Method for producing induced pluripotent stem cell

Abstract

To provide a method for producing an induced pluripotent stem cell (iPS cell) capable of initializing a somatic cell without using a feeder cell or without adhesion of a cell to a substrate.SOLUTION: In the production of an iPS cell, following steps are performed: I. an initializing gene is introduced into a somatic cell of a liquid medium; and II. the cell having the introduced gene is subjected to initialization and amplification culture in a non-gelling medium under a suspension culture condition with the medium being flowed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing induced pluripotent stem cells (iPS cells) under suspension culture conditions. [Background technology]

[0002] Pluripotent stem cells, such as induced pluripotent stem cells (iPS cells), have the capacity 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, in vitro differentiation of pluripotent stem cells into a wide variety of somatic cells, including neurons, cardiomyocytes, blood cells, and retinal cells, has already been developed. Furthermore, attempts are being made to create stocks of HLA-homozygous iPS cells, which are less likely to cause immune rejection in many patients when induced to differentiate into somatic cells. Furthermore, recent efforts are also being made to generate iPS cells from individual patients to minimize the risk of immune rejection during transplantation.

[0003] Cell reprogramming technologies, such as iPS cell generation, have rapidly advanced as innovative fundamental technologies in the fields of life science, drug discovery, and regenerative medicine. iPS cells are induced by introducing reprogramming factors, such as OCT3 / 4, SOX2, KLF4, and c-MYC, into somatic cells (Patent Documents 1 and 2 and Non-Patent Documents 1 and 2). These reprogramming factors are transcription factors that are thought to regulate the expression of genes involved in self-renewal and pluripotency, thereby inducing somatic cell reprogramming. However, iPS cells are not induced solely by introducing these reprogramming factors into somatic cells; rather, they are gradually induced into iPS cells by culturing them for a certain period of time after the introduction of the reprogramming factors, and establishment is complete. Furthermore, it may be necessary to select iPS cells induced to a desirable state by cloning or other methods. In other words, the establishment of iPS cells is influenced by various factors.

[0004] iPS cells established as described above are expanded and cultured, then aliquoted into vials or other storage containers and stored (e.g., frozen) for distribution or sale to various user institutions, such as universities and companies, as iPS cell stocks. Alternatively, they may be expanded and cultured and then subjected to differentiation induction to prepare pharmaceutical preparations for administration to individual patients. The iPS cell culture methods used in these expansion cultures can be broadly divided into adhesion culture, in which cells are cultured by adhering them to a flat substrate, and suspension culture, in which cells are cultured suspended in a liquid medium. Because the number of cells obtained in adhesion 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 contrast, suspension culture involves culturing cells suspended in a liquid medium, which facilitates scale-up and makes it suitable for mass production of cells. For example, Non-Patent Document 3 discloses a method for suspension culture of pluripotent stem cells using a spinner flask as a cell culture vessel for suspension culture while stirring the liquid medium. Furthermore, Patent Document 3 discloses a method in which a protein kinase C β (PKC β) inhibitor and a tankyrase inhibitor (TNKS inhibitor) are present in the culture medium to maintain an undifferentiated state when pluripotent stem cells are cultured in suspension. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2007 / 069666 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-283972 [Patent Document 3] International Publication No. 2021 / 162090 [Non-patent literature]

[0006] [Non-Patent Document 1] Takahashi K. and Yamanaka S., Cell, 2006, Aug 25;126(4):663-76. [Non-patent document 2] Takahashi K. et al., Cell, 2007, Nov 30;131(5):861-72. [Non-patent document 3] Sumi T, et al., PLos One, 2013; 8(5):e63378 Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, methods for maintaining established iPS cells in an undifferentiated state through suspension culture are known. However, no method for establishing highly pure iPS cells by performing the step of reprogramming somatic cells to obtain iPS cells under suspension culture conditions is known. Typically, iPS cell establishment is performed under adherent culture conditions, which facilitates the stabilization of cells undergoing iPS cell transformation and iPS cells. However, this requires the use of feeder cells and extracellular matrix. However, differences in the type of feeder cells and extracellular matrix used, as well as the culture conditions, can lead to problems such as inability to obtain high-quality iPS cells. Furthermore, establishing iPS cells under adherent culture conditions can be cumbersome, requiring steps such as colony selection, making it difficult to achieve high-throughput and automated iPS cell production for individual patients, as described above.

[0008] An object of the present invention is to provide a method for producing iPS cells that can reprogram somatic cells without using feeder cells or attaching cells to a substrate. [Means for solving the problem]

[0009] The present invention encompasses the following. (1) A method for producing induced pluripotent stem cells (iPS cells), comprising the following steps I and II: I. Introducing a reprogramming gene into a somatic cell; and II. A step of subjecting the transfected cells to reprogramming and amplification culture in a non-gelling medium under suspension culture conditions with the medium flowing. (2) The method according to (1), further comprising the following steps III and IV: III. A step of separating undifferentiated marker-positive cells from the cells obtained in the step II; and IV. A step of obtaining a cell population by suspension culture of the cells sorted in step III. (3) The method according to (1) or (2), wherein the amplification culture in step II is carried out by a suspension rotation method, and the rotation speed is 10 to 100 rpm. (4) The method according to (1) or (2), wherein the amplification culture in step II is carried out by a suspension stirring method, and the rotation speed of the stirring blade is in the range of 0.05 to 1.37 m / s in terms of blade tip speed. (5) The method according to (4), wherein the amplification culture includes subculture accompanied by a change in culture scale, and the rotation speed of the impeller is set so that the change in the required stirring power per volume or the impeller tip speed is less than 10% before and after the change in culture scale. (6) The method according to any one of (2) to (5), wherein the step III is a step of fractionating the separated cells. (7) The method according to any one of (2) to (6), wherein the undifferentiation marker is at least one selected from the group consisting of SSEA-4, TRA-1-60, TRA-1-81, and TRA-2-49. (8) The method according to any one of (1) to (7), wherein the somatic cells are blood mononuclear cells. (9) The method according to any one of (1) to (8), wherein the gene to be introduced into the somatic cells is incorporated into an episomal plasmid or a Sendai virus and then introduced into the somatic cells. (10) The method according to any one of (1) to (9), wherein the reprogramming gene to be introduced comprises at least one gene selected from the group consisting of the OCT3 / 4 gene, the KLF4 gene, and the L-MYC gene. (11) The method according to any one of (1) to (10), wherein the liquid medium used in the step II and subsequent steps contains at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate. (12) The method according to any one of (1) to (11), wherein the liquid medium used in the step II and subsequent steps contains FGF2 and / or TGF-β1. (13) The method according to any one of (1) to (12), wherein the liquid medium used in the step II and subsequent steps contains a ROCK inhibitor. (14) The method according to (13), wherein the ROCK inhibitor is Y-27632. (15) The method according to any one of (1) to (14), wherein the step II comprises recovering the cell aggregate. (16) The method according to any one of (2) to (15), wherein the step IV comprises recovering the cell aggregate. (17) The method according to any one of (2) to (16), wherein in the cell population obtained in step IV, the proportion of cells positive for OCT4 is 90% or more and the proportion of cells positive for NANOG is 90% or more. (18) A cell population comprising iPS cells produced by the method according to any one of (1) to (17). (19) The method according to any one of (1) to (17), wherein in the step II and / or the step IV, the cells are cultured in a liquid medium containing at least one selected from the group consisting of a protein kinase C β (PKC β) inhibitor and a WNT inhibitor. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a method for producing iPS cells that is capable of initializing somatic cells without using feeder cells or attaching cells to a substrate. [Brief explanation of the drawings]

[0011] [Figure 1]The results of flow cytometry using TRA-1-60 and SSEA-4 antibodies are shown for cells reprogrammed with episomal plasmids and cultured in suspension in the presence of a PKCβ inhibitor and a WNT inhibitor. (A) shows a dot plot of forward scatter (FSC) versus side scatter (BSC) for all events. The area enclosed by the line in the figure was used for gating. (B) shows a dot plot of TRA-1-60 fluorescence intensity and SSEA-4 fluorescence intensity for the cells gated in (A). The cells within the enclosed area were sorted. (C) shows a histogram plot of TRA-1-60 fluorescence intensity, and (D) shows a histogram plot of SSEA-4 fluorescence intensity. [Figure 2] The results of flow cytometry using TRA-1-60 and SSEA-4 antibodies are shown for cells reprogrammed with episomal plasmids and cultured in suspension in the absence of a PKCβ inhibitor or a WNT inhibitor. (A) shows a dot plot of forward scatter (FSC) versus side scatter (BSC) for all events. The area enclosed by the line in the figure was used for gating. (B) shows a dot plot of TRA-1-60 fluorescence intensity and SSEA-4 fluorescence intensity for the cells gated in (A). The cells within the enclosed area were sorted. (C) shows a histogram plot of TRA-1-60 fluorescence intensity, and (D) shows a histogram plot of SSEA-4 fluorescence intensity. [Figure 3] 1 shows micrographs of cell aggregates at passage 5, which were obtained by sorting cells by flow cytometry and reprogramming them using episomal plasmids, followed by suspension culture in the presence and absence of a PKCβ inhibitor and a WNT inhibitor in Example 2. (A) is a micrograph of a cell aggregate cultured in the presence of a PKCβ inhibitor and a WNT inhibitor, and (B) is a micrograph of a cell aggregate cultured in the absence of a PKCβ inhibitor and a WNT inhibitor. [Figure 4] Three clones of reprogrammed cells (#1, #2, and #3) were cultured in suspension in the presence of a PKCβ inhibitor and a WNT inhibitor. These are micrographs of cell aggregates at passage 11. (A) is a micrograph of the cell aggregate of clone #1, (B) is a micrograph of the cell aggregate of clone #2, and (C) is a micrograph of the cell aggregate of clone #3. DETAILED DESCRIPTION OF THE INVENTION

[0012] 1. Method for producing induced pluripotent stem cells (iPS cells) Overview The method for producing iPS cells of the present invention (hereinafter also referred to as "the method of the present invention") is characterized by comprising the following steps I and II. I. introducing a reprogramming gene into somatic cells in a liquid medium; and II. A step of subjecting the transfected cells to reprogramming and amplification culture in a non-gelling medium under suspension culture conditions with the medium flowing. Furthermore, the method of the present invention optionally comprises the following steps III and IV: III. A step of separating undifferentiated marker-positive cells from the cells obtained in the step II; and IV. A step of obtaining a cell population by suspension culture of the cells sorted in step III. The method of the present invention has the above-mentioned characteristics, and therefore makes it possible to obtain iPS cells under suspension culture conditions without using feeder cells or attaching cells to a substrate.

[0013] 1-2.Definition of Terms Terms used in this specification are defined below. <cell> As used herein, "pluripotent stem cells" refer to cells that possess the multipotency (multiple-potency) to differentiate into almost all types of cells (tissue cells and germ cells) 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). Known examples of such cells include embryonic stem cells (ES cells), embryonic germ stem cells (EG cells), germline stem cells (GS cells), and induced pluripotent stem cells (iPS cells, also known as induced pluripotent stem cells). "iPS cells" are the subject of the invention herein and refer to pluripotent stem cells that are reprogrammed from differentiated somatic cells to an undifferentiated state by introducing genes encoding a small number of reprogramming factors into the somatic cells.

[0014] As used herein, the term "somatic cells" refers to cells constituting an individual animal, other than germ cells. As used herein, somatic cells are not limited as long as they can acquire pluripotency through reprogramming induction. Furthermore, the species from which somatic cells are derived is not particularly limited as long as they are multicellular organisms, but animal-derived cells, particularly mammalian-derived cells, are preferred. Examples include cells derived from rodents such as mice, rats, hamsters, and guinea pigs; livestock and 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. The tissues and organs from which somatic cells are derived are not particularly limited, but those that are easy to collect and from which reprogramming can be efficiently induced are preferred. Examples include skin, organs such as liver, blood, urine, cancer tissue, and dental pulp cells. Somatic cells may be differentiated or undifferentiated, and may be established cell lines or primary cultured cells isolated from tissues. Differentiated cells are preferred. Examples of somatic cells in this specification include human fibroblasts, human epithelial cells, human hepatocytes, human blood cells, mesenchymal cells, nerve cells, muscle cells, and the like.

[0015] <Initialization (reprogramming)> As used herein, "reprogramming" refers to the operation or process of changing a somatic cell into a different cell type. Generally, it refers to dedifferentiating a differentiated cell to change it into an undifferentiated cell state. Unless otherwise specified, as used herein, it refers to the operation or process of changing a somatic cell into an iPS cell.

[0016] As used herein, "reprogramming induction" or "inducing reprogramming" means subjecting a cell to a manipulation that can cause reprogramming, thereby actually bringing the cell into an initialized state. In contrast, "performing reprogramming induction" means subjecting a cell to a manipulation that can cause reprogramming, regardless of whether or not reprogramming actually occurs. For example, "performing reprogramming induction" refers to introducing reprogramming factors necessary for reprogramming into somatic cells and culturing the introduced somatic cells under specified conditions.

[0017] As used herein, the term "reprogramming factor" refers to a factor that can induce somatic cell reprogramming by introducing it into a somatic cell, either alone or together with other factors. As used herein, the term "reprogramming factor" refers to any of the proteins to which the reprogramming factor corresponds, nucleic acids encoding the proteins, or gene expression vectors containing the nucleic acids, without specifying whether the reprogramming factor is a protein or a gene. In particular, nucleic acids encoding the proteins are also referred to as "reprogramming genes." Examples of reprogramming factors include any of the four factors OCT3 / 4, SOX2, KLF4, and C-MYC (often referred to herein as the "four reprogramming factors"), as well as factors related to any of the four reprogramming factors.

[0018] As used herein, the term "related factor" of the four reprogramming factors refers to a factor that can induce somatic cell reprogramming when introduced into a somatic cell in place of one of the four reprogramming factors.

[0019] In the present specification, the animal species from which the reprogramming factors are derived is not limited. The animal species from which the reprogramming factors are derived is, for example, a mammalian species. For example, any mammalian species may be used, such as a human, mouse, rat, hamster, guinea pig, dog, cat, rabbit, cow, horse, sheep, goat, rhesus monkey, gorilla, chimpanzee, etc. Preferably, the mammal is a human.

[0020] Examples of reprogramming factors and their associated factors are given below, but the reprogramming factors and their associated factors in this specification are not limited to the following examples.

[0021] A specific example of OCT3 / 4 is the human OCT3 / 4 protein consisting of the amino acid sequence shown in SEQ ID NO: 1. Factors related to OCT3 / 4 include NR5A2 (LRH1) and TBX3.

[0022] A specific example of KLF4 is the human KLF4 protein consisting of the amino acid sequence shown in SEQ ID NO: 4. Factors related to KLF4 include KLF1, KLF2, KLF5, and the mutant KLFs of the present invention. Examples include the human KLF1 protein consisting of the amino acid sequence shown in SEQ ID NO: 2, the human KLF2 protein consisting of the amino acid sequence shown in SEQ ID NO: 3, and the human KLF5 protein consisting of the amino acid sequence shown in SEQ ID NO: 5.

[0023] A specific example of C-MYC is the human C-MYC protein consisting of the amino acid sequence shown in SEQ ID NO: 6. Factors related to C-MYC include the T58A mutant of C-MYC, N-MYC, and L-MYC. Examples include the human N-MYC protein consisting of the amino acid sequence shown in SEQ ID NO: 7 and the human L-MYC protein consisting of the amino acid sequence shown in SEQ ID NO: 8.

[0024] A specific example of SOX2 is the human SOX2 protein consisting of the amino acid sequence shown in SEQ ID NO: 10. In addition, SOX2-related factors include SOX1, SOX3, SOX15, and SOX18. For example, these include the human SOX1 protein consisting of the amino acid sequence shown in SEQ ID NO: 9, the human SOX3 protein consisting of the amino acid sequence shown in SEQ ID NO: 11, the human SOX15 protein consisting of the amino acid sequence shown in SEQ ID NO: 12, and the human SOX18 protein consisting of the amino acid sequence shown in SEQ ID NO: 13.

[0025] Other examples of reprogramming factors and related factors include LIN28A, LIN28B, LIN41, GLIS1, FOXH1, and HMGA2.

[0026] <Cell aggregate> As used herein, a "cell aggregate" refers to a mass-shaped cell group formed by cell aggregation in suspension culture, and is also called a spheroid or aggregate. 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 the above-mentioned cells. For example, a cell aggregate made up of iPS cells such as human iPS cells or human embryonic stem cells contains cells that express undifferentiation markers and / or are positive for undifferentiation markers.

[0027] Undifferentiated markers are gene markers that are specifically or excessively expressed in iPS cells, and examples include alkaline phosphatase, NANOG, OCT4, SOX2, TRA-1-60, TRA-1-81, TRA-2-49, c-Myc, KLF4, LIN28, SSEA-4, and SSEA-1.

[0028] Undifferentiation 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 are 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.

[0029] When the cells constituting the cell aggregate are iPS cells, the positive rate of the undifferentiation 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 more than 99% but not more than 100%. A cell aggregate in which the rate of cells expressing and / or positive for an undifferentiation marker is within the above range is a highly undifferentiated and more homogeneous cell population.

[0030] <Culture and medium> Cell culture methods are broadly divided into "suspension culture" and "adhesion culture." As used herein, "suspension culture" refers to a method of culturing cells in suspension. In this method, cells may exist as single cells in the culture medium in the early stages of culture, but typically exist as aggregated cell clumps from the middle stage onward. "Suspension culture" refers to growing cells in a suspended state in a medium. As used herein, "suspension state" refers to a state in which cells are non-adherent to an external matrix, such as a culture vessel. "Adhesion culture" refers to a method of culturing cells in an adherent state. "Adhesion culture" refers to adhering cells to an external matrix, such as a culture vessel, and growing them, typically in a monolayer. As used herein, culture performed by suspending cells on microcarriers or the like to which they are attached is considered to be adherent culture because the cells themselves adhere to the external matrix. Note that the aforementioned adherent cells can generally be cultured not only in adherent culture but also in suspension culture.

[0031] 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.

[0032] 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. 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 serum-free medium.

[0033] 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.

[0034] Furthermore, it is preferable that the medium contains a ROCK inhibitor. In particular, when the cells are in a single-cell state, it is preferable that the medium contains a ROCK inhibitor. An example of a ROCK inhibitor is Y-27632. By containing a ROCK inhibitor in the medium, cell death in suspension culture of iPS cells can be significantly suppressed.

[0035] 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.

[0036] 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.), UltraCULTURE™ (BioWhittaker), UltraDOMATM (BioWhittaker), UltraCHOTM (BioWhittaker), UltraMDCK™ (BioWhittaker), STEMPRO (registered trademark) hESC SFM (Life Technologies Japan, Inc.), Essential8™ (Life Technologies Japan, Inc.), StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.), mTeSR1 (Veritas), and TeSR2 (Veritas).

[0037] The preferred medium for use in the present invention is a liquid medium containing at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate. The preferred medium for use in the present invention is a liquid medium containing at least one growth factor (preferably FGF2 and / or TGF-β1). 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 / or TGF-β1).

[0038] <PKCβ inhibitor> In this specification, the "protein kinase Cβ (PKCβ) inhibitor" means a substance that inhibits or suppresses the activity of PKCβ. Protein kinases have a catalytic region on the C-terminal side and a regulatory region on the N-terminal side. The catalytic region is composed of a sequence that recognizes phosphorylated residues on the substrate protein and a sequence that binds to ATP / Mg 2+ which binds. The regulatory region is composed of C1 and C2 domains.

[0039] PKC includes PKCα, PKCβI, PKCβII, and PKCγ as conventional isozymes. In addition, PKC includes PKCδ, PKCε, PKCθ, and PKCη as novel isozymes, and PKCζ, PKCλ, and PKCμ as atypical isozymes.

[0040] In this specification, when referring to PKCβ, it means both PKCβI and PKCβII or one of PKCβI and PKCβII. Also, in this specification, when referring to a PKCβ inhibitor, it means a substance that inhibits at least PKCβI and / or PKCβII among these conventional, novel, and atypical isozymes. That is, a PKCβ inhibitor means a substance that inhibits only the activity of PKCβI, a substance that inhibits only the activity of PKCβII, and a substance that inhibits or suppresses the activities of both PKCβI and PKCβII.

[0041] 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.

[0042] 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.

[0043] An example of a PKCβ inhibitor is a compound having the following structural formula [Formula I]:

[0044] [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 an alkyl group having 1 to 3 carbon atoms substituted with -N(RD) (preferably -(CH)-N(CH), RD is independently an ethyl group or a methyl group (preferably a methyl group).

[0045] 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.

[0046] 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.

[0047] [ka]

[0048] The structural formula of GF109203X (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide) is shown below.

[0049] [ka]

[0050] 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.

[0051] [ka]

[0052] 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.

[0053] [ka]

[0054] 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.

[0055] [ka]

[0056] 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.

[0057] [ka]

[0058] 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.

[0059]

Chemical formula

[0060] <WNT inhibitor> In this specification, the "WNT inhibitor" refers to all substances that inhibit the signaling pathway (WNT signaling pathway) by WNT signaling proteins. Any substance that inhibits the WNT signaling pathway is not particularly limited, but typical WNT inhibitors include tankyrase (TNKS) inhibitors and Porcupine (PORCN) inhibitors.

[0061] In this specification, the "tankyrase (TNKS) inhibitor" means a substance that inhibits or suppresses the activity of tankyrase. Tankyrase (sometimes referred to as tankylase) belongs to the poly(ADP-ribosyl)ation enzyme (PARP) family that 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 have a function of promoting telomere elongation by telomerase by poly(ADP-ribosyl)ating the telomere protein TRF1 and releasing it from the telomere.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] [ka]

[0067] 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.

[0068] [ka]

[0069] 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.

[0070] [ka]

[0071] 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.

[0072] [ka]

[0073] 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.

[0074] [ka]

[0075] As used herein, the term "porcupine (PORCN) inhibitor" refers to a substance that inhibits or suppresses the activity of porcupine. Porcupine is an enzyme that catalyzes the addition of palmitoylphosphate to serine residues in WNT proteins, a process necessary for their secretion. Known examples of porcupine (PORCN) inhibitors include IWP-2 and WNT-C59.

[0076] The structural formula of IWP-2 (N-(6-methyl-2-benzenethiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide) is shown below.

[0077] [ka]

[0078] The structural formula of WNT-C59 (4-(2-methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide) is shown below.

[0079] [ka]

[0080] <Other> In this specification, the term "plurality" refers to an integer of, for example, 2 to 50, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.

[0081] 1-3.Configuration As described above, the method of the present invention essentially comprises the following steps I and II. I. introducing a reprogramming gene into somatic cells in a liquid medium; and II. A step of subjecting the transfected cells to reprogramming and amplification culture in a non-gelling medium under suspension culture conditions with the medium flowing.

[0082] The method of the present invention further optionally comprises the following steps III and IV. III. A step of separating undifferentiated marker-positive cells from the cells obtained in the step II; and IV. A step of obtaining a cell population by suspension culture of the cells sorted in step III. The method of the present invention will be described below step by step.

[0083] 1-3-1 Reprogramming gene introduction step (step I) A gene expression vector can be used as a means for introducing reprogramming genes into somatic cells in step I of the method of the present invention. Step I comprises a procedure of contacting somatic cells with a reagent containing a gene expression vector equipped with reprogramming genes (hereinafter also referred to as a "reprogramming inducer").

[0084] (cell) As described in Section "1-2. Definition of Terms," ​​in the method of the present invention, "somatic cells" refers to cells other than germ cells that constitute an individual animal. Blood mononuclear cells, more specifically peripheral blood mononuclear cells, are particularly suitable for use because they are relatively less invasive, easier to collect, relatively easy to induce reprogramming, and are suspension cells. While not particularly limited, activated T cells are suitable for use as mononuclear cells. Here, cells into which reprogramming genes are introduced must be in a liquid medium or a dedicated solution for gene transfer by electroporation or the like. While cells in such a liquid may be suspended or adherent cells, suspended cells are preferred. Such suspended cells may be cells cultured in suspension in a liquid medium, or may be cells detached from adherent cultured cells and suspended.

[0085] (Gene expression vector) The gene expression vector used in step I contains a promoter and a reprogramming gene as essential components. As used herein, "gene expression vector" refers to a vector that contains a gene or a gene fragment in an expressible state and includes an expression unit that can control the expression of the gene, etc. As used herein, "expressible state" refers to the placement of a gene to be expressed downstream of a promoter under the control of the promoter. The gene expression vector that can be used in the method of the present invention is a vector that contains a reprogramming factor in an expressible state and is capable of expressing the reprogramming factor or a peptide fragment thereof in somatic cells.

[0086] Below, we will explain gene expression vectors that can be used in the method of the present invention, as well as promoters and other optional components contained in the vectors.Vectors that can be used as the gene expression vectors are not particularly limited as long as they can express reprogramming factors in somatic cells.For example, viral vectors, plasmid vectors, and artificial chromosome vectors are exemplified.

[0087] In the methods of the present invention, viral vectors that can be used as gene expression vectors are not particularly limited, as long as they can infect somatic cells to be reprogrammed and express reprogramming factors in the somatic cells. Examples include adenoviral vectors, adeno-associated viral (AAV) vectors, retroviral vectors, lentiviral vectors, and Sendai viral vectors. Different types of viral vectors vary in the size of DNA they can carry, the types of cells they can infect, cytotoxicity, whether they are integrated into the host genome, and the expression period. Therefore, they can be appropriately selected depending on the type of somatic cells to be reprogrammed. For example, the replication-defective and persistent Sendai virus vector (SeVdp vector) is highly safe and particularly suitable because it does not integrate into the host genome and remains persistently in the cytoplasm (Nishimura K., et al., J Biol Chem. 2011 Feb 11;286(6):4760-71.; Fusaki N., et al., Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(8):348-62.).

[0088] In the methods of the present invention, the plasmid vector usable as a gene expression vector is not particularly limited, as long as it is capable of expressing reprogramming factors in somatic cells when introduced into the somatic cells to be reprogrammed. The plasmid vector may also be a shuttle vector capable of replicating between mammalian cells and bacteria such as Escherichia coli. Specific examples of plasmid vectors include E. coli-derived plasmids (pBR322, pUC18, pUC19, pUC118, pUC119, pBluescript, etc.), actinomycete-derived plasmids (pIJ486, etc.), Bacillus subtilis-derived plasmids (pUB110, pSH19, etc.), yeast-derived plasmids (YEp13, YEp24, Ycp50, etc.), as well as commercially available vectors. Specific examples of commercially available vectors include CMV6-XL3 (OriGene), EGFP-C1, pGBT-9 (Clontech), pcDNA, pcDM8, and pREP4 (Thermo Fisher Scientific). In particular, an episomal plasmid that incorporates oriP-EBNA1 and is capable of autonomous replication without causing integration into the host genome can be preferably used. For example, the Human iPS Cell Generation Kit, which is an iPS cell production kit containing an episomal plasmid incorporating a reprogramming gene, is available. TM Episomal Vector Mix (manufactured by Takara) or the like can be used.

[0089] In the method of the present invention, examples of artificial chromosome vectors that can be used as gene expression vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC).

[0090] In the methods of the present invention, the promoter contained in the gene expression vector is a promoter that has the activity of inducing gene expression in somatic cells to be reprogrammed. Since the somatic cells into which the gene expression vector is introduced are, in principle, mammalian cells, particularly cells derived from humans, any promoter capable of expressing downstream genes in these cells may be used. Examples of such promoters include the CMV promoter (CMV-IE promoter), SV40 early promoter, RSV promoter, HSV-TK promoter, EF1α promoter, Ub promoter, metallothionein promoter, SRα promoter, and CAG promoter. Other examples include inducible promoters such as a heat shock promoter that can be controlled by temperature and a tetracycline-responsive promoter that can be controlled by the presence or absence of tetracycline.

[0091] In the method of the present invention, the gene expression vector may contain, as optional components, a regulatory sequence other than the promoter, a selection marker gene, and / or a reporter gene.

[0092] In the method of the present invention, control sequences other than promoters that can be contained in a gene expression vector include expression control sequences, intron sequences, nuclease recognition sequences, replication origin sequences, etc. Examples of expression control sequences include enhancers, ribosome binding sequences, terminators, poly(A) addition signals, etc. Examples of nuclease recognition sequences include restriction enzyme recognition sequences, loxP sequences recognized by Cre recombinase, sequences targeted by artificial nucleases such as ZFN and TALEN, or sequences targeted by the CRISPR / Cas9 system. Examples of replication origin sequences include the SV40 replication origin sequence.

[0093] For example, in the gene expression vector of the present invention, nuclease recognition sequences can be introduced before and after the coding region of the reprogramming factor, in which case, after somatic cell reprogramming is complete, the coding region of the reprogramming factor can be removed by introducing a nuclease.

[0094] In the method of the present invention, the selectable marker gene that can be contained in the gene expression vector is a selectable marker gene that allows selection of somatic cells into which the gene expression vector has been introduced. Specific examples of the selectable marker gene include drug resistance genes such as ampicillin resistance gene, kanamycin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, neomycin resistance gene, puromycin resistance gene, and hygromycin resistance gene.

[0095] In the method of the present invention, the reporter gene that can be contained in the gene expression vector is a gene encoding a reporter that can identify somatic cells into which the gene expression vector has been introduced. Examples of reporter genes include genes encoding fluorescent proteins such as GFP and RFP, and luciferase genes.

[0096] (reprogramming genes) In the method of the present invention, the reprogramming inducer contains, as an essential component, one or more (1) reprogramming genes incorporated into a gene expression vector. In addition, the reprogramming inducer may optionally contain one or more (2) cofactors. (1) and (2) are described in detail below.

[0097] (1) Regarding reprogramming genes, in the method of the present invention, the reprogramming inducer comprises one or more reprogramming genes. The reprogramming factors encoded by the reprogramming genes are not limited as long as they are capable of inducing somatic cell reprogramming. Examples include OCT3 / 4, SOX2, C-MYC, and KLF, as well as factors related to any of these. Examples of such related factors include SOX1, SOX3, SOX15, SOX18, the T58A mutant of C-MYC, N-MYC, and L-MYC, as well as KLF1, KLF2, KLF4, and KLF5. These reprogramming factors may be proteins corresponding to the reprogramming factors or peptide fragments thereof, but at least one reprogramming factor selected from the group consisting of OCT3 / 4, KLF4, and L-MYC is particularly preferred. That is, the reprogramming inducer preferably comprises at least one gene selected from the group consisting of the OCT3 / 4 gene, the KLF4 gene, and the L-MYC gene.

[0098] In the methods of the present invention, the number of reprogramming genes contained in the reprogramming inducer is not limited. For example, the reprogramming inducer may contain one, two, three, four, five, six, or more types of reprogramming genes.

[0099] Furthermore, when the reprogramming inducer contains two or more reprogramming genes, the two or more reprogramming genes may be contained in the same vector or in separate vectors.

[0100] (2) Regarding reprogramming cofactors, in the method of the present invention, the "reprogramming cofactors" that can be included as optional components in the reprogramming inducer are factors other than those described in (1) above that are not essential for the induction of somatic cell reprogramming but can increase the efficiency of reprogramming induction when introduced into somatic cells. Examples include NANOG, NR5A2, LIN28A, LIN28B, LIN41, GLIS1, TBX3, HMGA2, FOXH1, mir-302, mir-367, mir-106a, mir-363, shRNA or siRNA against TP53, dominant-negative TP53, and shRNA or siRNA against P21.

[0101] In step I of the present invention, the method for introducing the reprogramming gene contained in the reprogramming inducer into somatic cells is not particularly limited, and the introduction method may be selected appropriately depending on the type of gene transfer vector (plasmid vector, viral vector, etc.). For example, the reprogramming gene can be introduced into somatic cells by viral infection, lipofection, liposome method, electroporation, calcium phosphate method, or DEAE-Dextran method. Other gene transfer methods (transformation methods) known in the art, such as those described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, can also be used. Furthermore, when introducing the reprogramming gene, the somatic cells into which the reprogramming gene is introduced are not particularly limited, and may be, for example, in a liquid medium or in a solution specifically designed for electroporation.

[0102] 1-3-2. Reprogramming and amplification culture step (step II) Step II of the method of the present invention is a step of performing reprogramming and amplification culture on the cells into which the reprogramming gene has been introduced in step I. In the present invention, it is preferable to proceed to step II within at most 5 days, particularly within 3 days, and even within 1 day after the introduction of the reprogramming gene in step I. In step II of the present invention, the cell culture is carried out under suspension culture conditions in which the medium is flowing. The liquid medium used in step II is a non-gelling medium. Below, the animal cell culture method used in this step and the subsequent culture step will be described.

[0103] (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.

[0104] 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.

[0105] 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, 9.6cm2 Less than or equal to 3.5cm 2 It is preferable that:

[0106] This step is characterized by culturing in a medium containing a PKCβ inhibitor and / or a WNT inhibitor as defined in the above section "1-2. Definition of Terms." The type of medium is not limited as long as it contains a PKCβ inhibitor and / or a WNT inhibitor and is capable of growing and / or maintaining the cells.

[0107] (Culture medium) The medium used in this step is a non-gelling medium. In other words, solid and semi-solid media are not used, but rather a highly fluid liquid medium is used. As used herein, "non-gelling medium," i.e., liquid medium, refers to a medium with a viscosity of 0.90 mPa·S or less, preferably 0.85 mPa·S or less. Specifically, it refers to a liquid basal medium listed in Section "1-2. Definition of Terms," ​​optionally containing culture additives. A preferred medium for use in this step is a liquid medium containing at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate. A preferred medium for use in the present invention is a liquid medium containing at least one growth factor (preferably FGF2 and / or TGF-β1). 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 / or TGF-β1). Furthermore, the medium used in this step is preferably a liquid medium containing a ROCK inhibitor (preferably Y-27632).

[0108] (PKCβ inhibitor and WNT inhibitor) The liquid medium used in this step may contain a PKCβ inhibitor and / or a WNT inhibitor. The PKCβ inhibitor may be one type or a combination of two or more different types. The WNT inhibitor may be one type or a combination of two or more different types. Of the PKCβ inhibitor and the WNT inhibitor, only the PKCβ inhibitor may be contained, only the WNT inhibitor may be contained, or both may be contained. More preferably, a PKCβ inhibitor and a WNT inhibitor coexist.

[0109] When the culture medium contains a PKCβ inhibitor, the lower limit of the concentration is not particularly limited and can be determined according to the range in which adhesion of cells into which a reprogramming gene has been introduced (hereinafter also referred to as "reprogramming cells") is suppressed.

[0110] 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.

[0111] The upper limit of the concentration of the PKCβ inhibitor in the medium is not particularly limited, and can be determined depending on the range in which adhesion of reprogrammed cells is inhibited, the solubility of the PKCβ inhibitor, and the like.

[0112] 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.

[0113] When the PKCβ inhibitor is added to the culture medium as a composition, the concentration of the PKCβ inhibitor in the composition can be determined so that it falls within the above range when added to the culture medium. For example, when the composition is diluted 2-fold for use, 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.

[0114] 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.

[0115] The lower limit of the concentration of the WNT inhibitor is not particularly limited, and can be determined depending on the range in which adhesion of reprogrammed cells is suppressed.

[0116] For example, the final concentration of the WNT 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.

[0117] The upper limit of the concentration of the WNT inhibitor in the medium is not particularly limited, and can be determined depending on the range in which adhesion of reprogrammed cells is suppressed, the solubility of the WNT inhibitor, and the like.

[0118] For example, the final concentration of the WNT 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.

[0119] When the WNT inhibitor is added to the culture medium as a composition, the concentration of the WNT inhibitor in the composition can be determined so that it is within the above range when added to the culture medium. For example, when the composition is used after being diluted 2-fold, the lower limit of the TNKS inhibitor in the composition can be 180 nM or more, 200 nM or more, 300 nM or more, 400 nM or more, 600 nM or more, 800 nM or more, 1 μM or more, 1.2 μM or more, 1.4 μM or more, 1.6 μM or more, 1.8 μM or more, 2 μM or more, 3 μM or more, 6 μM or more, 10 μM or more, 20 μM or more, 30 μM or more, 60 μM or more, or 70 μM or more.

[0120] The upper limit of the WNT inhibitor concentration in the composition is not particularly limited and can be the solubility of the WNT inhibitor. Specifically, the upper limit of the WNT inhibitor concentration in the composition can be set to 113 mM.

[0121] When a PKCβ inhibitor and a WNT inhibitor are allowed to coexist in a liquid medium, the lower limit of the ratio of the concentrations of the PKCβ inhibitor to the WNT inhibitor is not particularly limited, and 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 WNT 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 WNT inhibitor in the liquid medium is not particularly limited, but can be, for example, in the range of 167:1 or more and 1:1600 or less. Furthermore, the ratio of the concentrations of the PKCβ inhibitor and the WNT inhibitor in the liquid 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 WNT 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.

[0122] (composition) Culture additives, PKCβ inhibitors, WNT inhibitors, etc. may be added to the medium as compositions containing these as active ingredients. In such cases, other components of the composition that are combined with the active ingredients include carriers. Carriers include solvents and / or excipients.

[0123] Examples of solvents include water, buffers (including PBS), saline, organic solvents (DMSO, DMF, xylene, lower alcohols), and the like.

[0124] 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.

[0125] (culture) In this step II, somatic cells into which reprogramming factors have been introduced in step I are used. If the somatic cells into which reprogramming factors have been introduced are in a floating state, they can be continuously cultured in suspension in this step II. If the somatic cells into which reprogramming factors have been introduced are in a state of adhering to a container or substrate, they can be detached by enzyme treatment or the like and then subjected to this step II. The amount of medium or culture solution may be adjusted appropriately depending on the culture container used. For example, in a 96-well plate (where the area of ​​the well bottom in a plan view is 0.35 cm per well), 2 When using a 12-well plate (where the area of ​​the well bottom in a plan view is 3.5 cm per well), the amount per well can be 0.05 mL or more and 0.3 mL or less, preferably about 0.1 mL. 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 a 30 mL spinner flask (a spinner flask with a capacity of 30 mL) is used, the amount per well can have a lower limit of 1.5 mL or more, 2 mL or more, or 3 mL or more, and an upper limit of 6.0 mL or less, 5 mL or less, or 4 mL or less. Furthermore, when a 30 mL spinner flask (a spinner flask with a capacity of 30 mL) is used, the amount per container can have a lower limit of 20 mL or more, 25 mL or more, or 30 mL or more, and an upper limit of 40 mL or less, 35 mL or less, or 30 mL or less. Furthermore, when a 125 mL Erlenmeyer flask (a Erlenmeyer flask with a capacity of 125 mL) is used, the amount per container can have a lower limit of 10 mL or more, 15 mL or more, 20 mL or more, 25 mL or more, or 30 mL or more, and an upper limit of 50 mL or less, 45 mL or less, or 40 mL or less. When a 500 mL Erlenmeyer flask is used, 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 a 1000 mL Erlenmeyer flask is used, 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, when a stirred reactor with a capacity of 1000 mL is used, the lower limit of the amount per container can be 300 mL or more, 310 mL or more, or 320 mL or more, and the upper limit can be 1000 mL or less, 900 mL or less, 800 mL or less, 700 mL or less, or 600 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.

[0126] Step II may include a medium replacement procedure and a procedure of seeding (subculture) in a new medium. In the subculture, the density of cells seeded in the new medium (seeding density) can be appropriately adjusted taking into consideration the culture time, the state of the cells after culture, and the number of cells required after culture. Although not limited thereto, the lower limit is usually 0.001 × 10 5 Cells / mL or more, 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 x 10 5 cells / mL or more, with an upper limit of 20 x 10 5 cells / mL or less, or 10 x 10 5 It is sufficient if it is in the range of cells / mL or less.

[0127] Culture conditions, such as culture temperature, time, and CO2 concentration, are not particularly limited. Culture may be performed 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 0.5%, 1%, 2%, 3%, 4%, or 4.5%, and 10% or lower or 5.5%, preferably 5%. Furthermore, the medium may be replaced at an appropriate frequency. 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. Medium changes can be performed by recovering cells in a manner similar to the recovery step, adding fresh medium, gently dispersing the cell aggregates, and then culturing them again. Alternatively, a constant amount of culture medium can be continuously aspirated and added, while fresh medium is continuously added. The frequency and method of medium changes are not limited to those described above, and any suitable method can be adopted.

[0128] 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 cells during culture and accumulates in the medium. Lactic acid produced by the cells or originally contained in the medium is known to damage the cells, thereby inhibiting the maintenance of the undifferentiated state of iPS 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.

[0129] (suspension culture) The culture in this step is both suspension culture and fluidized bed culture. As used herein, "fluidized bed culture" refers to culture under conditions in which the medium is fluidized. In the case of fluidized bed culture, a method in which the medium is fluidized so as to promote aggregation of single-cell cells and suppress excessive aggregation is preferred. Examples of such culture methods include a suspension rotation method, a suspension rocking method, a suspension stirring method, or a combination thereof. In particular, the suspension rotation method or the suspension stirring method can be preferably used.

[0130] The "swirl suspension method" (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 medium containing cells along a generally horizontal plane, tracing a closed orbit such as a circle, ellipse, flattened circle, or flattened ellipse.

[0131] The rotation speed is not particularly limited, but is preferably 10 to 100 rpm. The rotation speed can be 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. On the other hand, it can be 100 rpm or less, 95 rpm or less, or 90 rpm or less. The amplitude of the shaker used in the suspension rotation method 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. On the other hand, 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 culture in the suspension rotation method is also not particularly limited, but is preferably set so that the amplitude is within the above-mentioned range. The lower limit of the rotation radius can be, 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, by setting the rotation conditions within the above range, there is an advantage that the cell aggregates produced are likely to be of an appropriate size.

[0132] The "suspension stirring method" refers to a culture method in which the culture vessel is left stationary while the medium in the vessel is stirred using a stirring means such as a stirrer bar or stirring blades. For example, the suspension stirring method can be achieved by using a spinner flask-type culture vessel equipped with a stirring blade. Such culture vessels are commercially available, and these 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.

[0133] In the "suspension stirring method," it is preferable to control the shear stress applied to cells during culture. Animal cells, including iPS cells, are generally more susceptible to physical stress than other cells. Therefore, if the shear stress applied to cells during stirring culture is too great, the cells may suffer physical damage, resulting in a decrease in their proliferation ability or, in the case of iPS cells, in the inability to maintain their undifferentiated state. The rotation speed of the stirring blade 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. Meanwhile, the upper limit can be 200 rpm or less or 150 rpm or less.

[0134] Furthermore, the shear stress imposed on cells in the suspension stirring method 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] x pi x rotation speed [rps] = impeller tip speed [m / s]. Note that if multiple impeller diameters can be determined depending on the impeller tip shape, the largest distance can be used.

[0135] The tip speed of the blade 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 keeping the tip speed within this range, it is possible to suppress excessive aggregation of cells while maintaining the undifferentiated state of iPS cells.

[0136] 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, it is possible to stabilize the medium flow state in the culture system while maintaining the undifferentiated state of iPS cells.

[0137] As described above, step II may include a subculture procedure, and subculture may involve a change in culture scale, preferably an increase in culture scale (scale-up). In subculture involving a change in culture scale, the rotation speed of the impeller of the stirring means can be set before and after the change to match the culture scale so that the stirring power required per volume or the impeller tip speed remains the same, preferably within ±10%, more preferably within ±5%, depending on the culture scale. During iPS cell reprogramming and establishment, a small number of cells are cultured and gradually expanded to obtain a large number of cells. Therefore, it is necessary to increase the culture scale as the culture progresses. In this case, setting the stirring power required per volume of culture medium or the impeller tip speed to remain constant enables culture in a consistent culture environment regardless of the scale. For example, when changing the culture scale in a similar manner, the rotation speed of the impeller can be determined using the constant Pv equation. Pv refers to the stirring power required per unit volume. By keeping Pv constant, stirring culture can be performed under comparable conditions across different scales. The formula for constant Pv is the number of rotations per unit time [rpm or rps] x (blade diameter [m]) 2 / 3 = can be expressed as a constant.

[0138] The "suspension rocking method" refers to a culture method in which a rocking flow is imparted to the medium by linear reciprocating motion, such as rocking agitation. Specifically, the culture vessel containing the cell-containing medium is rocked 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 provide the culture vessel with 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 particular, by setting the rocking conditions within the above range, there is an advantage that the cell aggregates that are generated tend to be of an appropriate size.

[0139] Furthermore, the culture can be performed while stirring by a combination of the above-mentioned rotation and rocking movements.

[0140] The extent to which the number of cells should be increased and the state of the cells should be adjusted in this process can be determined appropriately depending on the type of cell to be cultured, the type of medium, and the culture conditions. This process can yield a cell population containing reprogrammed cells, iPS cells.

[0141] (collect) Step II preferably includes a procedure of recovering the cell population by separating the cells from the culture medium by a conventional method after culturing under the above conditions. At this time, the recovered cell population usually forms cell aggregates. The method of the present invention preferably includes a procedure of recovering the formed cell aggregates in Step II.

[0142] After culturing in step II, the cells typically exist as cell aggregates suspended in the culture medium. Therefore, the cells can be recovered by removing the liquid components of the supernatant by standing or centrifugation. They can also be recovered using a filtration filter or hollow fiber separation membrane. When removing the liquid components by standing, the container containing the culture medium can be left standing for about 5 minutes, and the supernatant can be removed, leaving 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. A buffer (including PBS buffer), saline, or a medium (basal medium is preferred) can be used as a washing solution.

[0144] (Effect of Step II) Somatic cells into which reprogramming genes have been introduced in Step I are then cultured in flow medium in Step II, allowing them to be reprogrammed, i.e., converted into iPS cells, under suspension culture conditions rather than adherent culture. Steps I and II enable the production of iPS cells, and after Step II, the purity of the resulting cell aggregates containing iPS cells can be increased by any known method. The present invention further provides Steps III and IV, which enable a cell population containing highly pure iPS cells to be obtained in a simple manner.

[0145] 1-3-3.Preparative separation process (Process III) Step III of the method of the present invention is a step of sorting undifferentiation marker-positive cells from the cells obtained in Step II. As used herein, "(cell) sorting" refers to a procedure for separating cells with a certain characteristic from other cells. Specifically, Step III refers to a procedure for separating undifferentiation marker-positive cells from a group of undifferentiation marker-negative cells. The cell sorting method may be a conventional method and is not particularly limited.

[0146] (single cell) Step III is preferably a step of fractionating cells. Therefore, step III preferably includes a procedure for disaggregating the collected cells into single cells. "Single cell disaggregation" 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, to form single, free cells. In this step, the number of fractionated cells is not particularly limited, but is usually 10 or less, preferably 5 or less, 4 or less, 3 or less, or 2 or less, and further preferably fractionated into single cells.

[0147] Dissociation into single cells involves the use of a detachment agent and / or a chelating agent. Dissociation agents include, but are not limited to, trypsin, collagenase, pronase, hyaluronidase, and elastase, as well as commercially available agents such as Accutase (registered trademark), Accumax (registered trademark), TrypLE™ Express Enzyme (Life Technologies Japan, Inc.), TrypLE™ Select Enzyme (Life Technologies Japan, Inc.), and Dispase (registered trademark). For example, when trypsin is used for dissociation into single cells, the lower limit of the trypsin concentration in the solution is not particularly limited as long as it is a concentration that can disperse cell aggregates, but may be, for example, 0.15% by volume or more, 0.18% by volume or more, 0.20% by volume or more, or 0.24% by volume or more. Meanwhile, the upper limit of the trypsin 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 lysis, but may be, for example, 0.30% by volume or less, 0.28% by volume or less, or 0.25% by volume or less. The treatment time depends on the trypsin concentration, but the lower limit is not particularly limited as long as the cell aggregates are sufficiently dispersed by the action of trypsin, and 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 lysed by the action of trypsin, and may be, for example, 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 disperses the cells into a single cell state, as described in the attached protocol. After treatment with the detachment agent and / or chelating agent, light physical treatment can promote the formation of single cells. 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.

[0148] 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.

[0149] (single cell sorting) Step III involves detecting the presence or absence of cell surface undifferentiation markers in the collected cell population, preferably the single-cell population. Methods for detecting cell surface undifferentiation 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 percentage 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.

[0150] As described in "1-2. Definition of Terms," ​​examples of cell surface undifferentiation markers to be detected include alkaline phosphatase, NANOG, OCT4, SOX2, TRA-1-60, TRA-1-81, TRA-2-49, c-Myc, KLF4, LIN28, SSEA-4, and SSEA-1. At least one undifferentiation marker selected from the group consisting of SSEA-4, TRA-1-60, TRA-1-81, and TRA-2-49 is particularly preferred. Specifically, cells can be fluorescently stained with a fluorescently labeled antibody that specifically binds to at least one of these undifferentiation markers, and then detected and separated, for example, individually by flow cytometry (single cell sorting).

[0151] (Effect of Step III) As described above, Step II and the subsequent Step III make it possible to generate and separate iPS cells by reprogramming somatic cells without the need for adherent culture using feeder cells or a substrate or colony selection. Furthermore, because the quality of reprogrammed iPS cells can vary slightly depending on the reprogramming efficiency of each cell, single-cell sorting makes it possible to selectively culture and expand iPS cells that are free of abnormalities during reprogramming, iPS cells with more stable quality, or iPS cells with a high ability to differentiate into specific cell types.

[0152] 1-3-4. Floating culture process (Step IV) Step IV of the method of the present invention is a step of suspension culture and proliferation of the undifferentiated marker-positive cells sorted in step III to obtain a cell population containing iPS cells. The cell culture method in this step basically conforms to the culture method described in the above section "1-3-2. Reprogramming and amplification culture step (step II)." Therefore, the explanation common to step II will be omitted here, and only the features characteristic of this step will be described in detail.

[0153] (cell) The cells cultured in this step are the undifferentiation marker-positive single cells sorted in step III, i.e., cells confirmed to express, on the cell surface, known undifferentiation markers such as alkaline phosphatase, NANOG, OCT4, SOX2, TRA-1-60, TRA-1-81, TRA-2-49, c-Myc, KLF4, LIN28, SSEA-4, and SSEA-1, preferably at least one undifferentiation marker selected from the group consisting of SSEA-4, TRA-1-60, TRA-1-81, and TRA-2-49.

[0154] (Culture medium) The medium used in this step may have the same composition as or a different composition from the medium used in step II. Other optional components are the same as those described in the section "1-3-2. Reinitialization and amplification culture step (step II)."

[0155] (suspension culture) In this step, suspension culture is preferably performed. Therefore, the culture method is preferably flow culture in which the medium is flowed. By culturing iPS cells in suspension using flow culture, the undifferentiated state of the iPS cells can be maintained.

[0156] (detection of undifferentiated markers) In this process, a portion of the cells can be removed during the culture process to confirm whether they maintain an undifferentiated state. For example, the maintenance of an undifferentiated state can be confirmed by measuring the expression of undifferentiation markers expressed in iPS cells removed during culture. As described above, examples of undifferentiation 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 undifferentiation markers include, for example, flow cytometry.

[0157] When the positive rate of undifferentiation markers among 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 more than 99% but less than 100%, it can be determined that the cells remain undifferentiated.

[0158] In this step, it is preferable that, in the cell population containing iPS cells, the proportion of cells that are positive for undifferentiation markers, particularly OCT4, is 90% or more, and the proportion of cells that are positive for NANOG is 90% or more.

[0159] (Detection of three germ layer markers) Furthermore, in this step, maintaining an undifferentiated state can be confirmed by measuring the expression of three germ layer markers (endodermal cell markers, mesodermal cell markers, and ectodermal cell markers) in some cells removed during the culture process. 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 cells have maintained an undifferentiated state.

[0160] 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.

[0161] Mesodermal cell markers are genes specific to mesodermal cells, and examples include T(BRACHYURY), SMA, 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).

[0162] Ectodermal cell markers are genes specific to ectodermal cells, such as FGF5, NESTIN, SOX1, PAX6, and TUJ1. 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 oral cavity, pharynx, nose, and rectum, as well as 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.

[0163] 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.

[0164] (Evaluation of differentiation potential into three germ layers) Alternatively, in this process, the differentiation potential of some cells removed during the culture process may be confirmed for the three germ layers. The method for evaluating the differentiation potential for the three germ layers is not particularly limited, but the following method can be used as a specific example. The removed cells are cultured in an undifferentiated state maintenance medium for several days to form embryoid bodies, and then cultured under adherent culture conditions using a standard animal cell culture medium, such as serum-containing DMEM medium, and feeder cells or a substrate. The culture is performed under standard culture conditions, for example, static culture in a 37°C, 5% CO2 incubator, with medium changes or subcultures every 2-3 days, for 16-21 days. The expression of three germ layer markers in the cell population obtained by culture can be measured to confirm whether the cells maintain their differentiation potential for the three germ layers. That is, if it is confirmed that cells positive for endodermal cell markers, mesodermal cell markers, and ectodermal cell markers are expressed in the cell population after culture without any significant bias, it can be said that the cells in the cell population maintain their differentiation potential for the three germ layers.

[0165] (collect) In step IV, it is preferable to provide a procedure for separating the culture medium and the cells by a conventional method and recovering the cell population. At this time, the recovered cell population preferably forms cell aggregates. The method of the present invention preferably includes a procedure for recovering the formed cell aggregates. Specific means, conditions, etc. for the cell population recovery procedure are in accordance with the matters described in the section "1-3-2. Reprogramming and amplification culture step (step II)."

[0166] The collected cells can be washed as needed. Detailed washing conditions are also in accordance with the items described in "1-3-2. Reprogramming and amplifying culture step (step II)."

[0167] 1-3-5. Other processes The method of the present invention may further include any step after step IV above, such as a step of storing the obtained iPS cells or a cell population containing iPS cells, a step of inducing differentiation into specific cells, or a step of reculturing the cells by thawing the frozen vial.

[0168] (Preservation process) The preservation process for iPS cells or cell populations containing iPS cells is not particularly limited under specific conditions, and any known method for preserving iPS cells can be used, as long as the iPS cells are preserved in an undifferentiated state. For example, iPS cells can be frozen by suspending them in a commercially available cryopreservation solution, such as Bambanker (registered trademark) hRM (GC Lymphotec, Inc.), CP-5E, CP-1 (Kyokuto Pharmaceutical Industries, Ltd.), StemCellKeep (BioVerde, Inc.), DAP213 (ReproCell, Inc.), or StemCellBanker (registered trademark) (Xenogen Pharma Co., Ltd.).

[0169] 2. Cell population containing iPS cells The cell population of the present invention is characterized by comprising iPS cells produced using the method for producing iPS cells (the method of the present invention) described in Section 1. Method for producing induced pluripotent stem cells (iPS cells). More specifically, the cell population of the present invention is a cell population obtained by suspension culture of iPS cells obtained using the method of the present invention.

[0170] The positive rate of undifferentiation markers of cells in the cell population of the present invention 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, and more preferably more than 99% but not more than 100%. Examples of undifferentiation markers referred to here include alkaline phosphatase, NANOG, OCT4, SOX2, TRA-1-60, c-Myc, KLF4, LIN28, SSEA-4, and SSEA-1. In particular, it is preferred that the rate of OCT4-positive cells is 90% or more and the rate of NANOG-positive cells is 90% or more, particularly 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0171] In the cell population of the present invention, it is preferred that the proportion of cells positive for undifferentiation markers, particularly OCT4, is 90% or more, and the proportion of cells positive for NANOG is 90% or more.

[0172] In the cell population of the present invention, it is preferable that the positive rates of endodermal cell markers, mesodermal cell markers, and ectodermal cell markers are all 20% or less, particularly 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or below the detection limit.

[0173] The cell population of the present invention preferably maintains the ability to differentiate into the three germ layers. Whether or not the ability to differentiate into the three germ layers is maintained can be confirmed, for example, by the method described in "1-3-4. Suspension culture step (step IV)."

[0174] The cell population of the present invention may typically take the form of a cell aggregate. The size of the cell aggregate is not particularly limited, but the minimum maximum width in an image observed under a microscope 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 maximum width in an image observed of a single human iPS cell is approximately 10 to 30 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.

[0175] Of the population of cell aggregates, it is preferred that 10% or more, 20% or more, 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% by volume are cell aggregates within the above size range. In a population of cell aggregates containing 20% ​​or more cell aggregates within the above size range, oxygen and nutrients are easily supplied to the cells inside each individual cell aggregate, making it a preferred cell growth environment.

[0176] The population of cell aggregates 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. [Example]

[0177] The method for producing iPS cells 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.

[0178] <Example 1: Establishment of iPS cells using Sendai virus> (1-1) Cultivation of blood cells Frozen vials of peripheral blood mononuclear cells (AccuCell® Human PBMC purified, 1 x 10 7The cells (per vial) were removed from the liquid nitrogen tank and thawed in a safety cabinet by opening the cryovial cap. The thawed cell suspension was transferred to a 15 mL tube containing 9 mL of mononuclear cell culture medium (StemSpan ACF (STEMCELL Technologies, #ST-09805) containing 100 ng / mL IL-6, 10 ng / mL IL-3, 300 ng / mL SCF, 300 ng / mL TPO, and 300 ng / mL Flt3 ligand) and centrifuged at 200 g for 10 min. After removing the supernatant, the cells were suspended in 4 mL of mononuclear cell culture medium and seeded into a 6-well suspension culture plate (Sumitomo Bakelite #MS-8006R). The cells were then cultured at 37°C with a gyratory chamber (90 rpm) for 24 hours under 5% CO2 conditions.

[0179] The mononuclear cells were transferred to a 15 mL tube using a 5 mL pipette and centrifuged at 200 g for 10 minutes. The pellet was washed with 5 mL of PBS and centrifuged again (200 g for 10 minutes). 1 mL of Accutase (Nacalai Tesque) was added to the pellet and incubated at 37°C for 5 minutes. The cells were dissociated by pipetting several times using a 1000 μL pipette tip, and then 4 mL of mononuclear cell culture medium was added. The cell number was then counted.

[0180] (1-2) Introduction of reprogramming factors by Sendai virus and initiation of reprogramming CytoTune TM Inducing factors were introduced into iPS cells using an EX-iPS kit (ID Pharma). Following the kit's protocol, 1 × 10 6 hKOS·hKLF4·hL-MYC was diluted with mononuclear cell culture medium to give a MOI of 5, and the virus solution was prepared. 6 The cell suspension containing mononuclear cells was dispensed into two 15 mL tubes and centrifuged at 200 g for 10 minutes.

[0181] The supernatant was removed, and the cells were suspended in 2 mL of mononuclear cell culture medium. 2 mL of the virus solution and 4 mL of mononuclear cell culture medium were added to a 15 mL tube containing 2 mL of this cell suspension (a total of 8 mL), and gently mixed with a 5 mL plastic pipette. The cell-virus mixture was quickly dispensed at 4 mL / well (5 × 10 5 Cells were seeded into two wells of a 6-well suspension culture dish (cells / well), and rotational culture was initiated at 90 rpm, 37°C, and 5% CO2. On days 3, 5, and 7 of culture, 2.0 mL of StemFit AK02N (Ajinomoto Co.) (previously mixed with solutions A, B, C, and 1% penicillin / streptomycin; hereafter referred to as "StemFit medium") was added to each well, and culture was continued. On days 9, 11, 13, and 15 of culture, half of the medium was replaced with StemFit medium (4 mL).

[0182] (1-3) Subculture (initialization culture) On day 16 of culture, suspension culture samples were transferred to 15 mL tubes and centrifuged (200 g x 1 min). The pellet was washed with 5 mL of PBS and centrifuged (200 g x 1 min). The pellet was suspended in Accutase and incubated at 37°C. The suspension culture samples were transferred to 15 mL tubes and centrifuged (200 g x 1 min). The pellet was washed with 5 mL of PBS, centrifuged (200 g x 1 min), resuspended in Accutase, and incubated at 37°C for 5–10 min. After pipetting several times with a pipette tip, an equal volume of StemFit medium was added and centrifuged at 200 g x 2 min. The pellet was suspended in 1 mL of StemFit medium (containing 10 μM ROCK inhibitor (Y-27632, Wako Pure Chemical Industries)). All cells were suspended in StemFit medium (+10 μM Y-27632) alone or in StemFit medium plus compounds (1 μM Ly333531, 10 μM IWR1-endo, 10 μM Y-27632) to a total volume of 4 mL and replated in one well of a 6-well plate. Rotating culture was initiated (90 rpm, 37°C, 5% CO2), and half of the medium was replaced every two days.

[0183] On day 23 of culture, half of the cells cultured under each of the above culture conditions were subcultured into two wells of a 6-well plate. At this time, half of the cells were suspended in StemFit medium (+10 μM Y-27632) alone, and the other half in StemFit medium + compounds (1 μM Ly333531, 10 μM IWR1-endo, 10 μM Y-27632) to a total volume of 4 mL, and culture was continued under the same conditions as before. Thereafter, the medium was changed every two days. Subculture was carried out in the same manner on days 28, 33, 38, and 43 of culture. On day 48 of culture, the cells in StemFit medium + compound-supplemented medium were suspended at a density of 1 × 10 6 Confirm the proliferation of approximately 4 x 10 cells / well. 5 On day 52 of culture, 1.6 × 10 cells were seeded at 1.6 × 10 cells / well. 6 Confirm proliferation of approximately 4 x 10 cells / well. 5 On day 57 of culture, 2.5 × 10 cells were seeded at 1000 cells / well. 6 Confirm proliferation of approximately 4 x 10 cells / well. 5 cells / well.

[0184] (1-4) Analysis of cell characteristics (1-4-1) Evaluation of cell undifferentiation marker expression by flow cytometry analysis The cells were collected on day 62 of the culture, and the expression of undifferentiated markers in the cells was evaluated by flow cytometry analysis according to the following procedure.

[0185] The cell masses in suspension culture were transferred to a 15 mL tube and centrifuged (200 g x 1 minute). The supernatant was aspirated, and the precipitated cell masses were washed with 5 mL of PBS and centrifuged again (200 g x 1 minute). The supernatant was aspirated, and 1 mL of Accutase was added to the precipitated cell masses and incubated at 37°C for 5-10 minutes. After dissociating the cells by pipetting several times with a pipette tip, an equal volume of StemFit medium was added and the mixture was centrifuged at 200 g x 2 minutes. The supernatant was aspirated, and the precipitate was suspended in 1 mL of StemFit medium (containing 10 μM Y-27632) and the cell number was counted.

[0186] After counting the number of cells, add 2.5 x 10 cells for checking (e.g., for negative control). 5 The cells were separated into three 15 mL tubes and centrifuged at 200 g for 3 minutes. After centrifugation, the supernatant was discarded and 1-2 mL of PBS was added. After centrifugation, the supernatant was discarded and 250 μL of flow cytometry buffer (PBS containing 0.1% BSA and 0.5 mM EDTA) was added and suspended by pipetting with a pipette tip.

[0187] The following primary antibodies were added to each sample tube, and the tubes were incubated on ice for approximately 1 hour in the dark. -No antibodies -TRA-1-60(Alexa Fluoro 488 anti-Human TRA-1-60 antibody:Biolegend #330614)(2.5μL) After incubation, 750 μL of flow cytometry buffer was added and the mixture was centrifuged at 200 g for 3 minutes. After centrifugation, the supernatant was removed, and 500 μL of flow cytometry buffer was added and the cells were suspended by pipetting. The cell suspension was passed through a cell strainer. The sample was then kept on ice and protected from light.

[0188] The cells in the cell suspension were counted using a flow cytometry device, Cell Sorter (Sony SH800). Cells were collected from the SSEA-4 and TRA-1-60 positive fractions and collected at a concentration of 1 × 10 5 The cells were directly collected in a 15 mL tube containing 4 mL of StemFit medium (containing 10 μM Y-27632, 10 μM IWR1-endo, and 1 μM LY-333531), transferred to a 6-well plate for suspension culture, and then cultured under rotation (90 rpm, 37°C, 5% CO). Subculture was continued every 5–6 days while monitoring the cell status, and subsequently frozen and stocked (5 × 10 5 ~1×10 6 Cells / vial, suspended in stem cell banker) were prepared.

[0189] On day 62 of culture, the TRA-1-60 positivity rate was measured by flow cytometry, and was found to be 98.27%, demonstrating that iPS cells can be generated under suspension culture conditions.

[0190] (1-4-2) Detection of undifferentiated markers by immunostaining Cells were harvested on day 57 of culture and immunostained to identify the expression of undifferentiated markers OCT4, NANOG, and SSEA-4 proteins. Immunostaining was performed as follows: The suspension-cultured cell masses were transferred to a 15 mL tube and centrifuged (200 g x 1 min). The supernatant was aspirated, and the precipitated cell masses were washed with 5 mL of PBS and further centrifuged (200 g x 1 min). The supernatant was aspirated, and 1 mL of Accutase was added to the precipitated cell masses and incubated at 37°C for 5–10 minutes. After dissociating the cells by pipetting several times with a pipette tip, an equal volume of StemFit medium was added and the mixture was centrifuged at 200 g x 2 minutes. The supernatant was then aspirated, and the precipitate was suspended in 1 mL of StemFit medium (containing 10 μM Y-27632) and the cell number was counted.

[0191] 1×10 5 Cells / well / 1 mL of medium was added to StemFit medium (10 μM Y-27632, 0.5 μg / cm 2 Cells were suspended in iMatrix-511 (containing Silk) and seeded into each well of a 24-well plate. The cells were cultured for 3 hours at 37°C and 5% CO2. The medium was removed from the 24-well plate and washed with 1 mL of PBS. After removing the PBS, 500 μL of PFA solution (PBS containing 4% paraformaldehyde) was added to each well and left to stand at room temperature for 10 minutes for fixation. The PFA solution was then removed, and 500 μL of 0.1% TritonX-100 / PBS solution was added to each well and incubated on a shaker for 10 minutes (this step was omitted for the wells used for SSEA-4 detection). The 0.1% TritonX-100 / PBS solution was discarded, and 500 μL of PBS was added to each well. The wells were then placed on a shaker for 5 minutes, after which the PBS was removed (washing). Washing was performed three more times.

[0192] After removing the PBS, 500 μL of 0.1% blocking buffer (PBS containing 0.1% BSA) was added to each well and the plate was placed on a shaker for 30-60 minutes for blocking. After blocking, 500 μL of the following primary antibody solution was added and the plate was left standing overnight at 4°C to allow the antibody to react. All primary antibodies were diluted as follows in PBS containing 0.1% BSA. -Mouse anti-human OCT3 / 4 antibody (Santa Cruz, #sc-5279, 1 / 400 dilution) -Rabbit anti-human NANOG antibody (ReproCell, #RCAB004P-F, 1 / 400 dilution) -APC-labeled rat anti-human SSEA antibody (Biolegend, #330418, 1 / 100 dilution) During the antibody reaction, the plate was covered with aluminum foil to block light.

[0193] After washing four times with PBS under light-shielded conditions, 500 μL of the following secondary antibody solution was added to each well (this step was not carried out for wells intended for SSEA-4 staining, and PBS was added instead), and the wells were incubated on a shaker at room temperature under light-shielded conditions for 1 to 2 hours. -Donkey anti-mouse IgG (H+L) antibody (Highly Cross-Absorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-21202)) -Donkey anti-rabbit IgG (DyLight TM 488, Biolegend, #406404) After washing four times with PBS under light-blocking conditions, one drop of DAPI solution (Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI (Nacalai Tesque, #12745-74)) was added to each well. The presence or absence of positive cells for nuclear staining with DAPI and immunostaining for each marker protein was observed under a fluorescence microscope (Keyence, BZ-X810).

[0194] The positivity rates of each undifferentiated marker (OCT4 positivity rate, NANOG positivity rate, and SSEA positivity rate among DAPI-positive cells) were calculated for suspension-cultured cells on day 57 of culture. As shown in Table 1, all showed very high positivity rates.

[0195] [Table 1]

[0196] (1-4-3) Evaluation of pluripotency by cell embryoid body formation (1-4-3-1) Embryoid body formation A portion of the suspension-cultured cells on day 57 of culture was evaluated for their ability to differentiate into three germ layers. The suspension-cultured cells were transferred to a 15 mL tube and centrifuged (200 g x 1 minute). The supernatant was aspirated, and the precipitated cell mass was washed with 5 mL of PBS and centrifuged again (200 g x 1 minute). The supernatant was aspirated, and 1 mL of Accutase was added to the precipitated cell mass and incubated at 37°C for 5–10 minutes. After dissociating the cells by pipetting several times with a pipette tip, an equal volume of StemFit medium was added and the mixture was centrifuged at 200 g x 2 minutes. The supernatant was aspirated, and the precipitate was suspended in 1 mL of StemFit medium (containing 10 μM Y-27632) and the cell number was counted.

[0197] 1.2 x 10 in a 15 mL tube 6 The cell number was adjusted to 12 mL of StemFit medium (containing 10 μM Y-27632) and suspended. The entire cell suspension was transferred to a reservoir (AS ONE, #2-7844-02) and 100 μL was dispensed into each well of a V-bottom 96-well plate for suspension culture (cell number: 1.0 × 10 4 After centrifugation at 200g for 3 minutes, the plate was incubated at 37°C with 5% CO2. On day 2 of incubation, 100µL of EB medium (DMEM + 10% FBS + 1% sodium pyruvate + 1% penicillin / streptomycin) was added to the plate at a rate of 100µL / well. On days 4 and 6 of incubation, the medium was replaced at a rate of 100µL / well.

[0198] Adherent culture was initiated on day 8 of culture. 500 μL of gelatin solution (0.1 w / v% solution, Fujifilm Wako Pure Chemical Industries, Ltd., #190-15805) was added to each of four wells of a 12-well plate and incubated at 37°C for approximately 1 hour. The gelatin solution was then removed to prepare gelatin-coated plates. Cells cultured in EB medium as described above were collected from each well and transferred to each well of a 12-well plate at 12–24 cells per well. Culture was initiated at 37°C in 5% CO2. The medium was replaced with 1 mL of EB medium every two days (days 10, 12, 14, etc.). Approximately 20 days after the start of culture, embryoid body differentiation was evaluated by immunostaining.

[0199] (1-4-3-2) Evaluation of three germ layer differentiation (immunostaining method) The EB medium was removed from the 12-well plate, and 2 mL of PBS was added for washing. After removing the PBS, 500 μL of PFA solution was added to each well and fixed for 10 minutes at room temperature. The PFA solution was then removed, and 1 mL of 0.1% TritonX-100 / PBS solution was added to each well and incubated on a shaker for 10 minutes. The 0.1% TritonX-100 / PBS solution was then removed, and 1 mL of PBS was added to each well. The plate was then placed on a shaker for 5 minutes, after which the solution was removed (washing). After 1-2 hours, the plate was washed four times, and 500 μL of 0.1% blocking buffer was added to each well and incubated on a shaker for 30-60 minutes (blocking).

[0200] After blocking, 500 μL of the following primary antibody solution was added to each well. The primary antibody solution was diluted with 0.1% BSA / PBS solution to the following concentrations. The plate was sealed with parafilm and left to stand overnight at 4°C. For detecting ectoderm marker TUJ1: Mouse anti-human β-3 tubulin antibody, R&D Systems, MAB1195, final concentration 2.5-5 μg / mL For detecting mesodermal marker SMA: (Mouse anti-human α-smooth muscle actin (SMA) antibody, R&D Systems, MAB1420, final concentration 2.5-5 μg / mL) For detecting the endoderm marker AFP: (Mouse anti-human alpha-fetoprotein (AFP) antibody, R&D Systems, MAB1368, final concentration 2.5-5 μg / mL)

[0201] After washing four times with PBS, 500 μL of secondary antibody solution (donkey anti-mouse IgG (H+L) antibody (Thermo Fisher Scientific, A-21202) diluted 500-1000 times with 0.1 BSA / PBS solution) was added to each well. The plate was then wrapped in aluminum foil to protect from light. After incubating the plate on a shaker at room temperature for 1-2 hours, it was washed four times with PBS and one drop of DAPI solution was added to each well. Nuclear staining with DAPI and immunohistochemistry were performed under a fluorescent microscope to observe the presence or absence of positive cells for each marker protein.

[0202] As a result, on day 23 of differentiation induction, the presence of cells positive for the germ layer markers TUJ1, SMA, and AFP was identified in the cultured cells, confirming that the suspension cultured cells maintained pluripotency.

[0203] (1-5) Save The suspension cultured cells were then passaged every 4-5 days, and the cells collected at each passage were diluted to 1 × 10 6 Cells / vial were suspended in a stem cell banker and frozen stocks were prepared.

[0204] <Example 2: Establishment of iPS cells using episomal plasmid vectors> (2-1) Cultivation of blood cells Blood cells were cultured in the same manner as in Example 1 (1-1).

[0205] (2-2) Introduction of reprogramming factors and initiation of reprogramming In a 1.5 mL tube, 81.8 μL of Nucleofector solution from the Amaxa® Human CD34+ Cell Nucleofector kit (Lonza Bioscience, VAPA-1003), 18.2 μL of Supplement solution I, and 3.02 μL of iPS cell Generation Episomal vector Mix (Takara Bio) were mixed to prepare 103 μL of gene transfer reagent.

[0206] 2.5 x 10 mononuclear cells cultured in 2-1 6 The cells were dispensed into 15 mL tubes so that the concentration of cells was 15 mL per tube. After centrifugation at 200 g for 10 minutes, the supernatant was removed, and 103 μL of the gene transfer reagent was added and carefully suspended. The prepared cell / plasmid mixture was set in a Nucleofector (Lonza Bioscience) and electroporation was carried out. After electroporation, the cell suspension was quickly transferred from the cuvette to a 1.5 mL tube containing mononuclear cell culture medium using the dropper provided with the kit and mixed, bringing the total volume to 1000 μL. The electroporated cell suspension was dispensed at approximately 5 × 10 per well. 5 The cells were seeded.

[0207] The cell suspension was seeded into two wells of a 6-well plate containing 4 mL of mononuclear cell culture medium per well, and rotational culture (90 rpm) was initiated at 37°C and 5% CO2. On days 3, 5, and 7 of culture, 2.0 mL of StemFit medium or StemFit medium + LY-333531 (1 μM) + IWR1-endo (10 μM) was added to each well. On day 9 of culture, the medium was completely replaced (4 mL / well). On days 11, 13, and 15 of culture, half of the medium was replaced (2 mL / well), and the culture was continued. Thereafter, the medium was replaced every 4–5 days.

[0208] (2-3) Subculture (initialization culture) Suspension culture samples were transferred to 15 mL tubes and centrifuged (200 g x 1 min). The pellets were washed with 5 mL of PBS, centrifuged (200 g x 1 min), resuspended in Accutase, and incubated at 37°C for 5–10 min. After pipetting several times with a pipette tip, an equal volume of StemFit medium was added and centrifuged at 200 g x 2 min. The pellets were suspended in 1 mL of StemFit medium (containing 10 μM Y27632). Total cells were suspended in a final 4 mL of StemFit medium (+ 10 μM Y27632) or StemFit medium + compound (1 μM LY-333531, 10 μM IWR1-endo, 10 μM Y27632). Each suspension was seeded into a 6-well plate and cultured with rotation (90 rpm, 37°C, 5% CO2). Thereafter, half of the medium was replaced every two days, and the cells were subcultured every four to five days.

[0209] 2 x 10 on day 31 of culture 5 After confirming the proliferation of approximately 2 × 10 cells / well, the entire amount was seeded. 6 Confirm proliferation of approximately 4 x 10 cells / well. 5 Cells were seeded at 1 x 10 cells / well. 6 The cells were subjected to flow cytometry analysis (see Example 3 below), and TRA-1-60 and SSEA-4 positive cells were isolated by cell sorting. The isolated cells were seeded in StemFit medium plus compounds (1 μM LY-333531, 10 μM IWR1-endo, 10 μM Y27632) and cultured in a rotary atmosphere (90 rpm, 37°C, 5% CO2). Half of the medium was replaced every two days, and the cells were subcultured every 5 to 6 days. After isolation, 7 × 10 cells were isolated at the third passage. 5 After confirming proliferation to approximately 4 × 10 cells, the entire amount was subcultured. 6 Check proliferation of 4 x 10 / well 5 The cells were subcultured at a concentration of 1 × 10 cells / well. 6 Frozen stocks were prepared at 100 cells / vial suspended in stem cell bankers.

[0210] (2-4) Analysis of cell characteristics (2-4-1) Evaluation of cell undifferentiation marker expression by flow cytometry analysis and single cell sorting (2-4-1-1) Procedure The cell masses in suspension culture were transferred to a 15 mL tube and centrifuged (200 g x 1 minute). The supernatant was aspirated, and the precipitated cell masses were washed with 5 mL of PBS and centrifuged again (200 g x 1 minute). The supernatant was aspirated, and 1 mL of Accutase was added to the precipitated cell masses and incubated at 37°C for 5-10 minutes. After dissociating the cells by pipetting several times with a pipette tip, an equal volume of StemFit medium was added and the mixture was centrifuged at 200 g x 2 minutes. The supernatant was aspirated, and the precipitate was suspended in 1 mL of StemFit medium (containing 10 μM Y-27632) and the cell number was counted.

[0211] After counting the number of cells, add 2.5 x 10 cells for checking (e.g., for negative control). 5 3 tubes for cells, 1 x 10 for sorting 6 The cells were separated into individual 15 mL tubes and centrifuged at 200 g for 3 minutes. After centrifugation, the supernatant was discarded and 1-2 mL of PBS was added. After centrifugation, the supernatant was discarded and 250 μL (for checking) or 1000 μL (for sorting) of flow cytometry buffer (PBS containing 0.1% BSA and 0.5 mM EDTA) was added, and the cells were suspended by pipetting using a pipette tip.

[0212] The following primary antibodies were added to each sample tube, and the tubes were incubated on ice for approximately 1 hour in the dark. -For checking -No antibodies -SSEA-4 (APC anti-human SSEA-4 antibody: Biolegend #330418) alone (2.5 μL) -TRA-1-60 (Alexa Fluoro 488 anti-Human TRA-1-60 antibody:Biolegend #330614) only (2.5μL) -For sorting -SSEA-4(10μL)+TRA-1-60(10μL) -TRA-1-60 only (10 μL) After incubation, 750 μL of flow cytometry buffer was added to the checking tube and 3 mL to the sorting tube, and the mixture was centrifuged at 200 g for 3 minutes. After centrifugation, the supernatant was removed, and 500 μL of flow cytometry buffer was added and suspended by pipetting. The cell suspension was passed through a cell strainer. The sample was then kept on ice and protected from light.

[0213] The cells in the cell suspension were counted and sorted using a flow cytometry device, Cell Sorter (Sony SH800). Cells were collected from the SSEA-4 and TRA-1-60 positive fractions and collected at a concentration of 1 × 10 5 The cells were directly collected in a 15 mL tube containing 4 mL of StemFit medium (containing 10 μM Y-27632, 10 μM IWR1-endo, and 1 μM LY-333531), transferred to a 6-well plate for suspension culture, and then cultured under rotation (90 rpm, 37°C, 5% CO). Subculture was continued every 5–6 days while monitoring the cell status, and subsequently frozen and stocked (5 × 10 5 ~1×10 6 Cells / vial, suspended in stem cell banker) were prepared.

[0214] Single-cell sorting was performed by preparing a 96-well plate for suspension culture, adding 200 μL of StemFit medium (containing 10 μM Y-27632, 10 μM IWR1-endo, and 1 μM LY-333531) to each well, collecting cells at 1 cell / well, and seeding them directly. After single-cell sorting, suspension culture (medium replacement and subculture) was performed under the same conditions as in (2-2) and (2-3) for approximately 1.5 months (approximately 8 passages) to establish clones. After single-cell sorting, cells expanded and cultured under suspension culture conditions were also subjected to detection of undifferentiated markers under the same conditions as above.

[0215] (2-4-1-2) Results After transfection with the reprogramming gene, cells were cultured in suspension with or without the addition of compound (LY-333531+IWR1-endo). Cells were harvested on day 35 of culture and counted for TRA-1-60- and SSEA-4-positive cells by flow cytometry. Figure 1 shows the results of flow cytometry for cells cultured in suspension with compound addition, while Figure 2 shows the results for cells cultured in suspension without compound addition. In both Figures 1 and 2, (A) shows a dot plot of forward scatter (FSC) versus side scatter (SSC) for all events. The area enclosed by the line in the figure was used for gating. (B) shows a dot plot of TRA-1-60 and SSEA-4 fluorescence intensities for the cells gated in (A). Cells within the enclosed area were sorted. (C) shows a histogram plot of TRA-1-60 fluorescence intensity, and (D) shows a histogram plot of SSEA-4 fluorescence intensity. Under the compound-added condition, 87% were TRA-1-60 positive and SSEA-4 positive, whereas under the compound-free condition, 72% were TRA-1-60 positive and SSEA-4 positive.

[0216] The cells on day 35 of culture were further subcultured. Figure 3 shows micrographs of cell aggregates at passage 5, cultured under compound-containing and compound-free conditions. (A) is a micrograph of a cell aggregate cultured under compound-containing conditions, and (B) is a micrograph of a cell aggregate cultured under compound-free conditions. It was demonstrated that uniform cell aggregates were obtained from cells cultured under compound-containing conditions. The number of TRA-1-60-positive and SSEA-4-positive cells in the same subculture was measured by flow cytometry, as described in (2-4-1-1) above. The results were 99% under compound-containing conditions and 39% under compound-free conditions.

[0217] On day 35 of culture, single-cell sorting of TRA-1-60 / SSEA-4-positive cells was performed using a cell sorter. One cell was seeded per well of a 96-well plate and cultured in suspension for 1.5 months (eight passages) under both compound-containing and compound-free conditions, yielding 10 clones. Figure 4 shows micrographs of cell aggregates from suspension-cultured cells of three of these clones (#1, #2, and #3). (A) shows a micrograph of clone #1, (B) shows a micrograph of clone #2, and (C) shows a micrograph of clone #3. The TRA-1-60 positivity rates of clones #1, #2, and #3 were measured using flow cytometry, and all showed high values: 93.66%, 94.18%, and 81.99%, respectively.

[0218] (2-4-2) Evaluation of cell undifferentiation marker expression by immunostaining Immunostaining was performed on 11th passage cells from three clones (#1-3) sorted and expanded as described in 2-4-1, as follows: The suspension-cultured cell masses of each clone were transferred to a 15 mL tube and centrifuged (200 g x 1 min). The supernatant was aspirated, and the precipitated cell masses were washed with 5 mL of PBS and further centrifuged (200 g x 1 min). The supernatant was aspirated, and 1 mL of Accutase was added to the precipitated cell masses and incubated at 37°C for 5-10 minutes. After dissociating the cells by pipetting several times with a pipette tip, an equal volume of StemFit medium was added and the mixture was centrifuged at 200 g x 2 minutes. The supernatant was aspirated, and the precipitate was suspended in 1 mL of StemFit medium (containing 10 μM Y-27632), and the cell number was counted.

[0219] 1×10 5 Cells / well / 1 mL of medium was added to StemFit medium (10 μM Y-27632, 0.5 μg / cm 2Cells were suspended in iMatrix-511 (containing Silk) and seeded into each well of a 24-well plate. The cells were cultured for 3 hours at 37°C and 5% CO2. The medium was removed from the 24-well plate and washed with 1 mL of PBS. After removing the PBS, 500 μL of PFA solution (PBS containing 4% paraformaldehyde) was added to each well and left to stand at room temperature for 10 minutes for fixation. The PFA solution was then removed, and 500 μL of 0.1% TritonX-100 / PBS solution was added to each well and incubated on a shaker for 10 minutes (this step was omitted for the wells used for SSEA-4 detection). The 0.1% TritonX-100 / PBS solution was discarded, and 500 μL of PBS was added to each well. The wells were then placed on a shaker for 5 minutes, after which the PBS was removed (washing). Washing was performed three more times.

[0220] After removing the PBS, 500 μL of 0.1% blocking buffer (PBS containing 0.1% BSA) was added to each well and the plate was placed on a shaker for 30-60 minutes for blocking. After blocking, 500 μL of the following primary antibody solution was added and the plate was left standing overnight at 4°C to allow the antibody to react. All primary antibodies were diluted as follows in PBS containing 0.1% BSA. -Mouse anti-human OCT3 / 4 antibody (Santa Cruz, #sc-5279, 1 / 400 dilution) -Rabbit anti-human NANOG antibody (ReproCell, #RCAB004P-F, 1 / 400 dilution) -Anti-SSEA antibody (APC anti-human SSEA-4 antibody, Biolegend, #330418, 1 / 100 dilution) During the antibody reaction, the plate was covered with aluminum foil to block light.

[0221] After washing four times with PBS under light-shielded conditions, 500 μL of the following secondary antibody solution was added to each well (this step was not carried out for wells intended for SSEA-4 staining, and PBS was added instead), and the wells were incubated on a shaker under light-shielded conditions at room temperature for 1-2 hours. -Donkey anti-mouse IgG (H+L) antibody (Highly Cross-Absorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-21202)) -Donkey anti-rabbit IgG (DyLight TM 488, Biolegend, #406404) After washing four times with PBS under light-blocking conditions, one drop of DAPI solution (Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI (Nacalai Tesque, #12745-74)) was added to each well. The presence or absence of positive cells for nuclear staining with DAPI and immunostaining for each marker protein was observed under a fluorescence microscope (Keyence, BZ-X810).

[0222] The cell aggregates (cell populations) obtained by culturing cells from three clones (#1 to #3) that had been initialized and amplified using episomal plasmids and then sorted were calculated for the positivity rates of each undifferentiated marker (OCT4 positivity rate, NANOG positivity rate, and SSEA positivity rate among DAPI-positive cells). As shown in Table 2, all of these showed very high positivity rates.

[0223] [Table 2]

[0224] (2-4-3) Evaluation of pluripotency by cell embryoid body formation (2-4-3-1) Differentiation of cells into three germ layers Cell clumps from the 12th passage of suspension culture of the three clones (same as clones #1-3 evaluated for undifferentiated marker expression in #2-4-2) isolated and expanded as described in 2-4-1 were transferred to a 15 mL tube and centrifuged (200 g x 1 min). The supernatant was aspirated, and the precipitated cell clumps were washed with 5 mL of PBS and further centrifuged (200 g x 1 min). The supernatant was aspirated, and 1 mL of Accutase was added to the precipitated cell clumps and incubated at 37°C for 5-10 minutes. After dissociating the cells by pipetting several times with a pipette tip, an equal volume of StemFit medium was added and the mixture was centrifuged at 200 g x 2 minutes. The supernatant was aspirated, and the precipitate was suspended in 1 mL of StemFit medium (containing 10 μM Y-27632). The cell number was counted.

[0225] 1.2 x 10 in a 15 mL tube 6 The cell number was adjusted to 12 mL of StemFit medium (containing 10 μM Y-27632) and suspended. The entire cell suspension was transferred to a reservoir (AS ONE, #2-7844-02) and 100 μL was dispensed into each well of a V-bottom 96-well plate for suspension culture (cell number: 1.0 × 10 4 After centrifugation at 200g for 3 minutes, the plate was incubated at 37°C with 5% CO2. On day 2 of incubation, 100µL of EB medium (DMEM + 10% FBS + 1% sodium pyruvate + 1% penicillin / streptomycin) was added to the plate at a rate of 100µL / well. On days 4 and 6 of incubation, the medium was replaced at a rate of 100µL / well.

[0226] Adherent culture was initiated on day 8 of culture. 500 μL of gelatin solution (0.1 w / v% solution, Fujifilm Wako Pure Chemical Industries, Ltd., #190-15805) was added to each of four wells of a 12-well plate and incubated at 37°C for approximately 1 hour. The gelatin solution was then removed to prepare gelatin-coated plates. Cells cultured in EB medium as described above were collected from each well and transferred to each well of a 12-well plate at 12–24 cells per well. Culture was initiated at 37°C in 5% CO2. The medium was replaced with 1 mL of EB medium every two days (days 10, 12, 14, etc.). Approximately 20 days after the start of culture, embryoid body differentiation was evaluated by immunostaining.

[0227] (2-4-3-2) Evaluation of three germ layer differentiation (immunostaining method) The EB medium was removed from the 12-well plate, and 2 mL of PBS was added for washing. After removing the PBS, 500 μL of PFA solution was added to each well and fixed for 10 minutes at room temperature. The PFA solution was then removed, and 1 mL of 0.1% TritonX-100 / PBS solution was added to each well and incubated on a shaker for 10 minutes. The 0.1% TritonX-100 / PBS solution was then removed, and 1 mL of PBS was added to each well. The plate was then placed on a shaker for 5 minutes, after which the solution was removed (washing). After 1-2 hours, the plate was washed four times, and 500 μL of 0.1% blocking buffer was added to each well and incubated on a shaker for 30-60 minutes (blocking).

[0228] After blocking, 500 μL of the following primary antibody solution was added to each well. The primary antibody solution was diluted with 0.1% BSA / PBS solution to the following concentrations. The plate was sealed with parafilm and left to stand overnight at 4°C. For detecting ectoderm marker TUJ1: Mouse anti-human β-3 tubulin antibody, R&D Systems, MAB1195, final concentration 2.5-5 μg / mL For detecting mesodermal marker SMA: (Mouse anti-human α-smooth muscle actin (SMA) antibody, R&D Systems, MAB1420, final concentration 2.5-5 μg / mL) For detecting the endoderm marker AFP: (Mouse anti-human alpha-fetoprotein (AFP) antibody, R&D Systems, MAB1368, final concentration 2.5-5 μg / mL)

[0229] After washing four times with PBS, 500 μL of secondary antibody solution (donkey anti-mouse IgG (H+L) antibody (Thermo Fisher Scientific, A-21202) diluted 500-1000 times with 0.1 BSA / PBS solution) was added to each well. The plate was then wrapped in aluminum foil to protect from light. After incubating the plate on a shaker at room temperature for 1-2 hours, it was washed four times with PBS and one drop of DAPI solution was added to each well. Nuclear staining with DAPI and immunohistochemistry were performed under a fluorescent microscope to observe the presence or absence of positive cells for each marker protein.

[0230] The presence of TUJ1-, SMA-, and AFP-positive cells was confirmed in the cell populations of all three clones, confirming that all of these clones maintained pluripotency.

Claims

1. A method for producing induced pluripotent stem cells (iPS cells), comprising the following steps I and II: I. Introducing a reprogramming gene into a somatic cell; and II. A step of subjecting the transfected cells to reprogramming and amplification culture in a non-gelling medium under suspension culture conditions with the medium flowing.

2. The method of claim 1 further comprising the following steps III and IV: III. A step of isolating undifferentiated marker-positive cells from the cells obtained in the step II; and IV. A step of obtaining a cell population by suspension culture of the cells separated in step III.

3. The method according to claim 1 or 2, wherein the amplification culture in step II is carried out in a suspension rotation mode, and the rotation speed is 10 to 100 rpm.

4. 3. The method according to claim 1 or 2, wherein the amplification culture in step II is carried out by a suspension stirring method, and the rotation speed of the stirring blade is in the range of 0.05 to 1.37 m / s in terms of blade tip speed.

5. The method according to claim 4, wherein the amplification culture includes subculture accompanied by a change in culture scale, and the rotation speed of the agitator blade is set so that the change in the agitation power required per volume or the blade tip speed is less than 10% before and after the change in culture scale.

6. The method according to any one of claims 2 to 5, wherein the step III is a step of fractionating the sorted cells.

7. The method according to any one of claims 2 to 6, wherein the undifferentiation marker is at least one selected from the group consisting of SSEA-4, TRA-1-60, TRA-1-81, and TRA-2-49.

8. The method according to any one of claims 1 to 7, wherein the somatic cells are blood mononuclear cells.

9. The method according to any one of claims 1 to 8, wherein the gene to be introduced into the somatic cell is incorporated into an episomal plasmid or a Sendai virus and then introduced into the somatic cell.

10. The method according to any one of claims 1 to 9, wherein the reprogramming gene to be introduced comprises at least one gene selected from the group consisting of an OCT3 / 4 gene, a KLF4 gene, and an L-MYC gene.

11. The method according to any one of claims 1 to 10, wherein the liquid medium used in the step II or later contains at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate.

12. The method according to any one of claims 1 to 11, wherein the liquid medium used after step II contains FGF2 and / or TGF-β1.

13. The method according to any one of claims 1 to 12, wherein the liquid medium used after step II contains a ROCK inhibitor.

14. The method of claim 13, wherein the ROCK inhibitor is Y-27632.

15. The method according to any one of claims 1 to 14, wherein step II comprises recovering the cell aggregates.

16. The method according to any one of claims 2 to 15, wherein step IV comprises recovering the cell aggregates.

17. The method according to any one of claims 2 to 16, wherein in the cell population obtained in step IV, the proportion of cells positive for OCT4 is 90% or more, and the proportion of cells positive for NANOG is 90% or more.

18. A cell population comprising iPS cells produced by the method according to any one of claims 1 to 17.

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