Method for improving differentiation efficiency of pluripotent stem cells

Abstract

Provided is a method for inducing the differentiation of pluripotent stem cells which are obtained by suspension culture with high quality and / or high efficiency.　The present invention pertains to a method for producing differentiated cells, the method comprising: a step in which thermal stimulation is applied to pluripotent stem cells after suspension culture of the pluripotent stem cells; a step in which the pluripotent stem cells are subsequently seeded in a culture medium that is different from a culture medium used in the suspension culture; and a step in which agregate mass formation and differentiation induction of the seeded pluripotent stem cells are performed.

Description

Method for improving differentiation efficiency of pluripotent stem cells

[0001] The present invention relates to a method for producing differentiated cells from pluripotent stem cells.

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

[0003] Pluripotent stem cell culture methods are broadly divided into adhesion culture, in which cells are attached to a flat substrate and cultured, and suspension culture, in which cells are suspended in a liquid medium and cultured. In general adhesion culture, approximately 1 to 2 × 10 ９ To obtain pluripotent stem cells, the total adhesion area must be approximately 10 (since the number of recovered cells depends on the culture area on the substrate surface). ４ cm ２ This corresponds to approximately 100 to 200 standard 10 cm dishes. Handling such a large number of substrates in a highly clean cell manufacturing environment is unproductive because it requires a large number of complicated procedures, and is unrealistic from the perspective of medical economics because it increases manufacturing costs.

[0004] On the other hand, in suspension culture, cells are cultured while suspended in a liquid medium, so the number of cells obtained depends on the volume of the medium. Therefore, compared to adherent culture, scaling up suspension culture does not require a large culture area, and cells can be cultured in a small area (cell preparation environment), enabling mass production of cells at reduced manufacturing costs. Therefore, suspension culture is also practical from the perspective of medical economics (Non-Patent Document 1). For example, Non-Patent Document 2 discloses a method for suspension culture of pluripotent stem cells while stirring the liquid medium using a spinner flask as a cell culture vessel. Non-Patent Document 3 discloses a method for improving cell proliferation of pluripotent stem cells by suspension culture using a medium perfusion method.

[0005] However, as mentioned above, even if pluripotent stem cells are mass-produced by suspension culture, unless the resulting pluripotent stem cells can be suitably induced to differentiate into the desired somatic cells (differentiated cells), therapies involving transplantation of somatic cells (differentiated cells) will not be widely adopted in society. For example, protocols for inducing differentiation of pluripotent stem cells are described in detail in Non-Patent Documents 4 and 5, but both of these differentiation induction protocols have been optimized using pluripotent stem cells obtained by adherent culture.

[0006] Cell culture technology for regenerative medicine and its industrial development CMC Publishing ISBN 978-4-7813-1480-8 Olmer R. et al., Tissue Engineering: Part C, Volume 18 (10): 772-784 (2012) Kropp C. et al., Stem Cells Translational Medicine, 5: 1289-1301 (2016) Mandy Kleinsorge and Lukas Cyganek. , STAR Protocols 1, 100026, June 19, (2020) Lukas Cyganek, Malte Tiburcy. , JCI Insight. Jun 21;3(12):e99941. (2018)

[0007] As a result of the inventors' investigation into the differentiation induction of pluripotent stem cells, they found that it was not easy to induce differentiation when a differentiation induction protocol optimized using pluripotent stem cells obtained by adherent culture was applied to pluripotent stem cells obtained by suspension culture.

[0008] The present invention aims to provide a method for inducing the differentiation of pluripotent stem cells obtained by suspension culture into differentiated cells (somatic cells) with high quality and / or high efficiency. More preferably, the differentiated cells required for industrial mass production are about 1 × 10 ９ Pluripotent stem cells (approximately 1 x 10 cells) required to produce more than 10 differentiated cells ６ The object of the present invention is to provide a method for inducing differentiation of cells (e.g., fibroblasts or more) into differentiated cells (somatic cells) with high quality and / or high efficiency.

[0009] As a result of intensive research aimed at solving the above-mentioned problems, the inventors discovered that by performing specific treatments on pluripotent stem cells after suspension culture and before differentiation induction, it is possible to induce differentiation with high quality and / or high efficiency even using a differentiation induction protocol optimized for pluripotent stem cells obtained by adherent culture, and thus completed the present invention.

[0010] That is, the present invention encompasses the following: (1) A method for producing differentiated cells, comprising: (a) a step of applying a temperature stimulus to pluripotent stem cells after suspension culture; (b) a step of subsequently seeding the pluripotent stem cells after step (a) in a medium different from the medium used for the suspension culture; and (c) a step of forming cell aggregates and inducing differentiation from the seeded pluripotent stem cells. (2) The production method according to (1) above, wherein step (a) includes a freezing step. (3) The production method according to (1) or (2) above, wherein the temperature of the different medium in step (b) is 32°C or higher and 38°C or lower. (4) The production method according to (1) above, wherein the number of cells seeded in step (b) is 1 x 10 ６ (5) The method of any one of (1) to (4) above, wherein the step (c) is performed without maintaining the pluripotent stem cells in an undifferentiated state. (6) In the step (c), the average number of cells in the aggregate is 1 x 10 ２ pcs or more, 1×10 ４ (7) The method for production according to any one of (1) to (5), wherein the medium is changed at least once in no more than three stages. (7) The method for production according to any one of (1) to (6), wherein the cell survival rate of the pluripotent stem cells after step (a) is 70% or higher. (8) The method for production according to any one of (1) to (7), wherein step (c) is a step of inducing suspension differentiation into endodermal cells. (9) The method for production according to any one of (1) to (7), wherein step (c) is a step of inducing suspension differentiation into ectodermal cells. (10) The method for production according to any one of (1) to (7), wherein step (c) is a step of inducing suspension differentiation into mesodermal cells. This specification incorporates the disclosures of Japanese Patent Application No. 2024-057242, from which the present application claims priority.

[0011] According to the present invention, pluripotent stem cells obtained by suspension culture can be induced to differentiate with high quality and / or high efficiency.

[0012] 1. Method for Producing Differentiated Cells from Pluripotent Stem Cells 1-1. Overview In the method for producing differentiated cells from pluripotent stem cells according to the present invention, a temperature stimulus is applied to pluripotent stem cells after suspension culture, which serve as starting materials, and the pluripotent stem cells are subsequently seeded in a medium different from the medium used for the suspension culture. The seeded pluripotent stem cells are then subjected to aggregate formation and differentiation induction, thereby enabling the production of high-quality and / or highly efficient differentiated cells from pluripotent stem cells obtained in suspension culture, which have been difficult to differentiate even with differentiation induction protocols optimized for pluripotent stem cells obtained in adherent culture. The method for producing differentiated cells from pluripotent stem cells according to the present invention enables the induction of high-quality and / or highly efficient differentiation of pluripotent stem cells obtained in suspension culture, which have been difficult to differentiate even with differentiation induction protocols optimized for pluripotent stem cells obtained in adherent culture, using a differentiation induction protocol optimized for pluripotent stem cells obtained in adherent culture.

[0013] 1-2. Definition of Terms The following terms used herein are defined. <<Cells>> As used herein, the term "pluripotent stem cells" refers to cells that have the multipotency (pluripotency) to differentiate into all types of cells that constitute a living organism and that 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 cells of all types of germ layers that constitute an individual (in vertebrates, the three germ layers are ectoderm, mesoderm, and endoderm). 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). "ES cells" refer to pluripotent stem cells prepared from early embryos. "EG cells" refer to pluripotent stem cells prepared from fetal primordial germ cells (Shamblott M.J. et al., 1998, Proc. Natl. Acad. Sci. USA., 95:13726-13731). "GS cells" refer to pluripotent stem cells prepared from testicular cells (especially spermatogonial stem cells) (Conrad S., 2008, Nature, 456:344-349). "iPS cells" refer to pluripotent stem cells obtained by reprogramming differentiated somatic cells to an undifferentiated state by introducing genes encoding a small number of reprogramming factors into the differentiated somatic cells.

[0014] The pluripotent stem cells used herein are not particularly limited as long as they are derived from a multicellular organism. Animal-derived cells or mammal-derived cells are preferred. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs; livestock or pets such as dogs, cats, rabbits, cows, horses, sheep, and goats; and primates such as humans, rhesus monkeys, gorillas, and chimpanzees. For example, human-derived cells can be preferably used.

[0015] As used herein, pluripotent stem cells may be classified into any type, such as naive pluripotent stem cells or primed pluripotent stem cells. Naive pluripotent stem cells are defined as cells in a state close to pluripotency found in the inner cell mass before implantation, while primed pluripotent stem cells are defined as cells in a state close to pluripotency found in the epiblast after implantation. Compared to naive pluripotent stem cells, primed pluripotent stem cells are characterized by a lower contribution to ontogeny, a single transcriptionally active X chromosome, and a high level of transcriptionally repressive histone modifications. Furthermore, the marker for primed pluripotent stem cells is the OTX2 gene, while the marker genes for naive pluripotent stem cells are REX1 and KLF family genes. Furthermore, the colonies formed by primed pluripotent stem cells are characterized by a flattened shape, while the colonies formed by naive pluripotent stem cells are dome-shaped.

[0016] As the pluripotent stem cells used in this specification, commercially available cells or cells provided by a supplier may be used as they are, or newly cultured cells may be used. Although not limited thereto, when used in each invention of this specification, the pluripotent stem cells are preferably iPS cells or ES cells.

[0017] When the iPS cells used herein are commercially available or research strains, they include, but are not limited to, the 253G1 strain, 253G4 strain, 201B6 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, TkDN4-M strain, TkDA3-1 strain, TkDA3-2 strain, TkDA3-4 strain, TkDA3-5 strain, TkDA3-9 strain, TkDA3-20 strain, and hiPSC 38-2 strain, MSC-iPSC1 strain, BJ-iPSC1 strain, RPChiPS771-2, WTC-11 strain, 1231A3 strain, 1383D2 strain, 1383D6 strain, 1210B2 strain, 1201C1 strain, 1205B2 strain, QHJI01s01 strain, QHJI01s04 strain, QHJI14s03 strain, QHJI14s04 strain, Ff-114s03 strain, Ff-114s04 strain, YZWI strain, and the like can be used.

[0018] As used herein, the combination of genes for reprogramming factors introduced into cells during the production of iPS cells is not limited. For example, a combination of the OCT3 / 4 gene, the KLF4 gene, the SOX2 gene, and the c-Myc gene (Yu J, et al. 2007, Science, 318:1917-20.), or a combination of the OCT3 / 4 gene, the SOX2 gene, the LIN28 gene, and the Nanog gene (Takahashi K, et al. 2007, Cell, 131:861-72.) can be used. The method for introducing these genes into cells is not particularly limited, and may include, for example, gene introduction using a plasmid such as an episomal vector, introduction as a nucleic acid such as introduction of synthetic RNA, or introduction as a protein. iPS cells generated by methods using Sendai virus vectors, non-translated RNA such as microRNA, low-molecular-weight compounds, etc. may also be used. Furthermore, universal iPS cells in which the HLA gene has been edited and deleted to suppress immune rejection may also be used.

[0019] When the ES cells used herein are commercially available, for example, but not limited to, KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SEES-1 strain, SEES-2 strain, SEES-3 strain, SEES-4 strain, SEES-5 strain, SEES-6 strain, SEES-7 strain, HUES8 strain, CyT49 strain, H1 strain, H9 strain, HS-181 strain, etc. can be used.

[0020] Pluripotent stem cell markers (also called undifferentiated markers) are gene markers that are specifically or excessively expressed in pluripotent stem cells, and examples thereof include alkaline phosphatase, NANOG, OCT4, SOX2, TRA-1-60, c-Myc, KLF4, LIN28, SSEA-4, and SSEA-1.

[0021] As used herein, "differentiated cells" refer to cells induced to differentiate from pluripotent stem cells into a wide variety of cells, including cardiomyocytes, neurons, blood cells, retinal cells, and adult stem cells, and are also called somatic cells. Adult stem cells are stem cells present in each tissue of an adult, have incomplete terminal differentiation, and have a certain degree of multipotency; they are also called somatic stem cells or tissue stem cells. Examples include mesenchymal stem cells, neural stem cells, intestinal epithelial stem cells, hematopoietic stem cells, hair follicle stem cells, melanocyte stem cells, and cancer stem cells. Other examples of differentiated cells include three-dimensional tissue-like structures (also called organoids) that have functions similar to those of various tissues or organs, which are formed by self-organizing the differentiated cells and / or adult stem cells described above. For example, organoids include cerebral organoids, cerebellar organoids, inner ear organoids, thyroid organoids, thymus organoids, T cell mature lymphoid organoids, myocardial organoids, lung organoids, liver organoids, pancreatic organoids, kidney organoids, gastric gland organoids, Foregutt organoids (organoids constituting the oral cavity and stomach), Midgut organoids (organoids constituting the small intestine and ascending colon), Hindgut organoids (organoids constituting parts other than the rectum and ascending colon), intestinal organoids, epithelial organoids, ovarian organoids, testicular organoids, etc. Furthermore, fusion organoids, which produce new differentiated cells by fusing these organoids together, can also be mentioned.

[0022] Differentiation cell markers are genes that are specifically expressed in a wide variety of somatic cells, and examples thereof include PAX6 for neural progenitor cells, CD31 for cardiovascular cells, troponin T (TnT) for cardiomyocytes, and CD34 for hematopoietic stem cells. Other examples of endodermal cell markers that form tissues of organs such as the digestive tract, lung, thyroid, pancreas, and liver, cells of secretory glands opening into the digestive tract, peritoneum, pleura, larynx, Eustachian tube, trachea, bronchi, and urinary tract (bladder, most part of the urethra, and part of the ureter), include cell markers such as SOX17, FOXA2, CXCR4, AFP, GATA4, and EOMES. Furthermore, examples of mesodermal cell markers that form 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, gonads (testes, uterus, gonadal epithelium), etc. include T (BRACHYURY), MESP1, MESP2, FOXF1, HAND1, EVX1, IRX3, CDX2, TBX6, MIXL1, ISL1, SNAI2, FOXC1, and PDGFRα. Furthermore, examples of ectodermal cell markers that form the epidermis of the skin, the epithelium of the distal part of the male urethra, hair, nails, skin glands (including mammary glands and sweat glands), sensory organs (including the epithelium of the distal parts of the oral cavity, pharynx, nose, and rectum, and salivary glands), lens, peripheral nervous system, etc. include FGF5, NESTIN, SOX1, and PAX6.

[0023] Cell markers, such as differentiated cell markers and pluripotent stem cell markers, can be detected by any detection method known in the art. Methods for detecting cell markers include, but are not limited to, flow cytometry. For example, when flow cytometry is used as the detection method and a fluorescently labeled antibody is used as the detection reagent, cells that exhibit stronger fluorescence compared to a negative control (isotype control) can be considered "positive" for the marker. The proportion of cells that exhibit positive results for a detection reagent (e.g., a fluorescently labeled antibody analyzed by flow cytometry) is sometimes referred to herein as the "positive rate." Furthermore, when a labeled antibody is used as the detection reagent, any antibody known in the art can be used. For example, fluorescently labeled antibodies include, but are not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc. For example, cells with a high positive rate for differentiated cell markers can be evaluated as a cell population enriched in differentiated cells, while cells with a high positive rate for pluripotent stem cell markers can be evaluated as a cell population enriched in undifferentiated cells. Other examples include quantitative real-time PCR analysis, RNA-Seq, Northern hybridization, and hybridization methods using DNA arrays. In quantitative real-time PCR analysis, the expression level of the marker 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 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 or bAct) gene. This detection method can also be used to analyze the expression of the above-mentioned pluripotent stem cell markers. For example, if the expression level of the pluripotent stem cell marker is high and the expression of the differentiated cell marker is undetectable (below the detection limit), the cells can be evaluated as undifferentiated pluripotent stem cells.

[0024] <<Adhesion Culture>> "Adhesion culture" is a cell culture method in which cells are cultured by adhering them to an external matrix or the like present on the surface of a culture vessel or the like. In typical adhesion culture, cells are grown in a monolayer. The external matrix is ​​not particularly limited, but examples thereof include laminin, vitronectin, gelatin, collagen, E-cadherin chimeric antibody, or combinations thereof. Cells grown in adhesion culture form dense cell colonies as they grow. Note that the aforementioned pluripotent stem cells can usually be cultured not only in adhesion culture but also in suspension culture.

[0025] "Suspension culture" refers to a cell culture method in which cells are cultured in a suspended state in a liquid medium. As used herein, "suspension state" refers to a state in which cells are not fixed by adhesion to an external matrix on the surface of a culture vessel (e.g., the inner surface of the wall, bottom, or underside of the lid, or the surface of a structure within the culture vessel (e.g., agitator blades)). "Suspension culture" refers to a method of culturing cells in suspension, in which cells exist as aggregated cell masses in the culture medium. Methods for suspending cells include, but are not limited to, stirring, swirling, and shaking. For example, a culture method in which cells are attached to microcarriers and cultured in a suspended state in the culture medium is considered suspension culture in this specification because, although the cells adhere to the microcarriers, the entire cell mass including the microcarriers floats without being fixed to the culture vessel. The aforementioned cells can generally be cultured not only in suspension culture but also in adherent culture.

[0026] "Maintenance culture" refers to culturing cells while maintaining their cell characteristics. For example, maintenance culture of undifferentiated pluripotent stem cells means culturing the cells while maintaining their undifferentiated state, regardless of the culture method, and may be either adherent culture or suspension culture.

[0027] <Culture Medium and Medium Exchange Method> As used herein, "culture 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. In this specification, liquid medium is often simply referred to as "culture medium."

[0028] As used herein, the term "basal medium" refers to a medium that is the basis for various animal cell culture media. It can be used alone for culture, and can also be prepared into a medium specific to various cells depending on the purpose by adding various culture additives. The basal medium used herein includes 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)). Examples of suitable basal media include, but are not limited to, DMEM / F12 medium and Ham's F12 medium. In the case of DMEM / F12 medium, the weight ratio of DMEM medium to Ham's F12 medium is not particularly limited. For example, it is preferable to use a DMEM / F12 medium in which DMEM medium and Ham's F12 medium are mixed at a weight ratio of 60 / 40 to 40 / 60, for example, 58 / 42, 55 / 45, 52 / 48, 50 / 50, 48 / 52, 45 / 55, or 42 / 58. Other preferred basal media that can be used in the present invention include, for example, serum-free media, and more preferably serum-free media.

[0029] As used herein, the term "culture additive" refers to a substance other than serum and gaseous components 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, antibiotics, or combinations thereof. Insulin, transferrin, and cytokines may be naturally occurring proteins isolated from tissues or serum of animals (e.g., humans, mice, rats, cows, horses, goats, etc.), or may be recombinant proteins produced by genetic engineering. Examples of 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, or combinations thereof. Examples of antibiotics that can be used include, but are not limited to, penicillin, streptomycin, amphotericin B, or combinations thereof.

[0030] The medium used in the present invention may contain one or more of the above culture additives. There is no limitation on the medium to which the above culture additives are added.

[0031] Culture additives can be added to the medium as they are, or 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 these culture additives, for example, 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), Hybridoma-SFM (Life Technologies Japan), eRDF Dry Powdered Media (Life Technologies Japan), and UltraCULTURE. ＴＭ (BioWhittaker), UltraDOMA ＴＭ (BioWhittaker), UltraCHO ＴＭ (BioWhittaker), UltraMDCK ＴＭ (BioWhittaker), STEMPRO (registered trademark) hESC SFM (Life Technologies Japan), Essential8 ＴＭ (Life Technologies Japan, Inc.), StemFit (registered trademark) AK02N (culture medium obtained by mixing 400 mL of solution A, 100 mL of solution B, and 2 mL of solution C) (manufactured by Ajinomoto Co., Inc.), StemFit (registered trademark) AK03N (culture medium obtained by mixing 400 mL of solution A, 100 mL of solution B, and 2 mL of solution C) (manufactured by Ajinomoto Co., Inc.), StemFit (registered trademark) Basic04 Complete Type (manufactured by Ajinomoto Co., Inc.), mTeSR1 (STEMCELL Technologies, Inc.), and TeSR2 (STEMCELL Technologies, Inc.).

[0032] As used herein, the term "medium exchange method" refers to a method of supplying cells with medium as a nutrient source for cell survival and proliferation, and a method of removing the medium in which nutrients have been consumed by cells and metabolic products have accumulated. Medium exchange methods are not particularly limited, but include, for example, a batch method and a perfusion method. A batch method refers to replacing a given amount (e.g., all or half) of the medium in the culture system (often referred to herein as "culture solution") with new medium at any given culture time interval. A perfusion method refers to continuous medium exchange by continuously removing and separately supplying the medium in the culture system, and the amount of medium removed and supplied per unit time is referred to as the medium perfusion rate. Medium perfusion may be performed continuously or intermittently in multiple batches. In suspension culture, medium exchange is preferably performed using a perfusion method.

[0033] <Gas Supply> As used herein, "gas supply" refers to the supply of oxygen and carbon dioxide necessary for cell survival and / or proliferation to the culture solution by aerating a gas through the culture solution during cell culture. Gas components used for gas supply include oxygen, nitrogen, carbon dioxide, and other gas components present in the atmosphere. Regarding the proportion of each component in the supply gas, the lower limit of the oxygen proportion is not particularly limited, but 1%, 2%, 3%, 4%, 5%, 10%, or 20% is preferred, and the upper limit is 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% is preferred. The lower limit of the carbon dioxide proportion is not particularly limited, but 0%, 1%, 2%, 3%, 4%, or 5% is preferred, and the upper limit is 20%, 10%, 9%, 8%, 7%, 6%, or 5% is preferred. The proportions of oxygen and carbon dioxide can be selected independently. The method for adjusting the proportions of oxygen and carbon dioxide is not particularly limited. For example, the oxygen and carbon dioxide concentrations in the gas can be adjusted by adding nitrogen as a component other than oxygen and carbon dioxide. The method for preparing the feed gas is not particularly limited. For example, purified oxygen, carbon dioxide, and nitrogen may be mixed, or air may be mixed with oxygen, carbon dioxide, and nitrogen. Examples of the ratio of oxygen, carbon dioxide, and nitrogen in the feed gas include, but are not limited to, 20:5:75, 20:4:76, 20:3:77, 20:2:78, 20:1:79, 20:0:80, 5:5:90, 5:0:95, 40:5:55, and 50:0:50. The ratio does not need to be constant during culture and may be changed as needed. Gas supply methods include connecting a tube to a bioreactor or the like to actively pump and aerate the gas, or filling an incubator with a gas of desired composition and supplying it to the culture vessel by natural diffusion. Typically, the gas supplied to the cell culture medium is preferably sterile, and although not limited thereto, it is preferable to supply the gas to the culture medium through a filter. In this specification, "carbon dioxide" may be referred to as "carbon dioxide gas," and "carbon dioxide concentration in the supplied gas" may be referred to as "carbon dioxide gas concentration."

[0034] 2. Further detailed description of the method for producing differentiated cells from pluripotent stem cells 2-1. Overview Below, the following steps, which are included in the method for producing differentiated cells from pluripotent stem cells according to the present invention, are described: (a) the step of applying a temperature stimulus to pluripotent stem cells after suspension culture; (b) the step of subsequently seeding the pluripotent stem cells after step (a) into a medium; (c) the step of inducing differentiation of the seeded pluripotent stem cells; and other optional steps.

[0035] 2-2. Method The method of this embodiment includes the essential steps of applying a temperature stimulus to pluripotent stem cells, seeding the temperature-stimulated pluripotent stem cells in a medium, and inducing differentiation of the seeded pluripotent stem cells, and also includes the optional steps of culturing the pluripotent stem cells in suspension and recovering them.

[0036] 2-2-0. Step of Suspension Culturing Pluripotent Stem Cells The step of suspension culturing pluripotent stem cells is an optional step in which the pluripotent stem cells are maintained and / or expanded in suspension culture. Note that adhesion culture may be performed as an optional additional step prior to the suspension culture step.

[0037] The basic details of suspension culture, culture medium, and culture medium exchange method are as described above in the section "1-2. Definition of terms."

[0038] 2-2-1. Step of applying a temperature stimulus to pluripotent stem cells The step of applying a temperature stimulus to pluripotent stem cells is a step of applying a temperature stimulus to pluripotent stem cells that have been maintained and / or expanded in suspension culture.

[0039] For the suspension culture, the medium and medium exchange method described above in "1-2. Definition of Terms" can be used. There are no particular limitations on the culture conditions, such as the medium, the components contained in the medium, and the medium exchange method.

[0040] The medium preferably contains, for example, a ROCK inhibitor. An example of a ROCK inhibitor is Y-27632. By including a ROCK inhibitor in the medium, cell death in a non-adherent state of pluripotent stem cells to a substrate or other cells and / or under high shear stress can be significantly suppressed. However, in adherent culture, continuous addition of Y-27632 causes cells to become dysmorphic, so it is preferable to use a medium that does not contain Y-27632 after the cells have formed colonies.

[0041] Furthermore, in order to maintain or improve the undifferentiated state of pluripotent stem cells in suspension culture, it is preferable to contain, for example, a protein kinase C β (PKCβ) inhibitor and / or a WNT inhibitor. PKCβ inhibitors include, for example, LY333531, Go6983, and GF109203X. WNT inhibitors include IWR-1-endo, XAV939, WNT-C59, IWP-2, and IWP-3. Addition of these inhibitors can suppress spontaneous differentiation and deterioration of the quality of pluripotent stem cells, thereby stabilizing the cells during culture.

[0042] Furthermore, when culturing primed pluripotent stem cells, the medium preferably does not contain, for example, LIF. Furthermore, when culturing primed pluripotent stem cells, the medium preferably does not contain, for example, either a GSK3 inhibitor or a MEK / ERK inhibitor, or both. A medium that does not contain any of LIF, GSK3 inhibitor, or MEK / ERK inhibitor allows primed pluripotent stem cells to be cultured without naivetization and while maintaining their undifferentiated state.

[0043] The composition of the cell suspension used for temperature stimulation is not particularly limited. Typically, it contains the medium components described above. Alternatively, for example, a cryopreservation solution can be used. The type of cryopreservation solution used is not particularly limited. For example, any cryopreservation solution known in the art can be used. Specific examples include STEM-CELLBANKER, NutriFreez, CryoStor, COS banker, CryoSolutions, Cryoscarless, Cell Reservoir One, or combinations thereof. The proportion of the cryopreservation solution is not particularly limited. For example, the cryopreservation solution may be added without removing the medium components, or the medium may be replaced (e.g., all or half) with the cryopreservation solution.

[0044] <<Temperature Stimulation>> In the present invention, the step of applying a temperature stimulus refers to a step of applying a stimulus that artificially changes the temperature, such as a cooling stimulus and / or a warming stimulus. The procedure, combination, and conditions of the temperature stimulus are not particularly limited. For example, the procedure of the temperature stimulus may be a cooling stimulus followed by a warming stimulus, a warming stimulus followed by a cooling stimulus, or a warming stimulus followed by a cooling stimulus, and the number of times the stimulus is applied is not particularly limited. Conditions for applying a temperature stimulus include, for example, a cooling rate in the case of a cooling stimulus. The cooling rate is not particularly limited, but is preferably, for example, −0.1° C. / min or more (rate of change in the absolute value of the temperature is 0.1° C. / min or more), −0.2° C. / min or more (rate of change in the absolute value of the temperature is 0.2° C. / min or more), −0.3° C. / min or more (rate of change in the absolute value of the temperature is 0.3° C. / min or more), −0.5° C. / min or more (rate of change in the absolute value of the temperature is 0.5° C. / min or more), or −1.0° C. / min or more (rate of change in the absolute value of the temperature is 1.0° C. / min or more), and is preferably −70° C. / min or less (rate of change in the absolute value of the temperature is 70° C. / min or less), −60° C. / min or less (rate of change in the absolute value of the temperature is 60° C. / min or less), or −50° C. / min or less (rate of change in the absolute value of the temperature is 1.0° C. / min or more). The temperature change rate is preferably in the range of -50°C / min or less), -45°C / min or less (rate of change in absolute temperature value is 45°C / min or less), or -40°C / min or less (rate of change in absolute temperature value is 40°C / min or less), -30°C / min or less (rate of change in absolute temperature value is 30°C / min or less), -20°C / min or less (rate of change in absolute temperature value is 20°C / min or less), -10°C / min or less (rate of change in absolute temperature value is 10°C / min or less), -5°C / min or less (rate of change in absolute temperature value is 5°C / min or less), -3°C / min or less (rate of change in absolute temperature value is 3°C / min or less), -2°C / min or less (rate of change in absolute temperature value is 2°C / min or less), or -1°C / min or less (rate of change in absolute temperature value is 1°C / min or less). In addition, in cooling and heating, the residence time in the maximum ice crystal formation temperature range of -1°C to -5°C is not particularly limited, but can be, for example, less than 30 minutes, 20 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 2 minutes or less, 1 minute or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, etc. Also, for example, it can be 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, etc. Another condition is the heating rate in the case of a heating stimulus.For example, the heating rate is not particularly limited, but is preferably +0.1°C / sec or more, +0.2°C / sec or more, or +0.3°C / sec or more, +5°C / sec or more, +10°C / sec or more, +20°C / sec or more, +40°C / sec or more, +60°C / sec or more, +70°C / sec or more, +75°C / sec or more, +80°C / sec or more, or +83°C / sec or more, and is preferably +120°C / sec or less, +100°C / sec or less, +90°C / sec or less, +85°C / sec or less, +84°C / sec or less, +83°C / sec or less, +50°C / sec or less, +45°C / sec or less, or +40°C / sec or less. The step of applying a temperature stimulus preferably includes a freezing step of freezing the cells by low-temperature stimulation to 0°C or below. When low-temperature stimulation of the cells to 0°C or below is performed, the temperature is preferably, for example, −10°C or below, −20°C or below, −30°C or below, or −50°C or below, and is preferably in the range of −220°C or above, −210°C or above, −200°C or above, or −195°C or above.

[0045] The freezing step can be carried out in multiple stages. For example, after cooling to a predetermined temperature (first temperature) at the above-mentioned cooling rate, further cooling can be carried out. In this case, the first temperature is not particularly limited, and can be, for example, −10°C or lower, −20°C or lower, −30°C or lower, −50°C or lower, −60°C or lower, −70°C or lower, −75°C or lower, or −80°C or lower. Furthermore, for example, the first temperature can be −196°C or higher, −190°C or higher, −150°C or higher, −120°C or higher, −100°C or higher, −90°C or higher, −85°C or higher, or −80°C or higher. When further cooling is carried out thereafter, the temperature to be reached (final temperature) is not particularly limited. For example, the temperature can be −220°C or higher, −210°C or higher, −200°C or higher, −196°C or higher, etc., and can also be, for example, −80°C or lower, −85°C or lower, −90°C or lower, −100°C or lower, −120°C or lower, −150°C or lower, −170°C or lower, −190°C or lower, −195°C or lower, −196°C or lower, etc. The rate of further cooling is not particularly limited. For example, it may be the same as or different from the cooling to the first temperature, and the cooling rate does not need to be controlled. When the cooling rate is not controlled, for example, it can be achieved by exposing the pluripotent stem cells to an environment with the final temperature. Alternatively, the pluripotent stem cells may be frozen at the freezing rate described above until the temperature reaches the first temperature or the final temperature exemplified above.

[0046] Furthermore, in the temperature stimulation step, when cells are stimulated by heating to 0°C or higher, the temperature reached after heating is not particularly limited, but is preferably 1°C or higher, 5°C or higher, 10°C or higher, 15°C or higher, 20°C or higher, 25°C or higher, 30°C or higher, 35°C or higher, or 37°C or higher, and is preferably in the range of 50°C or lower, 45°C or lower, or 40°C or lower, or 37°C or lower.

[0047] The temperature stimulation step can also be carried out by combining the various temperature stimulations described above. For example, after performing a cooling stimulation, the sample can be maintained under specific temperature conditions (preferably 0 ° C or less) for a certain period of time, followed by a heating stimulation. The specific temperature conditions in this case are not particularly limited, but can be, for example, the temperatures exemplified as the first temperature reached or the final temperature reached above. The retention period is not particularly limited, but is preferably 1 day or more, 2 days or more, 3 days or more, 4 days or more, 10 days or more, 20 days or more, 1 month or more, 1.5 months or more, or 2 months or more, and preferably 3 years or less, 2 years or less, 1 year or less, 6 months or less, 4 months or less, 3 months or less, or 2 months or less.

[0048] The cell density of the pluripotent stem cells before the step of applying the temperature stimulation, i.e., the cell density to be subjected to the temperature stimulation step, is not particularly limited, but may be, for example, 2 × 10 ４ cells / mL or more, 5 x 10 ４ cells / mL or more, 1×10 ５ cells / mL or more, 5 x 10 ５ cells / mL or more, and ６ cells / mL or more, 2 x 10 ６ Preferably, the concentration is 5 x 10 cells / mL or more. ６ More preferably, the concentration is 1 x 10 cells / mL or more. ７ More preferably, the concentration is 2 x 10 cells / mL or more. ７ The upper limit of the cell density is not particularly limited, but it is preferably 1×10 １２ cells / mL, 1×10 １１ cells / mL, 1×10 １０ cells / mL, 1×10 ９ cells / mL, 1×10 ８ cells / mL, 1×10 ７ cells / mL, 5×10 ６ cells / mL, 2×10 ６ It can be expressed as cells / mL, etc.

[0049] The cell survival rate of the pluripotent stem cells after the step of applying a temperature stimulus is not particularly limited, but is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more.

[0050] 2-2-2. Step of seeding temperature-stimulated pluripotent stem cells into a medium The step of seeding temperature-stimulated pluripotent stem cells into a medium is a step of seeding the pluripotent stem cells into a medium after the step of applying the temperature stimulus.

[0051] For example, the medium is different from the medium used for the suspension culture performed before the step (a).

[0052] The "medium different from the medium used for suspension culture" is not particularly limited as long as it has a different medium composition from the medium used for suspension culture, but is preferably a medium containing a differentiation-inducing factor, and is a medium that can induce differentiated cells according to the purpose by adding a differentiation-inducing factor to a basal medium that serves as the basis for various animal cell culture media. For example, a medium containing multiple types of differentiation-inducing factors can be used to induce the desired differentiated cells.

[0053] The number of pluripotent stem cells to be seeded is not particularly limited, but in order to produce differentiated cells that require industrial mass production, 1 × 10 ４ pcs or more, 1×10 ５ More than 5 x 10 ５ pcs or more, 7×10 ５ pcs or more, 9×10 ５ pcs or more, 1×10 ６ It is preferable that the number of particles is 2×10 or more. ６ pcs or more, 2.5×10 ６ pcs or more, 1×10 ７ More preferably, it is 1×10 or more. ８ More preferably, it is equal to or greater than 100.

[0054] The temperature of the medium in the seeding step is not particularly limited, but it is preferable that the lower limit is 25°C, 30°C, 32°C, 35°C, 36°C, or 37°C, and the upper limit is 45°C, 40°C, 38°C, or 37°C.

[0055] The cell seeding time is the time from when the pluripotent stem cells are recovered after the temperature stimulation step until when they are seeded into a medium different from the medium used to culture the pluripotent stem cells, and is not particularly limited, but is preferably 10 minutes or more, 15 minutes or more, 20 minutes or more, or 25 minutes or more, and is preferably in the range of 9 hours or less, 8 hours or less, 7 hours or less, or 6 hours or less.

[0056] 2-2-3. Step of inducing differentiation of seeded pluripotent stem cells The step of inducing differentiation of seeded pluripotent stem cells is a step of inducing differentiation of the pluripotent stem cells seeded in step (b) above. In this step, differentiation may be induced after forming cell aggregates from single cells, or differentiation may be induced before forming cell aggregates, or cell aggregates and differentiation induction may proceed simultaneously, as long as a differentiation-induced cell aggregate is ultimately obtained.

[0057] This step is preferably carried out without maintaining the seeded pluripotent stem cells in an undifferentiated state. Surprisingly, the pluripotent stem cells that have undergone the step of applying a temperature stimulus have a high differentiation induction efficiency even without maintenance culture.

[0058] <<Formation of Aggregates>> In this specification, the "formation of aggregates" as the subject of the present invention preferably refers to seeding the single pluripotent stem cells recovered after the temperature stimulation step into a medium different from the medium used for suspension culture, and gradually forming aggregates under suspension culture conditions. Until aggregates begin to form in suspension culture, this does not include any other culture procedures such as medium replacement and / or medium addition, recovery culture, subculture, etc.

[0059] The aggregates are clump-like cell masses formed by cell aggregation in suspension culture. These aggregates usually have a roughly spherical shape. During the process of aggregate formation, the cells constituting the aggregates may be pluripotent stem cells or differentiated cells, but the cells that ultimately constitute the aggregates are differentiated cells generated by differentiation induction from pluripotent stem cells, and the type of differentiated cells is not particularly limited.

[0060] When differentiation is induced after forming a cell aggregate from a single cell, the culture period for forming the cell aggregate is not particularly limited, and can be, for example, 6 hours or more, 12 hours or more, 16 hours or more, 20 hours or more, 1 day or more, etc. Also, it can be, for example, 7 days or less, 5 days or less, 3 days or less, 2 days or less, 1 day or less, etc.

[0061] In the aggregates, the average number of cells per aggregate is not particularly limited, but is preferably 1 x 10 ２ pcs or more, 2×10 ２ pcs or more, 3×10 ２ 10 or more, and 4 x 10 ２ Preferably, 1×10 or more ４ Less than or equal to 0.9×10 ４ Less than or equal to 0.8×10 ４ Less than or equal to 0.7×10 ４ Less than or equal to 0.6×10 ４ In this step, the average number of cells per aggregate is preferably in the range of 1 × 10 or less. ２ pcs or more, 1×10 ４ It is preferable to exchange the medium at least once at each stage or less.

[0062] The size of the aggregates formed within 20 to 24 hours after seeding the cells in the medium is not particularly limited, but an average particle diameter of 20 μm or more, 30 μm or more, or 40 μm or more is preferred, and 500 μm or less, 400 μm or less, or 300 μm or less is preferred. Cell aggregates within this range are preferred as a differentiation-inducing environment for cells, as differentiation-inducing factors, oxygen, and nutrients can be easily supplied to the cells inside the aggregates. Typically, aggregates of 300 μm or less within one day after the start of aggregate formation are particularly preferred. Note that it is not necessary for the size of all cell aggregates in the culture medium to be within the above range; for example, it is sufficient if the number-average size is within the above range.

[0063] <<Differentiation Induction>> The differentiation induction according to the present invention is characterized by the use of pluripotent stem cells obtained through the above-described steps (a) and (b). The pluripotent stem cells obtained through the above-described steps (a) and (b) have excellent differentiation induction efficiency, so that maintenance culture before differentiation induction is not necessary, and the obtained pluripotent stem cells or pluripotent stem cells after storage for a predetermined period can be directly subjected to (aggregate formation and) differentiation induction.

[0064] Differentiation induction refers to the phenomenon in which pluripotent stem cells produced as described above are caused to differentiate into different cells upon contact with other cells or upon stimulation with a differentiation-inducing factor or the like.

[0065] The differentiation induction is preferably carried out in suspension culture, and the medium and medium exchange method described above in "1-2. Definition of Terms" can be used, but it is preferable to culture in a medium containing a differentiation induction factor. The medium to which the differentiation induction factor is added is not particularly limited, and any medium conventionally used for differentiation induction can be used as appropriate. Examples of the medium include StemFit (registered trademark) AK02N minus C liquid medium (a culture medium comprising 400 mL of liquid A and 100 mL of liquid B) (manufactured by Ajinomoto Co., Inc.), Stem Pro34 (registered trademark), Neurobasal (registered trademark), and medium.

[0066] The term "differentiation inducer" refers to a substance used for the purpose of promoting differentiation induction, and examples thereof include, but are not limited to, in vivo gene products such as growth factors, low-molecular-weight compounds that control the activity of in vivo gene products, hormones, vitamins, and other physiologically active substances. Specific examples of differentiation inducers include, but are not limited to, Activin / TGFβ signal activators or inhibitors, BMP signal activators, BMP signal inhibitors, retinoic acid signal activators, Hedgehog signal activators, Hedgehog signal inhibitors, Notch signal inhibitors, WNT / β-catenin signal activators, JNK signal inhibitors, Src signal inhibitors, AMPK signal inhibitors, EGF signal activators, EGF signal inhibitors, HGF signal activators, and VEGF signal activators. The differentiation inducer can be appropriately selected depending on, for example, the type of differentiated cell of interest, and is not particularly limited. For example, any differentiation inducer known in the art can be used depending on the type of differentiated cell of interest.

[0067] The differentiation induction preferably involves inducing pluripotent stem cells to differentiate into endodermal cells, ectodermal cells, or mesodermal cells under suspension conditions. Specifically, for example, differentiation into endodermal or mesodermal cells can be induced under suspension conditions. The proportion of pluripotent stem cells constituting the aggregate can be determined, for example, by the positivity rate of a pluripotent stem cell marker. The positivity rate of a pluripotent stem cell marker in the cells constituting the final aggregate is not particularly limited, but can also be considered the contamination rate of pluripotent stem cells during the differentiation induction stage. Therefore, it is preferably 55% or less, 52% or less, 51% or less, 50% or less, more preferably 40% or less, for example, 30% or less, for example, 29% or less, for example, 28% or less, for example, 27% or less, for example, 26% or less, for example, 25% or less, for example, 24% or less, for example, 23% or less, for example, 22% or less, for example, 21% or less, or for example, 20% or less. Aggregates with a high proportion of cells expressing pluripotent stem cell markers and / or a high proportion of cells that are positive for pluripotent stem cell markers are cell populations that contain a high rate of undifferentiated pluripotent stem cells and have not been induced to differentiate appropriately.

[0068] The proportion of differentiated cells constituting the aggregate can be determined, for example, by the positive rate of a differentiated cell marker. The positive rate of a differentiated cell marker in the cells constituting the finally obtained aggregate is not particularly limited, but is preferably 30% or more, more preferably 40% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, for example, 50% or more, for example, 60% or more, for example, 70% or more, for example, 80% or more. An aggregate having a high proportion of cells expressing a differentiated cell marker and / or a high proportion of cells positive for a differentiated cell marker is a cell population with a high proportion of differentiated cells.

[0069] Aggregates with a high proportion of cells expressing differentiated cell markers and / or a high proportion of cells that are positive for differentiated cell markers, and with a low proportion of cells expressing pluripotent stem cell markers and / or a low proportion of cells that are positive for pluripotent stem cell markers, are cell populations that have a high proportion of differentiated cells and a low rate of contamination with undifferentiated cells, making them extremely safe and suitable for clinical use.

[0070] The type of differentiated cells contained in the aggregate can be determined by detecting the expression of one or more, two or more, or three or more types of differentiated cell markers. In this case, the type of differentiated cell marker is not particularly limited. For example, a differentiated cell marker for endodermal cells includes SOX17, a differentiated cell marker for ectodermal cells includes Pax6, and a differentiated cell marker for mesodermal cells includes Brachyury, troponin T (TnT), CD31, etc.

[0071] In the differentiation induction step, some of the cells can be removed during the culture to confirm the cell number and cell aggregate size. The cell aggregates removed during culture can be loosened into single cells, for example, by enzyme treatment, and the number of viable cells can be measured using a method such as the trypan blue method. Alternatively, the cell number can be estimated from the number and size of the cell aggregates removed during culture. The size or volume of the cell aggregates can be measured by, but is not limited to, laser size measurement, image acquisition and size calculation from the image, or the like.

[0072] 2-2-4. Recovery Step The "recovery step" is a step of recovering cultured cells or cell clusters from the culture medium after the step of inducing differentiation of the seeded pluripotent stem cells, and is a selection step in the method of the present invention.

[0073] As used herein, the term "recovery (of cells)" refers to obtaining cells by separating the cells from the culture medium. The method for recovering cells may be any conventional method used in cell culture methods in the art, and is not particularly limited.

[0074] After the differentiation induction step, the cells remain suspended in the culture medium. Therefore, cell recovery can be achieved by removing the liquid components of the supernatant by standing or centrifugation. Cells can also be recovered using a filtration filter, hollow fiber separation membrane, or the like. When removing the liquid components by standing, the container containing the culture medium can be left standing for approximately 5 minutes, and the supernatant can be removed, leaving the settled cells and cell clumps. 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 any speed at which the cells are not or are least likely to be damaged by centrifugal force, such as 16,000 × g or less, 15,000 × g or less, or 14,000 × g or less. The lower limit of the treatment time is not particularly limited as long as it allows the cells to settle due to the centrifugal acceleration, but 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, 100 minutes or less, 80 minutes or less, 60 minutes or less, or 30 minutes 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).

[0075] The collected cells and cell aggregates can be washed as needed. The washing method is not limited. A buffer (e.g., PBS buffer), physiological saline, or a medium (preferably a basal medium) may be used as a washing liquid.

[0076] 3. Other Aspects The present invention further provides a method for enhancing differentiation induction efficiency, comprising the step of applying a temperature stimulus to pluripotent stem cells after suspension culture. The step of applying a temperature stimulus to pluripotent stem cells after suspension culture is as described in Section "2-2-1. Step of applying a temperature stimulus to pluripotent stem cells." The method for enhancing differentiation induction efficiency further comprises, as an optional step, a step of suspension culturing the pluripotent stem cells.

[0077] The present invention further provides a method for producing pluripotent stem cells for differentiation induction, which includes a step of applying a temperature stimulus to pluripotent stem cells after suspension culture. The step of applying a temperature stimulus to pluripotent stem cells after suspension culture is similar to the description in Section "2-2-1. Step of applying a temperature stimulus to pluripotent stem cells." The method for producing pluripotent stem cells for differentiation induction further includes, as optional steps, a step of suspension culturing the pluripotent stem cells and a step of recovering them.

[0078] The present invention further provides pluripotent stem cells produced by the above-mentioned method for producing pluripotent stem cells for differentiation induction.

[0079] The method for producing differentiated cells according to the present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0080] (Production Example 1: Adherent culture of human iPS cells (QHJI strain)) Frozen human iPS cells QHJI strain (Center for iPS Cell Research and Application, Kyoto University) were thawed, and then iMatrix-511MG (Matrixome, Inc.) was added at 0.5 μg / cm ２ Coated 25cm ２ In a culture flask, 6000 cells / cm ２ and inoculated at 37°C and 5% CO ２Maintenance adherent culture was performed under a humid atmosphere. StemFit® AK03N (Ajinomoto Co.) medium was used, and the day the cells were seeded was designated culture day 0. The entire medium was replaced on culture days 1, 4, and 5. The medium volume was 5 mL on culture days 0, 4, and 5, and 10 mL on culture day 1 only. Y-27632 (Fujifilm Wako Pure Chemical Industries) was added to the medium to a final concentration of 10 μM only at the time of cell seeding, and LY333531 (Cayman) was added to a final concentration of 1 μM and IWR-1endo was added to a final concentration of 20 μM only on culture days 4 and 5.

[0081] On the sixth day of culture, TrypLE was added with 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) for subculture. ＴＭ The cells were treated with Select Enzyme (Life Technologies Japan, Inc.) for 15 minutes, and dispersed into single cells by pipetting while detaching the cells from the culture surface. The cells were suspended and collected in StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) containing Y-27632 at a final concentration of 10 μM.

[0082] A portion of the recovered cells was subjected to adherent subculture in StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) using iMatrix-511MG (Matrixome, Inc.) as a substrate by repeating the same procedure as the maintenance adherent culture and subsequent subculture described above. The cells recovered after adherent subculture were used for temperature stimulation in Comparative Example 1.

[0083] In addition, the cells dispersed into single cells and collected as described above were diluted to 0.5 μg / cm ２ Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Thermo Fisher Scientific) coated 300 cm ２ In a culture flask, 12,000 cells / cm ２ and inoculated at 37°C and 5% CO ２Adherent expansion culture was performed under a 500-μm atmosphere. StemFit® AK03N (Ajinomoto Co.) was used as the medium, and the entire medium was replaced on the second and third days of culture. The medium volume was 60 mL. Y-27632 (Fujifilm Wako Pure Chemical Industries) was added to the medium to a final concentration of 10 μM only at the time of cell seeding, and LY333531 (Cayman) was added to a final concentration of 1 μM and IWR-1endo was added to a final concentration of 20 μM only on the second and third days of culture. TrypLE containing 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries) was added on the fourth day of culture. ＴＭ The cells were treated with Select Enzyme (Life Technologies Japan, Inc.) for 3 minutes, detached from the culture surface by tapping, and dispersed into single cells by pipetting. The cells were suspended and collected in StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) containing Y-27632 at a final concentration of 10 μM.

[0084] (Production Example 2: Suspension Culture of Human iPS Cells) The cells recovered from adherent expansion culture in StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) in Production Example 1 were seeded and cultured in suspension using Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Thermo Fisher Scientific) as a substrate. First, a BioBlu 1c Single-Use Vessel (Eppendorf) was used as the culture vessel, and a Bioflo (Eppendorf) was used as the reactor system for controlling the culture.

[0085] The medium used for cell seeding was StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) supplemented with Y-27632 at a concentration of 10 μM and IWR-1 endo at a final concentration of 20 μM. The medium temperature at cell seeding was 32°C, the culture temperature during culture was 37°C, and the supply gas volume was maintained at 0.2 L / min, with aeration from the top of the culture solution. The supply gas was prepared by mixing an arbitrary amount of carbon dioxide gas with air. The pH sensor and medium perfusion pump installed in Bioflo were calibrated according to the method specified by the manufacturer. The total medium volume was 500 mL, and the cell density at the start of culture was 5.0 x 10 ４The carbon dioxide concentration in the supply gas was set to 5% at the start of the culture, and was adjusted to a range of 0% to 5% so as to maintain the pH of the culture solution at around 7.15 (to prevent a decrease in pH), and the pH was maintained at around 7.15±0.15.

[0086] The stirring speed of the reactor system was 87 rpm until 48 hours into the culture, and 79 rpm thereafter. The start of the culture was designated as time 0, and the perfusion of the medium was started at time 24. The perfusion rate F per unit time was controlled every hour to control the culture environment. The perfusion rate F per unit time at the start of the perfusion was ０ is the culture volume (500 mL) divided by 24 hours. ０ The time point at which the medium perfusion rate per unit time began to be changed was 64 hours into the culture, and the medium perfusion rate per unit time thereafter was determined by the formula 3 [F = M × K × F] described in WO 2022 / 203051. ０ × (C / C ０ where F is the perfusion rate per unit time; M is a correction coefficient; K is a variable that varies depending on the amount of lactate produced per cell by metabolism per unit time; F ０ is the medium perfusion rate per unit time at the start of perfusion rate control; C is the culture variable; C ０ indicates the culture variable at the start of perfusion control], and Equation 4 [K = L / L ０ where L is the amount of lactic acid produced by metabolism per cell per unit time; L ０ represents the amount of lactic acid produced per cell by metabolism per unit time at a certain point in time]. The culture variable was the cell density, and C ０ The value of the seeding density 5 × 10 ４ cells / mL, the percentage of cells forming aggregates at 24 hours of culture for the assumed seeding amount is 160%, and the assumed specific cell growth rate is 0.90 days. －１ The cell number at 64 hours after culture was calculated from the estimated cell density transition. C was also calculated from the estimated cell density transition. The constant M for correcting the influence of cell line, etc. was set to 1. The amount of lactic acid produced per cell by metabolism per unit time, L ０The amount of lactic acid produced by metabolism per cell per unit time at each culture time, L, was determined from the value of general pluripotent stem cells, and K (= L / L) of the formula 4 described in WO 2022 / 203051 was calculated. ０ ) was calculated, and the medium perfusion rate F per unit time was set from the formula 3 described in WO 2022 / 203051. Furthermore, as the perfusion medium, StemFit (registered trademark) AK03N (manufactured by Ajinomoto Co.) supplemented with Y-27632 at a final concentration of 2.5 μM, IWR-1 endo at a final concentration of 20 μM, and LY333531 at a final concentration of 1 μM was used. Furthermore, in order to remove the cell aggregates from the culture medium and aspirate only the medium, the medium was removed through a sintered wire mesh filter with a mesh size of 30 μm.

[0087] After 96 hours of culture, the entire suspension culture medium was collected, and the cell aggregates and culture supernatant were separated by centrifugation. ＴＭ The cells were treated with Select Enzyme (Life Technologies Japan, Inc.) for 5 minutes and dispersed into single cells by pipetting. The cells were then suspended in StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) and collected.

[0088] (Production Example 3: Suspension Subculture of Human iPS Cells) The cells cultured in suspension and collected in Production Example 2 were further subcultured in suspension. The culture volume was adjusted to 682 mL, and the seeding density was 7.5 × 10 ４ The amount of medium perfusion per unit time at the start of perfusion was F ０ The cells were subcultured and recovered in the same manner as in Production Example 2, except that the volume of the medium perfusion per unit time was set to 28.4 mL, the time point at which the medium perfusion rate per unit time began to be changed was 54 hours into the culture, and LY333531 was added to the medium at the time of seeding to a final concentration of 1 μM.

[0089] (Production Example 4: Suspension Subculture of Human iPS Cells) The cells cultured in suspension and collected in Production Example 3 were further subcultured in suspension. The culture volume was adjusted to 907 mL, and the seeding density was 1.0 × 10 ５ The amount of medium perfusion per unit time at the start of perfusion was F ０= 37.8 mL, the next point at which the medium perfusion rate per unit time was to be changed was 46 hours into the culture, and StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) supplemented with Y-27632 at a final concentration of 2.5 μM and LY333531 at a final concentration of 1 μM was used as the perfusion medium, and the cells were subcultured and recovered in the same manner as in Production Example 3, except that the volume of the medium perfusion rate per unit time was set to 37.8 mL, the next point at which the medium perfusion rate per unit time was to be changed was 46 hours into the culture, and StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) supplemented with Y-27632 at a final concentration of 2.5 μM and LY333531 at a final concentration of 1 μM was used as the perfusion medium.

[0090] (Example 1: Temperature stimulation of suspension human iPS cells) The cells collected from suspension culture in Production Example 4 were cultured in a STEM-CELLBANKER (Xenogen Pharma) cooled to about 4°C in advance, at a cell density of 5.0 × 10 ７ The cells were suspended in 39 mL of STEM-CELLBANKER solution to a concentration of 1000 cells / mL. The STEM-CELLBANKER solution containing the suspended cells was then dispensed into 39 1 mL aliquots using a pipette into NUNC cryotubes (Thermo Fisher Scientific) that had been kept cool on a cooling core (Corning Incorporated). The cells were then cooled to -80°C using a programmable freezer at a cooling rate of -1°C / min. The cells were then maintained at a low temperature of -196°C. After two months, the NUNC cryotubes containing the cell-suspended STEM-CELLBANKER solution were heated in a 37°C incubator for 2 minutes at a rate of approximately +83°C / min, and the cell suspension was then collected.

[0091] Comparative Example 1: Temperature Stimulation of Adherent Human iPS Cells Using the same procedure as in Example 1, the cells recovered from the adherent subculture in Production Example 1 were placed in a STEM-CELLBANKER (Xenogen Pharma) that had been cooled to about 4°C in advance, at a cell density of 1.0 × 10 ６ The cells were then dispensed into NUNC cryotubes at 1 mL each so as to achieve a concentration of 100 cells / mL. The subsequent cooling stimulation, low temperature maintenance, and heating stimulation were carried out in the same manner as in Example 1, and the cell suspension was collected.

[0092] Example 2: Counting the number of human iPS cells after thermal stimulation 10 mL of StemFit (registered trademark) AK02N minus C liquid medium (Ajinomoto Co., Inc.) containing 10 μM Y-27632 was transferred to a 15 mL centrifuge tube. The cells recovered after thermal stimulation in Example 1 were seeded into the medium in the 15 mL centrifuge tube. The NUNC cryotube was washed with StemFit (registered trademark) AK02N minus C liquid medium (Ajinomoto Co., Inc.) containing 10 μM Y-27632, and the cells remaining in the centrifuge tube were transferred. The centrifuge tube was centrifuged at 300 × g for 3 minutes, after which the entire supernatant was discarded, and the precipitated cells were resuspended and recovered in 10 mL of StemFit (registered trademark) AK02N minus C liquid medium (Ajinomoto Co., Inc.) containing 10 μM Y-27632. 160 μL of the homogenous cell suspension was sampled and appropriately diluted in a 1.5 mL tube, and the cell count of the resulting cell suspension was calculated using an NC200 (Chemometec).

[0093] Comparative Example 2: Counting the number of adherent human iPS cells after thermal stimulation After thermal stimulation in Comparative Example 1, the number of cells collected was calculated in the same manner as in Example 2.

[0094] (Comparative Example 3: Suspension culture of human iPS cells without temperature stimulation) The culture volume was 30 mL, and the cell density was 2 × 10 ５ The cells were cultured in a StemFit (registered trademark) AK03N culture vessel using Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Thermo Fisher Scientific) as a substrate in Preparation Example 1 to a concentration of 1000 cells / mL, and the collected cells were seeded in a culture vessel. The culture vessel was maintained at 37°C and 5% CO using an ABLE 30 mL Disposable Bioreactor (ABLE). ２The cells were cultured in suspension in an incubator under the specified conditions at an agitation speed of 100 rpm. The medium used at the time of seeding was StemFit® AK03N (Ajinomoto Co., Inc.) containing a final concentration of 10 μM Y-27632 and a final concentration of 20 μM IWR-1 endo. The day of cell seeding was designated day 0, and on the following day (culture day 1), the previous day's medium was completely replaced with StemFit® AK02N (Ajinomoto Co., Inc.) containing a final concentration of 5 μM Y-27632, a final concentration of 20 μM IWR-1 endo, and a final concentration of 1 μM LY333531. Furthermore, on the second day after (the second day of culture), the medium from the previous day was completely replaced with StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) containing 2.5 μM Y-27632, a final concentration of 20 μM IWR-1 endo, and a final concentration of 1 μM LY333531. On the third day of culture, cell aggregates were collected and used for the TrypLE ＴＭ The cells were treated with SELECT (Thermo Fisher Scientific) and spun into single cells by pipetting. A predetermined amount of cell suspension was prepared from the cells as a control without temperature stimulation, and used in the following study of suspension differentiation induction.

[0095] Comparative Example 4: Suspension culture of human iPS cells without temperature stimulation The cells collected in Comparative Example 3 were further passaged twice in suspension culture and used in the following investigation of induction of suspension differentiation.

[0096] (Evaluation Example 1: Quality Evaluation of Human iPS Cells from Production Example 1, Example 2, Comparative Example 2, Comparative Example 3, and Comparative Example 4) Of the cells collected in Production Example 1, Example 2, Comparative Example 2, Comparative Example 3, or Comparative Example 4, 4 × 10 ５The cell suspension containing 100 or more cells was transferred to a 1.5 mL tube, centrifuged, and the supernatant was discarded. 500 μL of TRIzol (Invitrogen) was added to the 1.5 mL tube to lyse the cells. RNA was extracted from the cell lysate using a PureLink® RNA Mini kit (Invitrogen) according to the manufacturer's instructions. The gene expression levels of Oct4 and Nanog, pluripotent stem cell markers, were quantitatively evaluated using quantitative PCR with RNA from cells induced to differentiate into suspension cells (Table 1). The viability (ratio of viable cells to the total number of cells collected) of the cells collected in Production Example 1, Example 2, Comparative Example 2, Comparative Example 3, or Comparative Example 4 was measured using an NC-200 (ChemoMetec) (Table 2).

[0097]

[0098]

[0099] From Tables 1 and 2, it was found that all cells had high gene expression of pluripotent stem cell markers Oct4 and Nanog, as well as high survival rates, and no difference was observed in the quality of human iPS cells.

[0100] (Example 3: Induction of floating differentiation from suspension human iPS cells to ectodermal differentiated cells after temperature stimulation) ６ The cell suspensions of the cells were seeded into the reactors at a medium temperature of 37°C, and the volume of the liquid in the reactor was adjusted to 5 mL with StemFit (registered trademark) AK02N minus C liquid medium containing 10 μM Y-27632, and the cell density was adjusted to 1.5 × 10 ６ The reactor was stirred at 45 rpm, at 37°C, and in 5% CO ２ Then, floating differentiation induction was initiated under the conditions.

[0101] The reactor stirring was stopped within 21 to 24 hours (day 1 after the start of differentiation induction), and after confirming the formation of aggregates, the entire cell suspension in the reactor was transferred to a 15 mL tube and centrifuged at 300 × g for 3 minutes. The entire supernatant was then removed and replaced with 5 mL of StemFitAK02 minus C medium containing 1 μM DMH1 (Fujifilm Wako Pure Chemical Industries, Ltd.) and 1 ng / mL SB431542 (Fujifilm Wako Pure Chemical Industries, Ltd.). The reactor was again stirred at 45 rpm, 37 °C, 5% CO ２ The induction of floating differentiation was continued for 2 days under the conditions.

[0102] Next, on days 3, 5, and 7 after the start of differentiation induction, the reactor stirring was stopped, and the entire cell suspension in the reactor was transferred to a 15 mL tube and centrifuged at 300 × g for 3 minutes. The supernatant was then removed and replaced with 5 mL of StemFitAK02 minus C medium containing 1 μM DMH1 (Fujifilm Wako Pure Chemical Industries, Ltd.) and 1 ng / mL SB431542 (Fujifilm Wako Pure Chemical Industries, Ltd.). The reactor was again stirred at 45 rpm, 37 °C, 5% CO. ２ The induction of floating differentiation was continued under the conditions.

[0103] On the 9th day after the start of differentiation induction, cells cultured in the reactor were transferred to a 15 mL centrifuge tube and centrifuged at 300 x g for 3 minutes. The supernatant was then discarded. 1 mL of Accutase (Innovative Cell Technologies) was added to the 15 mL centrifuge tube and allowed to stand in a 37°C water bath for 10 minutes. After the enzyme reaction, the aggregates were pipetted and the cells were dispersed into a single cell. Then, 1 mL of 10 μM Y-27632-containing StemFitAK02 minus C medium was added and centrifuged at 300 x g for 3 minutes. The entire supernatant was removed, and 3 mL of StemFitAK02 minus C medium containing 10 μM Y-27632 was added and pipetted. 200 μL of the homogenous cell suspension was collected in a 1.5 mL tube, and the cell number was calculated using NC200 (ChemoMetec).

[0104] (Comparative Example 5: Induction of suspension differentiation of adherent human iPS cells into ectodermal differentiated cells after temperature stimulation) Suspension culture differentiation induction was carried out using the same reagents and procedures as in Example 3, except that the cell suspension recovered in Comparative Example 2 was used as the cell suspension.

[0105] (Comparative Example 6: Induction of suspension differentiation from suspension human iPS cells to ectodermal differentiated cells without temperature stimulation) Suspension culture differentiation induction was carried out using the same reagents and procedures as in Example 3, except that the cell suspension recovered in Comparative Example 3 was used as the cell suspension.

[0106] (Evaluation Example 2: Comparison of Quality of Ectodermally Differentiated Cells Obtained in Example 3, Comparative Example 5, and Comparative Example 6) In order to confirm that differentiation was induced by the treatment in Example 3, 4 × 10 cells collected in Example 2 or Example 3 were cultured at 4 × 10 ５ The cell suspension containing the cells was transferred to a 1.5 mL tube, centrifuged, the supernatant discarded, and 500 μL of TRIzol (Invitrogen) was added to the 1.5 mL tube to lyse the cells. RNA was extracted from the cell lysate using a PureLink® RNA Mini kit (Invitrogen) according to the manufacturer's instructions. Using the RNA, quantitative PCR was used to confirm the gene expression levels of pluripotent stem cell markers Oct4 and Nanog, the ectodermal differentiation cell marker Pax6, the endodermal differentiation cell marker SOX17, and the mesodermal differentiation cell marker Brachyury.

[0107] Furthermore, the cells collected in Example 3, Comparative Example 5, or Comparative Example 6 were treated with TrypLE. ＴＭ After single cell treatment with Select Enzyme (Life Technologies Japan, Inc.), the Pax6 positivity rate of the cells induced to differentiate into floating cells was evaluated by FCM (flow cytometry) according to the following procedure.

[0108] First, fixation, permeabilization, and blocking were performed using eBioscience Foxp3 Transcription factor staining buffer set (Thermo Fisher Scientific). Then, cell samples were divided into 50 μL aliquots and resuspended in the buffer provided with the eBioscience Foxp3 Transcription factor staining buffer set (Thermo Fisher Scientific). Fluorescently labeled anti-Pax6 antibody was added and mixed. FMO control or ISOtype antibody was used. Staining was performed at 4°C for 1 hour in the dark. Table 3 shows the antibodies used and the amounts added.

[0109]

[0110] The results of the quantitative PCR are shown in Table 4.

[0111]

[0112] As can be seen from Table 4, it was confirmed by quantitative PCR that the cells recovered in Example 3 had lower gene expression levels for pluripotent stem cell markers Oct4 and Nanog than in Example 2, that the gene expression level for Pax6, an ectodermal differentiation cell marker, was increased, and that the gene expression levels for SOX17, an endodermal differentiation cell marker, and Brachyury, a mesodermal differentiation cell marker, were undetectable (1.0E-05), confirming that they had been induced to differentiate into ectodermal cells.

[0113] The Pax6 positivity rate of the cells recovered in Example 3, Comparative Example 5, or Comparative Example 6 was confirmed using a flow cytometer. The results are shown in Table 5.

[0114]

[0115] When the cells prepared in suspension culture and subjected to temperature stimulation in Example 3 were induced to differentiate into ectodermal cells, the Pax6 positivity rate was confirmed to be 49.1%, demonstrating that they were induced to differentiate into neural progenitor cell-like ectodermal cells. Furthermore, the Pax6 positivity rate of ectodermal cells induced to differentiate in Comparative Example 5 was 25.9%, demonstrating a significantly lower differentiation induction efficiency than in Example 3. In Comparative Example 6, the Pax6 positivity rate of cells induced to differentiate into ectodermal cells without temperature stimulation was 44.1%, which was lower than the result in Example 3, and the Pax6 positivity rate of the cells recovered in Example 3 was the most efficiently induced to differentiate.

[0116] These results demonstrate that even when there is no difference in the quality of iPS cells, the culture method can result in differences in the efficiency of differentiation induction. Furthermore, it was found that applying a temperature stimulus to suspension-cultured iPS cells further increases the efficiency of inducing differentiation into ectodermal cells compared to when no temperature stimulus is applied.

[0117] (Example 4: Induction of floating differentiation of human iPS cells into mesodermal differentiated cells (TnT-positive cells) after temperature stimulation) To induce differentiation, the human iPS cells obtained in Example 2 were seeded into a reactor at a medium temperature of 35°C. A 5 mL reactor (manufactured by ABLE) was used as the culture vessel, the culture medium volume was 5 mL, and the number of seeded cells was 1 x 10 ６ The culture conditions were 37°C, 5% CO ２ , 5% O ２ The cells were seeded in an incubator at a stirring speed of 56 rpm. The medium used for seeding was StemFit® AK02N minus C liquid medium supplemented with Y-27632 at a final concentration of 10 μM, L-ascorbic acid (Sigma) at a final concentration of 50 μg / mL, Glutamax (Thermo Fisher Scientific) at a final concentration of 2 mM, and BMP4 at a final concentration of 2 ng / mL.

[0118] The day the cells were seeded was designated day 0, and on the following day (culture day 1), the cells were cultured in StemFit (registered trademark) AK02N minus C liquid medium supplemented with L-ascorbic acid (Sigma) at a final concentration of 50 μg / mL, Glutamax (Thermo Fisher Scientific) at a final concentration of 2 mM, BMP4 (R&D Systems) at a final concentration of 10 ng / mL, Activin A (R&D Systems) at a final concentration of 6 ng / mL, and FGF (R&D Systems) at a final concentration of 5 ng / mL.

[0119] Furthermore, on day 3 of culture, the entire medium was replaced with StemFit (registered trademark) AK02N minus C liquid medium supplemented with L-ascorbic acid (Sigma) at a final concentration of 50 μg / mL, Glutamax (Thermo Fisher Scientific) at a final concentration of 2 mM, VEGF (Fujifilm Wako Pure Chemical Industries) at a final concentration of 10 ng / mL, SB431542 (Fujifilm Wako Pure Chemical Industries) at a final concentration of 2.1 μg / mL, Dorsomorphin (Fujifilm Wako Pure Chemical Industries) at a final concentration of 0.23 μg / mL, and IWP-3 (Stemgent) at a final concentration of 0.48 μg / mL.

[0120] Furthermore, on day 7 of culture, the entire medium was replaced with StemFit® AK02N minusC liquid medium supplemented with L-ascorbic acid at a final concentration of 50 μg / mL, Glutamax® (Thermo Fisher Scientific) at a final concentration of 2 mM, and VEGF at a final concentration of 5 ng / mL. From day 7 onwards, the entire medium was replaced every two or three days with a medium of the same composition (StemFit® AK02N minusC liquid medium supplemented with L-ascorbic acid (Sigma) at a final concentration of 50 μg / mL, Glutamax® (Thermo Fisher Scientific) at a final concentration of 2 mM, and VEGF (Fujifilm Wako Pure Chemical Industries) at a final concentration of 5 ng / mL).

[0121] Furthermore, from the 13th day of culture onwards, the cells were incubated at 37°C and 5% CO ２ The cells were cultured in an incubator under the following conditions. On day 15 of culture, the entire culture medium was collected from the culture vessel. The resulting cell aggregates were then enzymatically treated with Accumax (Innovative Cell Technologies) to dissociate them into single cells.

[0122] (Comparative Example 7: Induction of suspension differentiation of suspension human iPS cells into mesodermally differentiated cells (TnT-positive cells) without temperature stimulation) Suspension culture differentiation induction was carried out using the same reagents and procedures as in Example 4, except that the cell suspension recovered in Comparative Example 3 was used as the human iPS cells.

[0123] (Evaluation Example 3: Comparison of the quality of mesodermal differentiated cells (TnT positive cells) obtained in Example 4 and Comparative Example 7) The cells obtained in Example 4 or Comparative Example 7 were treated with TrypLE. ＴＭ After single cell treatment with Select Enzyme (Life Technologies Japan), the cells were fixed with 4% PFA and then suspended in PBS supplemented with 3% FBS. Fluorescently labeled anti-troponin T (TnT) antibody was added and mixed, and the mixture was allowed to stand at 4°C in the dark for 30 minutes. An isotype antibody was used as a negative control. The antibodies used and the amounts added are shown in Table 6.

[0124]

[0125] After washing once with 3% FBS (fetal bovine serum) / PBS, the cells were analyzed using a Guava easyCyte 8HT (Lumix). In the isotype control sample, a region was selected from the cell population extracted based on the FSC / SSC dot plot, where the proportion of cells with stronger fluorescence intensity was 0.5% or less. In the sample treated with anti-TnT antibody, the proportion of cells contained within this region was calculated from the cell population extracted based on the FSC / SSC dot plot, and this was taken as the proportion of TnT-positive cells. The results are shown in Table 7.

[0126]

[0127] The TnT positivity rate of the cells recovered in Example 4 was as high as 50.2%, while the TnT positivity rate of the cells recovered in Comparative Example 7 was 35.7%.

[0128] Even when the same differentiation induction protocol was applied, as can be seen from the results of Comparative Example 7 and Example 4, it was revealed that the efficiency of differentiation induction changes when iPS cells are subjected to a temperature stimulus, and it was found that the cells recovered in Example 2 can be induced to differentiate more efficiently than the cells recovered in Comparative Example 3.

[0129] From the above results, it was found that the efficiency of inducing differentiation of iPS cells is increased by applying a temperature stimulus.

[0130] (Example 5: Induction of floating differentiation of floating human iPS cells into mesodermal differentiated cells (CD31-positive cells) after temperature stimulation) ６ The cell suspension was seeded into the reactor at a medium temperature of 35°C, and the volume of the liquid in the reactor was adjusted to 5 mL with StemFit (registered trademark) AK02N minus C liquid medium containing 10 μM Y-27632, and the cell density was adjusted to 5.0 × 10 ５ The reactor was stirred at 85 rpm, at 37°C, and in 5% CO ２ Then, floating differentiation induction was initiated under the conditions.

[0131] Agitation of the reactor was stopped within 21 to 24 hours (1 day after the start of differentiation induction) and on the 3rd day after the start of differentiation induction. After confirming the formation of aggregates, the entire cell suspension in the reactor was transferred to a 15 mL tube and centrifuged at 300 × g for 3 minutes. The entire supernatant was then removed and replaced with 5 mL of StemFitAK02 minus C medium containing 1 μM CHIR99021 (Fujifilm Wako Pure Chemical Corporation) and 10 ng / mL BMP-4 (Fujifilm Wako Pure Chemical Corporation). The reactor was again agitated at 85 rpm, 37°C, 5% CO ２ The induction of floating differentiation was continued under the conditions.

[0132] On the fifth day after the start of differentiation induction, cells cultured in the reactor were transferred to a 15 mL centrifuge tube and centrifuged at 300 x g for 3 minutes. The supernatant was then discarded. 1 mL of Accutase (Innovative Cell Technologies) was added to the 15 mL centrifuge tube, and the tube was left to stand in a 37°C water bath for 10 minutes to allow the enzyme reaction to proceed. The aggregates were then pipetted to disperse the cells. Then, 1 mL of 10 μM Y-27632-containing StemFitAK02 minus C medium was added, and the tube was centrifuged at 300 x g for 3 minutes. Thereafter, the entire supernatant was removed, and 3 mL of StemFitAK02 minus C medium containing 10 μM Y-27632 was added and pipetted. 200 μL of the homogenous cell suspension was collected in a 1.5 mL tube, and the cell number was calculated using NC200 (manufactured by ChemoMetec).

[0133] (Evaluation Example 4: Quality Comparison of Mesodermally Differentiated Cells (CD31 Positive Cells) Obtained in Example 5) The cells recovered in Example 5 were cultured at 4 × 10 ５ The cell suspension containing the cells was transferred to a 1.5 mL tube, centrifuged, and the supernatant was discarded. 500 μL of TRIzol (Invitrogen) was added to the 1.5 mL tube to lyse the cells. RNA was extracted from the cell lysate using a PureLink® RNA Mini kit (Invitrogen) according to the manufacturer's instructions. The gene expression levels of CD31, a cardiovascular cell marker, in the cells induced to differentiate into suspension cells, were determined by quantitative PCR using the RNA (Table 8).

[0134]

[0135] As shown in Table 8, the cells recovered in Example 5 were cells induced to differentiate into mesodermal lines from the temperature-stimulated iPS cells prepared in Example 2, and quantitative PCR confirmed that they expressed CD31, a cardiovascular cell marker. These results demonstrate that even temperature-stimulated iPS cells can be induced to differentiate into CD31-positive cells.

[0136] (Example 6: Induction of floating differentiation of human iPS cells into endoderm-differentiated cells after temperature stimulation) ６The cell suspension was seeded into the reactor at a medium temperature of 35°C, and the volume of the liquid in the reactor was adjusted to 5 mL with StemFit (registered trademark) AK02N minus C liquid medium containing 10 μM Y-27632, and the cell density was adjusted to 1.5 × 10 ６ The reactor was stirred at 45 rpm, at 37°C, and in 5% CO ２ Then, floating differentiation induction was initiated under the conditions.

[0137] The reactor stirring was stopped within 21 to 24 hours (day 1 after the start of differentiation induction). After confirming the formation of aggregates, the entire cell suspension in the reactor was transferred to a 15 mL tube and centrifuged at 300 × g for 3 minutes. The supernatant was then removed and replaced with 5 mL of StemFitAK02 minus C medium containing 10 μM CHIR99021 (Fujifilm Wako Pure Chemical Industries, Ltd.) and 100 ng / mL Activin A (Fujifilm Wako Pure Chemical Industries, Ltd.). The reactor was again stirred at 45 rpm, 37 °C, 5% CO ２ The induction of floating differentiation was continued for 2 days under the conditions.

[0138] Next, on the third day after the start of differentiation induction, the reactor stirring was stopped, and the reactor was left to stand for 5 minutes. After that, 4.5 mL of the supernatant was removed, and 4.5 mL of StemFitAK02 minus C liquid medium containing 100 ng / mL Activin A (Fujifilm Wako Pure Chemical Industries, Ltd.) was added. The reactor was again stirred at 45 rpm, 37°C, 5% CO ２ The induction of floating differentiation was continued under the conditions.

[0139] On the fifth day after the start of differentiation induction, cells cultured in the reactor were transferred to a 15 mL centrifuge tube and centrifuged at 300 x g for 3 minutes. The supernatant was then discarded. 1 mL of Accutase (Innovative Cell Technologies) was added to the 15 mL centrifuge tube and allowed to stand in a 37°C water bath for 10 minutes to allow the enzyme reaction to occur. The aggregates were then pipetted to disperse the cells. Then, 1 mL of 10 μM Y-27632-containing StemFitAK02 minus C medium was added, and the mixture was centrifuged at 300 x g for 3 minutes. Thereafter, the entire supernatant was removed, and 3 mL of StemFitAK02 minus C medium containing 10 μM Y-27632 was added and pipetted. 200 μL of the homogenous cell suspension was collected in a 1.5 mL tube, and the cell number was calculated using NC200 (manufactured by ChemoMetec).

[0140] (Comparative Example 8: Induction of suspension differentiation from human iPS cells to endoderm-differentiated cells without temperature stimulation) Suspension culture differentiation induction was carried out using the same reagents and procedures as in Example 6, except that the cell suspension recovered in Comparative Example 4 was used as the cell suspension.

[0141] (Evaluation Example 5: Quality evaluation of endodermally differentiated cells obtained in Example 6 and Comparative Example 8) The cells collected in Example 6 or Comparative Example 8 were collected in a 4×10 ５ The cell suspension containing the cells was transferred to a 1.5 mL tube, centrifuged, and the supernatant was discarded. 500 μL of TRIzol (Invitrogen) was added to the 1.5 mL tube to lyse the cells. RNA was extracted from the cell lysate using a PureLink® RNA Mini kit (Invitrogen) according to the manufacturer's instructions. The RNA was used to quantitatively analyze the gene expression levels of Oct4, a pluripotent stem cell marker, and SOX17, an endoderm differentiation cell marker, in cells induced to differentiate into suspension cells (Table 9).

[0142]

[0143] From Table 9, it was confirmed that the cells recovered in Example 6 expressed higher levels of SOX17, an endoderm differentiation cell marker, than the cells recovered in Comparative Example 8. Furthermore, the cells recovered in Example 6 also expressed lower levels of Oct4, an undifferentiated cell marker, than the cells recovered in Comparative Example 8, confirming that the temperature-stimulated cells were induced to differentiate more efficiently.

[0144] These results demonstrate that applying a temperature stimulus to pluripotent stem cells obtained by suspension culture can induce differentiation with high quality and / or high efficiency.

[0145] Example 7: Temperature stimulation of suspension human iPS cells In the same manner as in Example 1, the cells that were cultured in suspension and collected in Production Example 4 were placed in a STEM-CELLBANKER (Xenogen Pharma) that had been cooled to about 4°C in advance, at a cell density of 2.0 × 10 ７ cells / mL, 2.0×10 ６ cells / mL, 2.0×10 ４ The cells were suspended to a concentration of 1000 cells / mL. The STEM-CELLBANKER solution containing the suspended cells was then dispensed in 1 mL aliquots using a pipette into NUNC cryotubes (Thermo Fisher Scientific) that had been kept cool on a cooling core (Corning). The cells were then cooled to -80°C using a programmable freezer at a cooling rate of -1°C / min. The cells were then maintained at a low temperature of -196°C. After two months, the NUNC cryotubes containing the cell-suspended STEM-CELLBANKER solution were heated in a 37°C incubator for 2 minutes at a rate of approximately +83°C / min, and the cell suspension was then collected.

[0146] (Evaluation Example 6: Measurement of the rate of aggregation of suspended human iPS cells subjected to temperature stimulation) The cells of Example 7 were placed in a 5 mL reactor (ABLE) at 2.0 × 10 ４ The cells were seeded at 1000 cells / mL and incubated at 37°C, 5% CO ２The suspension culture was performed at 100 rpm under a 100% RT atmosphere. The medium used at the time of seeding was StemFit (registered trademark) AK02N medium (Ajinomoto Co., Inc.) containing 10 μM Y-27632. The day the cells were seeded was designated as culture day 0, and on culture day 1, the entire culture medium was collected, and the cell aggregates and culture supernatant were separated by centrifugation. The cells were then treated with Accutase (Innovative Cell Technology, Inc.) for 10 minutes, and the cell aggregates were dispersed into single cells by pipetting. The cells were suspended in StemFit (registered trademark) AK02N medium containing 10 μM Y-27632, and the number of viable cells was counted using an NC-200 (MS Techno Systems Co., Ltd.). The aggregate formation rate (number of seeded cells (1.0 x 10)) was calculated. ５ The ratio of the number of cells surviving as aggregates on day 1 of culture to the number of cells surviving as aggregates on day 1 of culture was calculated.

[0147] The results are shown in Table 10.

[0148]

[0149] As shown in Table 10, 2.0 × 10 ７ cells / mL, 2.0×10 ６ The cells were subjected to a temperature stimulus by cooling and heating a suspension containing cells at 2.0 × 10 cells / mL. ４ The cell suspension containing cells at 2.0 × 10 cells / mL showed a higher rate of aggregate formation than the cells subjected to temperature stimulation by cooling or heating. ７ cells / mL and 2.0 x 10 ６ In the case of 0.1% cells / mL, the rate of aggregate formation exceeded 100%, and cells were already proliferating on the first day of culture after temperature stimulation. This suggests that aggregates can be formed more efficiently when the cell density is high when subjected to temperature stimulation. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A method for producing differentiated cells, comprising: (a) applying a temperature stimulus to pluripotent stem cells after suspension culture; (b) subsequently seeding the pluripotent stem cells after step (a) into a medium different from the medium used for the suspension culture; and (c) forming cell aggregates and inducing differentiation from the seeded pluripotent stem cells.

2. The method of claim 1, wherein step (a) comprises a freezing step.

3. The method of claim 1, wherein the temperature of the different culture media in step (b) is 32°C or higher and 38°C or lower.

4. The number of cells to be seeded in step (b) is 1 x 10 ６ The method according to claim 1 , wherein the number of the particles is 1 or more.

5. The method of claim 1, wherein step (c) is carried out without maintaining the pluripotent stem cells in an undifferentiated state.

6. In the step (c), the average number of cells in the aggregate is 1 x 10 ２ pcs or more, 1×10 ４ The method of claim 1, wherein the medium is changed at least once at a stage of 10 or less.

7. The method of claim 1, wherein the cell survival rate of the pluripotent stem cells after step (a) is 70% or higher.

8. The method of claim 1, wherein step (c) is a step of inducing suspension differentiation into endodermal cells.

9. The method of claim 1, wherein step (c) is a step of inducing floating differentiation into ectodermal cells.

10. The method of claim 1, wherein step (c) is a step of inducing floating differentiation into mesodermal cells.

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