30 μM, 10 μM, 3 μM, 1 μM, 300 nM, or 100 nM, and Go6983 or LY-333531 was added as a PKCβ inhibitor at a final concentration of 10 μM, 100 nM, or 30 nM.

[0221] <Example 10: Quantitative real-time PCR analysis> Quantitative real-time PCR analysis was performed on the cells on day 4 of culture in Reference Example 5 and the cells on day 10 of culture in Comparative Example 10 and Example 9 using the same procedure as in Example 4. The results of measuring gene expression are shown in Tables 13 and 14 and Figures 9 to 11.

[0222] [Table 13]

[0223] [Table 14]

[0224] As shown in Tables 13 and 14 and Figures 9 to 11, in Reference Example 5, where adherent culture was performed, the expression levels of T, SOX17, and PAX6 were low, and the undifferentiated state of pluripotent stem cells was maintained. On the other hand, in Comparative Example 10, where suspension culture was performed, the expression levels of T, SOX17, and PAX6 increased, indicating spontaneous differentiation into mesoderm, endoderm, and ectoderm, and the undifferentiated state of pluripotent stem cells was not maintained. In Example 9, where suspension culture was performed with the addition of a TNKS inhibitor and a PKCβ inhibitor, the addition of a TNKS inhibitor at a final concentration range of 30 μM or less and 100 nM or more reduced the expression levels of T and SOX17 compared to Comparative Example 10, suppressing spontaneous differentiation into mesoderm and endoderm. Furthermore, in Example 9, the addition of a PKCβ inhibitor at a final concentration range of 10 μM or less and 30 nM or more reduced the expression level of PAX6 compared to Comparative Example 10, suppressing spontaneous differentiation into ectoderm.

[0225] Comparative Example 11: Addition of TNKS inhibitor and PKCβ inhibitor in adherent culture of human iPS cell line 201B7 In Reference Example 2, adhesion culture was performed under two conditions: one with and one without the addition of XAV939 and GF109203X to the seeding medium and medium replacement medium at a final concentration of 10 μM and 5 μM, respectively. Figure 12 shows a phase-contrast image of the cells on day 4 of culture. Figure 12 confirms that most cells died under the condition where XAV939 and GF109203X were added. On day 4 of culture, the cells were treated with Accutase for 5 minutes to detach them from the culture surface and dispersed into single cells by pipetting. These cells were suspended in StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM, and a portion of the suspension was stained with trypan blue to measure the viable cell count. The amplification factor, defined as the number of viable cells on day 4 of culture relative to the number of viable cells seeded, was 2.11 × 10 -2 This indicates that the addition of a TNKS inhibitor and a PKCβ inhibitor to adherent cultures has a negative effect on the survival and proliferation of pluripotent stem cells.

[0226] Example 11: Addition of TNKS inhibitor and PKCβ inhibitor to suspension culture of human iPS cell line 201B7 In Comparative Example 4, suspension culture was performed under two conditions: one in which XAV939 and GF109203X were added to the seeding medium and medium exchange medium at a final concentration of 10 μM and 5 μM, respectively, and the other in which they were not added. Figure 13 shows a phase-contrast image of cells on day 4 of culture. As shown in Figure 13, the condition with XAV939 and GF109203X added formed cell aggregates and exhibited the same morphology as the condition without addition. On day 4 of culture, the cells were detached by treatment with Accutase for 10 minutes and dispersed to single cells by pipetting. These cells were suspended in StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM, and a portion was stained with trypan blue to measure the viable cell count. The amplification rate was calculated as in Comparative Example 9, and the amplification rate with the addition of XAV939 and GF109203X was 2.68. Figure 14 compares the amplification rates of Comparative Example 11 and Example 11. From these findings, it became clear that when the TNKS inhibitor and PKCβ inhibitor of Example 11 were added to suspension culture, the adverse effects on survival and proliferation observed in the adherent culture of Comparative Example 11 were significantly improved.

[0227] Comparative Example 12: Long-term suspension culture of human iPS cell line 1231A3 Human iPS cell line 1231A3, which had been cultured in adherent culture using the same procedure as in Reference Example 2, was treated with Accutase (Innovative Cell Technology, Inc.) for 5 minutes to detach it from the culture surface and dispersed into single cells by pipetting. The cells were suspended in StemFit (registered trademark) AK02N medium containing Y-27632 at a final concentration of 10 μM, and a portion of the suspension was stained with trypan blue to measure the number of viable cells. The cell suspension was diluted to 2 × 10 per mL using StemFit (registered trademark) AK02N medium containing Y-27632 at a final concentration of 10 μM. 5 The cells were prepared to contain 1000 cells. 4 mL of cell suspension was seeded per well in a 6-well suspension culture plate. The plate with the seeded cells was rotated on a rotary shaker at 90 rpm in a circle with a diameter of 25 mm along the horizontal plane, and suspension culture was carried out in a 37°C, 5% CO2 environment. The day the cells were seeded was designated day 0 of culture. Subculture was performed on days 4, 8, 16, and 20 of culture, and suspension culture was continued until day 24 of culture. Medium changes were performed daily. To change the medium, the entire volume of medium containing the cell aggregates was collected in a centrifuge tube and allowed to stand for approximately 5 minutes to allow the cell aggregates to settle. The culture supernatant was then removed, and the cells were gently resuspended in StemFit® AK02N medium and returned to the original well for medium change. During medium changes, Y-27632 was added to the medium at a final concentration of 5 μM on days 1, 5, 9, 13, 17, and 21 of culture, and Y-27632 was added to the medium at a final concentration of 2 μM on days 2, 6, 10, 14, 18, and 22 of culture. On days 4, 8, 12, 16, and 20 of culture, the cell aggregates and culture supernatant were collected from the wells into centrifuge tubes and allowed to stand for approximately 5 minutes to allow the cell aggregates to settle, after which the culture supernatant was removed. 1 mL of Accutase was added to the cell aggregates, treated at 37°C for 10 minutes, and then dispersed into single cells by pipetting. The cells were suspended in StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM, and a portion of the suspension was stained with trypan blue to measure the number of viable cells. The cell suspension was diluted to 2 × 10 per mL using StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM. 5 The suspension was prepared to contain 100 cells, and cells were seeded in the same manner as on day 0 of culture, and suspension culture was continued. Phase-contrast images of the cell aggregates were taken using a phase-contrast microscope on days 8, 16, and 24 of culture. The phase-contrast images of the cell aggregates are shown in Figure 15. Figure 15 confirms that cell aggregates had been formed, but their shapes were distorted.

[0228] Example 12: Addition of TNKS inhibitor and PKCβ inhibitor to long-term suspension culture of human iPS cell line 1231A3 The suspension culture medium was supplemented with 20 μM IWR-1-endo as a TNKS inhibitor and 1 μM LY-333531 as a PKCβ inhibitor, and the culture supernatant was sampled immediately before medium replacement and subculture, and the lactate concentration was measured using the multifunctional biosensor BF-7D manufactured by Oji Scientific Instruments. Except for this, the culture was carried out in the same manner as in Comparative Example 12. A phase-contrast image of the cell aggregate is shown in Figure 16. Figure 16 confirms the formation of beautiful spherical cell aggregates. Figure 17 also shows the time course of the results of quantitative real-time PCR analysis performed in Comparative Example 12 and Example 12 (see Example 4 for the primers and analysis conditions used). As shown in Figure 17, in Example 12, it was found that the relative gene expression level of OCT4, an undifferentiation marker, was maintained high even in long-term culture. In addition, in Example 12, it was found that the relative gene expression level of SOX17, an endoderm marker, was maintained low even in long-term culture. Meanwhile, Figure 18 shows the time course of the positivity rate of OCT4, an undifferentiation marker, in Comparative Example 12 and Example 12. Figure 18 also shows that in Example 12, the positivity rate of OCT4, an undifferentiation marker, was maintained high even in long-term culture.

[0229] The results of Comparative Example 12 and Example 12 shown in Figures 15 to 18 demonstrate that the use of a medium containing a TNKS inhibitor and a PKCβ inhibitor allows pluripotent stem cells to maintain their undifferentiated state even when cultured in suspension for an extended period of time. Furthermore, Figure 19 shows the time course of the attained viable cell density in Comparative Example 12 and Example 12. Figure 19 demonstrates that in Example 12, proliferation was maintained even when pluripotent stem cells were cultured in suspension for an extended period of time. Furthermore, Figure 20 shows the results of measuring the lactate concentration in the medium immediately before medium change and passaging in Example 12. Figure 20 demonstrates that in Example 12, the lactate concentration in the medium immediately before passaging exceeded 10 mM, reaching approximately 12 mM. The results shown in Figures 17 to 20 demonstrate that the use of a medium containing a TNKS inhibitor and a PKCβ inhibitor allows pluripotent stem cells to maintain their undifferentiated state and proliferation ability even when the lactate concentration in the medium is relatively high.

[0230] <Example 13: Long-term suspension culture using a stirred reactor> A single-use bioreactor (30 mL) for iPS cell culture manufactured by ABLE was used as the culture vessel for suspension culture. 30 mL of the cell suspension was inoculated and stirred at 135 rpm, i.e., an impeller tip speed of 0.23 m / s. Subculture was performed on days 3, 6, 9, and 12 of culture, and suspension culture was continued until day 15. In this example, culture was performed in the same manner as in Example 12, except that when changing the medium, Y-27632 was added to the medium to a final concentration of 6.9 μM on days 1, 4, 7, 10, and 13 of culture, and Y-27632 was added to the medium to a final concentration of 3.71 μM on days 2, 5, 8, 11, and 14 of culture.

[0231] <Reference Example 6: Long-term adhesion culture of human iPS cell line 1231A3> Human iPS cell line 1231A3, which had been cultured in an adhesion culture using the same procedure as in Reference Example 2, was treated with Accutase (Innovative Cell Technology, Inc.) for 5 minutes to detach it from the culture surface and disperse it into single cells by pipetting. Thereafter, the cells were cultured and passaged repeatedly using the same procedure as in Reference Example 2, except that the number of culture days and passage number were the same as in Example 13. Figure 21 shows the time course of the results of quantitative real-time PCR analysis performed in Example 13 and Reference Example 6 (see Example 4 for the primers and analysis conditions used). Note that missing data in Figure 21 indicates that the data was below the detection limit. As shown in Figure 21, even in Example 13, despite the suspension culture, the relative gene expression levels of undifferentiated markers were maintained at a high level, even in long-term culture, comparable to that in the adherent culture of Reference Example 6. Furthermore, in Example 13, the relative gene expression levels of representative three germ layer markers were maintained at a low level, even in long-term culture, comparable to that in the adherent culture of Reference Example 6. Furthermore, Figure 22 shows the results of G-Band analysis of the cells obtained in Example 13 (commissioned to Nihon Gene Research Institute, Inc.). As shown in Figure 22, it was found that no new karyotype abnormalities occurred in either the suspension culture of Example 13, even after three or five generations. From the results shown in Figures 21 and 22 above, it was found that by using a medium containing a TNKS inhibitor and a PKCβ inhibitor, it is possible to maintain the undifferentiated state of pluripotent stem cells without causing karyotypic abnormalities, even under agitated culture conditions that impose high shear stress on the pluripotent stem cells.

[0232] <Example 14: Evaluation of the differentiation potential of iPS cells after long-term suspension culture> The cryopreserved cells of Example 13 were thawed and suspended in StemFit (registered trademark) AK02N medium containing 10 μM Y-27632 at a final concentration. A portion of the suspension was stained with trypan blue to measure the number of viable cells. The cell suspension was diluted to 2 × 10 cells per mL using StemFit (registered trademark) AK02N medium containing 10 μM Y-27632, 20 μM IWR-1-endo, and 1 μM LY-333531 at a final concentration of 10 μM. 5 The cells were prepared to contain 1000 cells. 4 mL of the cell suspension was seeded per well in a 6-well suspension culture plate. The plate with the seeded cells was rotated on a rotary shaker at 90 rpm in a horizontal plane, drawing a circle with a diameter of 25 mm, and suspension culture was carried out for 4 days at 37°C in a 5% CO2 environment. The medium was changed daily. The entire medium containing the cell aggregates was collected in a centrifuge tube and allowed to settle for approximately 5 minutes. The culture supernatant was then removed, the cells gently resuspended in medium, and returned to the original well. However, during the medium change, the final concentration of Y-27632 in the medium was reduced to 6.9 μM after 1 day of culture, to 3.71 μM after 2 days, and to 2.53 μM after 3 days. Next, differentiation into each of the three germ layers was induced while changing the medium and the additives in the medium as shown in Table 15.

[0233] [Table 15]

[0234] In this example, quantitative real-time PCR analysis was performed on CDX2 as a mesoderm marker, PDGFRa as a cardiogenic mesoderm marker, SOX17 as an endodermal marker, and PAX6 as an ectodermal marker. The primers and analysis conditions used for quantitative real-time PCR analysis were the same as those in Example 4. For PDGFRa, a cardiogenic mesoderm marker, the following primer set was used. PDGFRa (Forward): 5'-GCTGAGCCTAATCCTCTGCC-3' (SEQ ID NO: 15) PDGFRa (Reverse): 5'-ACTGCTCACTTCCAAGACCG-3' (SEQ ID NO: 16)

[0235] <Reference Example 7: Evaluation of the differentiation potential of adherently cultured iPS cells> The frozen cells of Reference Example 6 were thawed and subjected to adhesion culture in the same manner as in Reference Example 2, except that the culture dish had an inner diameter of 6 cm, the medium volume was 4.2 mL, and the seeding density was 1.5 × 10 4 cells / cm 2 The cells were cultured for 4 days. After the adherent culture, the human iPS cell line 1231A3 was treated with Accutase (Innovative Cell Technology, Inc.) for 5 minutes to detach the cells from the culture surface and disperse them into single cells by pipetting. Then, differentiation into each of the three germ layers was induced in the same manner as in Example 14. The results of measuring the relative gene expression levels of three germ layer markers by quantitative real-time PCR analysis are shown in Figure 23. As shown in Figure 23, it was revealed that the iPS cells cultured in long-term suspension culture in Example 14 have the ability to induce differentiation into each of the three germ layers.

[0236] <Regarding Examples 12 to 14> As shown above, the use of a medium containing a TNKS inhibitor and a PKCβ inhibitor has been shown to enable long-term culture of suspension cultures, which are expected to be a method for mass-producing pluripotent stem cells, while maintaining their proliferation, undifferentiated state, and differentiation-inducing ability. The pluripotent stem cells used in Examples 12 to 14 include those established in a feeder-free manner or otherwise provided by methods equivalent to those used for clinical pluripotent stem cells. Therefore, as shown in Examples 12 to 14, it is expected that clinical pluripotent stem cells can also be mass-produced while maintaining their proliferation, undifferentiated state, and differentiation-inducing ability.

Claims

1. A method for producing a pluripotent stem cell population, comprising a step of suspension culturing pluripotent stem cells in a liquid medium containing a PKCβ inhibitor and a TNKS inhibitor, the concentration of the PKCβ inhibitor in the liquid medium is 25 nM or more and 15 μM or less; The concentration of the TNKS inhibitor in the liquid medium is 90 nM or more and 40 μM or less, and The step of suspension culture includes a step of forming cell aggregates. The manufacturing method.

2. The method described in claim 1, wherein the PKCβ inhibitor is LY333531.

3. 2. The method according to claim 1, wherein the ratio of the concentrations of the PKCβ inhibitor and the TNKS inhibitor contained in the liquid medium is in the range of 167:1 or more and 1:1600 or less.

4. 2. The method according to claim 1, wherein the liquid medium contains at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate.

5. The method according to claim 1, wherein the liquid medium contains FGF2 and / or TGF-β1.

6. The method according to claim 1, wherein the liquid medium contains a ROCK inhibitor.

7. The method according to claim 6, wherein the ROCK inhibitor is Y-27632.

8. The method according to claim 1 , wherein the step of suspension culture includes a step of recovering cell aggregates.

9. The method of claim 1, wherein the pluripotent stem cell population has a proportion of cells that are positive for OCT4 of 90% or more, a proportion of cells that are positive for SOX2 of 90% or more, and a proportion of cells that are positive for Nanog of 90% or more.

10. The method according to claim 1, wherein the pluripotent stem cells are ES cells and / or induced pluripotent stem cells.

11. 2. The method according to claim 1, wherein the suspension culture is carried out until the lactic acid concentration in the liquid medium reaches 5 to 15 mM.

12. 2. The method according to claim 1, wherein the suspension culture is an agitation culture with an impeller tip speed of 0.05 m / s or more and 1.37 m / s or less.

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