Culture medium composition and culture method

Abstract

The present invention provides an improved culture medium composition that can be used for culturing mammalian pluripotent stem cells, and preferably can be used for establishing and maintaining mammalian pluripotent stem cells useful for the production of cultured meats such as beef and pork, and a culture method using the same. A culture method according to the present invention includes a culture medium composition for a mammalian pluripotent stem cell culture that is a serum-free culture medium containing (1) a CDK8 / 19 inhibitor, (2) a ROCK inhibitor, (3) an SMAD activator, and (4) a STAT3 activator and includes a step for culturing mammalian pluripotent stem cells in the culture medium composition.

Description

Medium composition and culture method

[0001] The present invention relates to a medium composition for culturing mammalian pluripotent stem cells (embryonic stem cells, induced pluripotent stem cells, etc.), and a method for culturing mammalian pluripotent stem cells using the same.

[0002] In recent years, the demand for meat has been increasing rapidly due to global population growth and economic growth, and research and development of cultured meat is being conducted as one way to meet this demand. Cultured meat can be produced by artificially culturing muscle cells, fat cells, etc. contained in edible meat from cows, pigs, etc., and giving them a three-dimensional structure resembling muscle tissue and / or fat tissue. The muscle cells, fat cells, etc. required for this can be produced from stem cells that have the ability to differentiate into these cells, such as embryonic stem cells (ES cells) derived from cows, etc.

[0003] Methods for producing and culturing pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, derived from bovine or other mammalian animals, and medium compositions used for these methods are known, for example, from the following patent documents:

[0004] Patent Document 1 describes a method for producing a formative stem cell line from a pluripotent stem cell precursor, which includes, for example, culturing currently pluripotent stem cell precursors in a medium containing less than 5 ng / mL of activin (SMAD activator), less than 10 ng / mL of exogenous FGF, a Wnt inhibitor, and a retinoic acid receptor inhibitor, and not containing serum or a serum substitute (claim 1), or in a medium containing 1 to 4 ng / mL of activin, less than 10 ng / mL of exogenous FGF, and a Wnt inhibitor (claim 2). Patent Document 1 describes that formative stem cell lines derived from humans and various non-human mammals, as well as birds, can be produced, but the examples disclosed only involve the production of formative stem cells or formative-induced pluripotent stem cells derived from mice, humans, or pigs, and does not disclose those derived from bovines.

[0005] Patent Document 2 describes a method for inducing naive pluripotent stem cells (a method for reverting primed pluripotent stem cells to naive pluripotent stem cells) or a method for maintaining naive pluripotent stem cells in their naive state, which comprises culturing these predetermined cells in a culture medium containing an effective amount of a lysophosphatidic acid receptor (LPAR) agonist and which may further contain a STAT3 activator (e.g., LIF), an ERK1 / 2 inhibitor, a GSK3β inhibitor, a PKC inhibitor, activin A (SMAD activator), TGF-β, bFGF, etc. Patent Document 2 describes that cells derived from humans or various mammals other than humans can be used, but the examples disclosed are embryonic stem cells or induced pluripotent stem cells derived from mice or humans, and no cells derived from bovines are disclosed.

[0006] Patent Document 3 describes a method for producing embryonic stem cells from ungulates (such as cattle), which comprises culturing ungulate blastocysts and pluripotent stem cells established therefrom in a medium (e.g., serum-free medium) containing inactivated feeder cells, FGF2, and a Wnt inhibitor, and which may further contain a ROCK inhibitor, activin A (an SMAD activator), etc. Patent Document 3 describes that such a method can yield stable ungulate embryonic stem cells that proliferate for a long period of time while maintaining stable cell morphology and a normal karyotype, have transcriptomes and epigenomes that indicate pluripotency, and are capable of forming teratomas containing three germ layers.

[0007] Patent Document 4 describes a method for producing expanded potential stem cells (EPSCs), which comprises culturing pluripotent cells in a medium containing two or more selected from a Ras-ERK inhibitor, an SKF (Src Kinase family) inhibitor, a GSK3 inhibitor, and a Wnt inhibitor, and which may further contain a JNK (Jun N-Terminal Kinase) inhibitor, a p38 inhibitor, a ROCK inhibitor, LIF, activin (SMAD activator), etc. EPSCs are said to resemble naive embryonic stem cells and to be capable of differentiating into three germ layers, including the placenta. Patent Document 4 describes the production of EPSCs derived from humans or various non-human mammals, but the examples disclosed only involve EPSCs produced from mouse-, human-, or porcine-derived embryonic stem cells or induced pluripotent stem cells, and does not disclose bovine-derived EPSCs.

[0008] Patent Document 5 describes a cell culture medium composition for extending the cell potential of pluripotent stem cells, in other words, for obtaining chemically induced extended pluripotent stem cells (ciEPSCs), which contains a cytokine (e.g., LIF), a GSK inhibitor, a GPCR inhibitor, and a PARP1 inhibitor, and may optionally further contain a ROCK inhibitor, end-IWR1, or XAV939 (an Axin stabilizer). Patent Document 5 describes that ciEPSCs derived from any mammal can be produced, but the examples disclosed are those in which ciEPSCs were produced from embryonic stem cells or induced pluripotent stem cells derived from mice, humans, or pigs, and does not disclose bovine ciEPSCs.

[0009] Patent Document 6 describes a culture medium that can be used for culturing pluripotent stem cells and the like, which contains a STAT3 activator, an ERK1 / 2 inhibitor, and an Axin stabilizer, and may further contain a PKC inhibitor, a ROCK inhibitor, an SRC inhibitor, a GSK3b inhibitor, an SMAD activator, etc. Patent Document 6 describes that embryonic stem cells or induced pluripotent stem cells derived from various mammals can be used as pluripotent stem cells, but the examples disclosed are culturing human-derived embryonic stem cells or induced pluripotent stem cells in the above medium, and transplanting the pluripotent stem cells obtained thereby into a host mouse embryo to form chimeras, but does not disclose the culturing of bovine-derived pluripotent stem cells.

[0010] Patent Document 7 describes a method for inducing, maintaining, or passage of pluripotent stem cells, which does not use an MEK inhibitor, but comprises culturing pluripotent stem cells in the presence of at least one of a TNKS (tankyrase) inhibitor, a PKC (protein kinase C) inhibitor, and a ROCK inhibitor, as well as a RAF inhibitor, an FGFR1 inhibitor, and an ERK inhibitor (claim 1); and a method for inducing the resetting of pluripotent stem cells from a primed type to a naive type, which comprises culturing primed pluripotent stem cells in a medium containing a MEK inhibitor, a TNKS inhibitor, a PKC inhibitor, an ERK inhibitor, a ROCK inhibitor, activin A (a SMAD activator), LIF, a GSK3 inhibitor, and an Src inhibitor (claim 13). Patent Document 7 describes that embryonic stem cells or induced pluripotent stem cells derived from non-human animals or humans can be induced from primed to naive types, but the examples disclosed are embryonic stem cells derived from humans, and bovine embryonic stem cells are not disclosed.

[0011] WO2018 / 138281, Patent Publication No. 2018-514220 (corresponding to WO2016 / 179243), WO2019 / 140260, WO2016 / 079146, Patent Publication No. 2018-527024 (corresponding to WO2017 / 025061), Patent Publication No. 2017-525351 (corresponding to WO2016 / 016894), US2022 / 0364060

[0012] Pluripotent stem cells such as embryonic stem cells derived from bovine or other animals, which are useful for producing cultured meat, have been difficult to produce even using the production methods, culture methods, medium compositions, etc. described in the prior art documents mentioned above. For example, the invention described in Patent Document 3 relates to a method for producing embryonic stem cells from ungulates (bovine or other animals), but even with this method, it is not easy to produce embryonic stem cells from bovine or other animals, and improvements have been desired.

[0013] An objective of the present invention is to provide an improved medium composition that can be used to culture mammalian embryonic stem cells, preferably to produce mammalian pluripotent stem cells (e.g., embryonic stem cells) useful for producing cultured meat from cattle, pigs, etc., specifically to establish and maintain cell lines, and a culture method using the same.

[0014] The present inventors discovered that the efficiency of establishing bovine embryonic stem cells is improved by using a serum-free medium containing at least a CDK8 / 19 inhibitor, a ROCK inhibitor, an SMAD activator, and a STAT3 activator, and that such a serum-free medium may be usable for establishing embryonic stem cells, induced pluripotent stem cells, etc. not only from bovine but also from porcine and other mammalian sources, and have arrived at the present invention.

[0015] That is, in one aspect, the present invention provides the following medium composition and culture method. [Item 1] A medium composition for culturing mammalian pluripotent stem cells, which is a serum-free medium containing (1) a CDK8 / 19 inhibitor, (2) a ROCK inhibitor, (3) an SMAD activator, and (4) a STAT3 activator. [Item 2] The medium composition of Item 1, wherein the serum-free medium further contains (5) an Src inhibitor. [Item 3] The medium composition of Item 1 or 2, wherein the serum-free medium further contains (6) a GSK3 inhibitor. [Item 4] The medium composition of any one of Items 1 to 3, wherein the serum-free medium does not contain (7) a Wnt inhibitor. [Item 5] The medium composition of any one of Items 1 to 4, wherein the mammal is an Artiodactyla. [Item 6] The medium composition of Item 5, wherein the Artiodactyla is a bovine. [Item 7] The medium composition of any one of Items 1 to 6, wherein the pluripotent stem cells are embryo-derived stem cells, including embryonic stem cells. [Item 8] A culture method comprising the step of culturing mammalian pluripotent stem cells in the medium composition according to any one of Items 1 to 7.

[0016] The medium composition and culture method according to the present invention improve the efficiency of establishing embryonic stem cells from bovine and other mammals, and make it possible to establish and maintain embryonic stem cells derived from desired individuals.

[0017] Optical microscope images of cultured cells in step (2.4) of Example [1] (Establishment of bovine embryonic stem cells). [A] When medium 1 was used, [B] When medium 2 was used, [C] When medium 3 was used. Immunostained images showing desmin expression in skeletal muscle cells obtained by Example [2] (Induction of skeletal muscle cell differentiation). [A] DAPI-stained image, [B] Desmin-stained image, [C] DAPI / desmin-stained image. Immunostained images showing MYH1 expression in skeletal muscle cells obtained by Example [2] (Induction of skeletal muscle cell differentiation). [A] DAPI-stained image, [B] MYH1-stained image, [C] DAPI / MYH1-stained image.

[0018] -Culture Medium Composition- The culture medium composition of the present invention is for culturing mammalian pluripotent stem cells, and is a serum-free medium containing at least (1) a CDK8 / 19 inhibitor, (2) a ROCK inhibitor, (3) a STAT3 activator, and (4) an SMAD activator, each in an effective amount.

[0019] "Mammals" include humans and non-human mammals. Non-human mammals include, for example, Primates such as chimpanzees, gorillas (Hominidae), orangutans (Pongoidae), and monkeys (Cercopithecidae); Rodents such as mice and rats (Muridae); Artiodactyla such as pigs (Suidae), cattle, sheep, and goats (Bovidae); Perissodactyla such as horses (Equidae); and Carnivora such as dogs (Canidae) and cats (Felidae). In the present invention, "mammals" are preferably mammals of the Artiodactyla order such as pigs (Suidae), cattle, sheep, and goats (Bovidae), more preferably pigs and cattle, and particularly preferably cattle.

[0020] As a term well known to those skilled in the art, "pluripotent stem cells" refers to cells that have the ability to self-proliferate and pluripotency (the ability to differentiate into all three germ layers: endoderm, mesoderm, and ectoderm). Pluripotent stem cells include, for example, embryonic stem cells (ES cells, etc.), induced pluripotent stem cells (iPS cells, etc.), embryonic germ stem cells (EG cells), and other cells that can be collected from living organisms or cells that can be prepared therefrom. Pluripotent stem cells may be in a naive state or a primed state (such as common ES cells and iPS cells). Whether a cell is a "pluripotent stem cell" can be confirmed by known techniques, for example, by determining whether the cell has at least one of the characteristics of a "pluripotent stem cell," such as the ability to self-proliferate (colony-forming ability) or the ability to differentiate pluripotently.

[0021] Serum-free medium: As the "serum-free medium" of the present invention, various serum-free media known in the technical fields related to cell culture can be used. Such serum-free media may be commercially available or may be prepared by adding appropriate components to a basal medium.

[0022] Examples of the basal medium include DMEM (Dulbecco's modified Eagle's medium), αMEM (α-modified Eagle's minimum essential medium), Ham's F-10 medium, Ham's F-12 medium, and mixtures thereof.

[0023] In one embodiment of the present invention, the serum-free medium is "mTeSR™1" (STEMCELL Technologies, Basal Medium + 5X Supplement) or "ON2 / AscleStem PSC medium" (DOI: 10.1186 / s13287-022-02879-z).

[0024] (1) CDK8 / 19 Inhibitors "CDK (Cyclin-Dependent Kinase) 8 / 19 inhibitors" are drugs that selectively inhibit the function of CDK8 and its isoform CDK19. These drugs are known in the art related to cell culture, and similar drugs can be used in the present invention. Examples of CDK8 / 19 inhibitors include, but are not limited to, ETP-47799 (CAS No. 923596-52-5), AS2863619 (CAS No. 2241300-51-4), and SEL120 (SEL120-34A) hydrochloride (CAS No. 1609452-30-3). The type and concentration of the drug used as the CDK8 / 19 inhibitor can be appropriately selected and set while taking into account the effects of the present invention.

[0025] In one embodiment of the present invention, the medium composition of the present invention contains ETP-47799 as a CDK8 / 19 inhibitor. The concentration of ETP-47799 in the medium composition can be adjusted as appropriate, but can be, for example, 0.01 to 10 μM, and preferably 0.1 to 2.0 μM.

[0026] (2) ROCK Inhibitors "ROCK (Rho-associated protein kinase) inhibitors" are known drugs in the technical field related to cell culture, and similar drugs can be used in the present invention. Examples of ROCK inhibitors include, but are not limited to, Y-27632 (CAS No. 146986-50-7), thiazovivin (CAS No. 1226056-71-8), and GSK429286A (CAS No. 864082-47-3). The type and concentration of the drug used as the ROCK inhibitor can be appropriately selected and set taking into consideration the effects of the present invention.

[0027] In one embodiment of the present invention, the medium composition of the present invention contains Y-27632 as a ROCK inhibitor. The concentration of Y-27632 in the medium composition can be adjusted as appropriate, but can be, for example, 0.1 to 100 μM, and preferably 1 to 50 μM.

[0028] (3) STAT3 Activators "STAT (Signal Transducer and Activator of Transcription) 3 activators" are known drugs in the technical field related to cell culture (see, for example, Patent Document 6), and similar drugs can be used in the present invention. Examples of STAT3 activators include, but are not limited to, interferon (IFN), epidermal growth factor (EGF), interleukin 5 (IL5), interleukin 6 (IL6), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), and bone morphogenetic protein 2 (BMP2). The type and concentration of the drug used as the STAT3 activator can be appropriately selected and set taking into consideration the effects of the present invention, etc.

[0029] In one embodiment of the present invention, the medium composition of the present invention contains LIF as a STAT3 activator. The concentration of LIF in the medium composition can be adjusted as appropriate, but can be, for example, 1 to 200 ng / mL, and preferably 3 to 50 ng / mL.

[0030] (4) SMAD activator "SMAD activator" is a drug known in the technical field related to cell culture (see, for example, Patent Document 6), and similar drugs can be used in the present invention. Examples of SMAD activators include, but are not limited to, activin A, TGFβ1, and bone morphogenetic protein 4 (BMP4). The type and concentration of the drug used as the SMAD activator can be appropriately selected and set taking into consideration the effects of the present invention, etc.

[0031] In one embodiment of the present invention, the medium composition of the present invention contains, as an SMAD activator, activin A. The concentration of activin A in the medium composition can be adjusted as appropriate, but can be, for example, 0.1 to 200 ng / mL, and preferably 5 to 100 ng / mL.

[0032] The medium composition of the present invention may further contain components other than the components (1) to (4) in the serum-free medium. For example, the serum-free medium may further contain components such as glutathione, L-glutamine, lipids, thiamine, β-mercaptoethanol, BSA, pipecolic acid, GABA, bFGF, and L-ascorbic acid. The types and concentrations of these additional components can be appropriately selected and set taking into consideration the effects of the present invention.

[0033] The serum-free medium product "mTeSR1" contains glutathione, L-glutamine, lipids, thiamine, trace elements B and C, β-mercaptoethanol, BSA, pipecolic acid, LiCl (a GSK3 inhibitor), GABA, bFGF, and TGFβ1 (a SMAD activator), among others. One or more of these components may be added as needed. For example, β-mercaptoethanol may be added to "mTeSR1" to a concentration of 100 mM. Furthermore, while "mTeSR1" itself contains LiCl and TGFβ1, these components may be further added to adjust the concentration within the preferred ranges described herein.

[0034] The medium composition of the present invention, containing the above-mentioned components (1) to (4), exhibits the desired effect even without containing the (5) Src inhibitor and the (6) GSK3 inhibitor. However, if necessary, in addition to the above-mentioned components (1) to (4), the (5) SFK inhibitor may be further added, or the (5) Src inhibitor and the (6) GSK3 inhibitor may be further added.

[0035] (5) Src Inhibitors "Src inhibitors" are drugs known in the art related to cell culture, and similar drugs can be used in the present invention. Src inhibitors may be drugs known as "SFK (Src Family Kinase) inhibitors," "Lck / Src inhibitors," etc. Examples of Src inhibitors include, but are not limited to, WH-4-023 (CAS No. 837422-57-8), bosutinib (also known as SKI-606, CAS No. 380843-75-4), dasatinib (also known as BMS-354825, CAS No. 302962-49-8), and saracatinib (also known as AZD0530, CAS No. 379231-04-6). The type and concentration of the drug used as the Src inhibitor can be appropriately selected and set taking into consideration the effects of the present invention.

[0036] In one embodiment of the present invention, the medium composition of the present invention contains WH-4-023 as an Src inhibitor. The concentration of WH-4-023 in the medium composition can be adjusted as appropriate, but can be, for example, 0.1 to 20 μM, and preferably 0.1 to 5 μM.

[0037] (6) GSK3 Inhibitors "GSK (Glycogen synthase kinase) 3 inhibitors" are drugs known in the technical field related to cell culture, and similar drugs can be used in the present invention. The GSK3 inhibitor may be a drug that inhibits the functions of GSK-3α and GSK-3β (GSK-3α / β inhibitor), or a drug that selectively inhibits the function of GSK-3β (GSK-3β inhibitor). Examples of GSK3 inhibitors include, but are not limited to, CHIR99021 (CAS No. 252917-06-9), CHIR98014 (CAS No. 252935-94-7), BIO (CAS No. 667463-62-9), SB216763 (CAS No. 280744-09-4), LY2090314 (CAS No. 603288-22-8), and LiCl (lithium chloride). The type and concentration of the drug used as the GSK3 inhibitor can be appropriately selected and set while taking into consideration the effects of the present invention.

[0038] In one embodiment of the present invention, the medium composition of the present invention contains CHIR99021 as a GSK3 inhibitor. The concentration of CHIR99021 in the medium composition can be adjusted as appropriate, but can be, for example, 0.1 to 20 μM, and preferably 0.3 to 10 μM.

[0039] The medium composition of the present invention contains the above-mentioned components (1) to (4), and thereby exhibits the desired effect even without containing (7) a Wnt inhibitor. In one embodiment of the present invention, the medium composition of the present invention does not contain (7) a Wnt inhibitor, which is not necessary.

[0040] (7) Wnt Inhibitors "Wnt inhibitors" are drugs known in the art related to cell culture, and similar drugs can be used in the present invention. Wnt inhibitors may be drugs known as "tankyrase inhibitors," "axin activators," "axin-β-catenin complex stabilizers," etc. Examples of Wnt inhibitors include, but are not limited to, XAV939 (CAS No. 284028-89-3), IWR-1 (CAS No. 1127442-82-3), and IWP-2 (CAS No. 686770-61-6).

[0041] -Culturing Method- The culturing method of the present invention comprises the step of culturing mammalian pluripotent stem cells in a medium composition for culturing mammalian pluripotent stem cells according to the present invention, i.e., a serum-free medium supplemented with at least (1) a CDK8 / 19 inhibitor, (2) a ROCK inhibitor, (3) a STAT3 activator, and (4) an SMAD activator.

[0042] For details (specific examples, embodiments, etc.) of the medium composition used in the culture method of the present invention, please refer to the items described in this specification in relation to the medium composition of the present invention.

[0043] Known methods can be applied to culture mammalian pluripotent stem cells. For example, embryonic stem cells can generally be produced by recovering the inner cell mass (ICM) from a late-stage blastocyst of a donor mammal and culturing it in an appropriate medium. In addition, induced pluripotent stem cells can generally be produced by introducing a predetermined gene (e.g., the four classical genes Oct3 / 4, Sox2, Klf4, and c-Myc, or a combination of improved genes) into donor mammalian somatic cells (e.g., fibroblasts) using a retroviral vector or other vector or plasmid, or by introducing a predetermined compound, followed by culturing in an appropriate medium. The culture method of the present invention can also be basically carried out in accordance with such known methods, adjusting the procedures, conditions, etc. as necessary.

[0044] In one embodiment of the present invention, the culture method of the present invention comprises culturing the inner cell mass and proliferated embryonic stem cells in the medium composition of the present invention from seeding the recovered inner cell mass in the medium composition of the present invention until the first passage is performed. In this embodiment, the medium composition of the present invention can be replaced at appropriate times and intervals (e.g., every other day after 3 days). Furthermore, in the culture method of this embodiment, the medium composition of the present invention can be modified to an embodiment that does not contain a ROCK inhibitor. The culture period of the culture method of this embodiment can be, for example, 3 to 10 days.

[0045] In one embodiment of the present invention, the culture method of the present invention comprises culturing embryonic stem cells (typically obtained by the above-described embodiment) in the medium composition of the present invention for a desired number of passages. In this embodiment, the medium composition of the present invention can be replaced at appropriate times and intervals (e.g., daily). The culture period of the culture method of this embodiment can be, for example, the period from the appearance of embryonic stem cell colonies and the establishment of embryonic stem cells to the proliferation of the desired cell number.

[0046] In another embodiment of the present invention, the culture method of the present invention includes temporarily maintaining the blastocyst or the like in the medium composition of the present invention while treating the blastocyst with a protease (e.g., pronase) to remove the zona pellucida in order to recover the ICM.

[0047] The uses of pluripotent stem cells established and maintained according to the present invention are not particularly limited and can be used for a variety of purposes, but in a preferred embodiment of the present invention, they are used to obtain cells useful for the production of cultured meat. That is, to produce cultured meat, pluripotent stem cells established and maintained according to the present invention can be used to obtain cells that make up tissues contained in meat, such as skeletal muscle, smooth muscle, cardiac muscle, fat, blood vessels, and blood, i.e., muscle cells, adipocytes, vascular endothelial cells, vascular wall cells, blood cells, etc., or stem cells or precursor cells that can induce these. Methods for inducing the above-mentioned muscle cells, adipocytes, etc. from pluripotent stem cells are known, and known methods can also be applied to the present invention.

[0048] [1] Establishment of bovine embryonic stem cell lines Bovine embryonic stem cells were established and maintained according to the following procedure. 1. Outgrowth (1.1) A 4-well plate seeded with mouse embryonic fibroblasts (MEF cells) or bovine-derived cells was removed from the incubator and seeded with inner cell masses recovered from bovine late blastocysts. (1.2) ROCK inhibitor "Y-27632" (Wako) was added to the medium, and the plate was incubated at 38.5°C and 5% CO. ２ Placed in an incubator.

[0049] 2. Establishment of bovine embryonic stem cells (2.1) The cultured cells as described above were observed for three days without changing the medium, and then the medium was changed with one of the following three types of medium for bovine embryonic stem cells (containing no ROCK inhibitor "Y-27632"):

[0050]

[0051] (2.2) The medium was changed every other day with the bovine embryonic stem cell medium described above, and the cells were cultured until confluent. (2.3) After about 10 days, the cells grew to confluence. Single cells were harvested using "TrypLE select" (Gibco) and then seeded into the bovine embryonic stem cell medium described above supplemented with the ROCK inhibitor. The cells were passaged at a 1:1 ratio from one 4-well plate to another. (2.4) After two days, colonies appeared, confirming the establishment of bovine embryonic stem cells. (2.5) After the establishment of bovine embryonic stem cells, the medium was changed daily and the cells were passaged at a ratio of 1:5 to 1:30 every 2 to 5 days.

[0052] 3. Cryopreservation (3.1) Bovine embryonic stem cells were prepared in a 10 cm dish at a confluence of 70% or more. (3.2) After washing twice with PBS, "TrypLE select" (Gibco) was added and the cells were cryopreserved at 38°C and 5% CO. ２ (3.3) After confirming cell detachment, the above-mentioned pluripotent stem cell medium was added, and the mixture was centrifuged at 1000 rpm for 3 minutes. (3.4) After centrifugation, 1 x 10 ６The cells were suspended in a cryopreservation solution so that there were one cell per tube, and stored overnight at −80° C. (3.5) The next day, the cells were transferred to a liquid nitrogen tank and stored in the liquid phase at −196° C.

[0053] The images observed with an optical microscope in the above step (2.5) are shown in Figure 1. It can be seen that colonies were formed in all of the above media 1 to 3, and bovine embryonic stem cells were successfully established.

[0054] [2] Induction of Differentiation into Skeletal Muscle Cells The bovine embryonic stem cells prepared in step 2 of [1] above were induced to differentiate into skeletal muscle cells according to the following procedure. (1) Bovine embryonic stem cells at 70% confluence or higher were prepared. (2) Treatment with the cell detachment enzyme "Accutase" (Innovative Cell Technologies, Inc.) was carried out at 38.5°C for 5 minutes. (3) The cells were collected in skeletal muscle induction medium, centrifuged at 300g for 4 minutes, and the number of cells was counted. (4) The cells collected in (3) above were collected at a concentration of 5 x 10 ３ cells / cm ２ The cells were seeded in the skeletal muscle induction medium to a density of 5 × 10. (5) The cells were cultured for 6 to 10 days in the skeletal muscle induction medium used in (3) above, with the medium replaced every two days. (6) The cells were treated with 0.05% trypsin at 38.5°C for 5 minutes. (7) The cells were recovered in a medium containing 10% serum and centrifuged at 300 g for 4 minutes. (8) The cells recovered in (7) above were cultured at a density of 5 × 10 ３ cells / cm ２ The cells were seeded onto type I collagen-coated plates at a density of 1000 x g. (9) The cells were cultured for 6 to 8 days in the serum-containing medium used in (7) above, with medium changes every 2 days. (10) The medium was switched to "Myotube Medium" or "Myotube Fusion Medium" (Myocea, Inc.), with medium changes every 3 to 4 days.

[0055] The cells cultured according to the above procedure were immunostained according to the following procedure and observed under a fluorescence microscope. (1) The cultured cells were washed twice with PBS and then fixed with 4% paraformaldehyde (PFA) at room temperature for 10 minutes. (2) The cells treated in (1) above were washed twice with PBS and then treated with 0.1% Triton-X100 (Sigma-Aldrich). (3) The cells treated in (2) above were washed twice with PBS and then blocked with 5% normal goat serum at room temperature for 30 minutes. (4) Immunostaining was performed for 1 hour at room temperature using anti-desmin mouse monoclonal antibody (DAKO "IR606", undiluted) or anti-MYH1 rabbit monoclonal antibody (PGI "25182-1-AP", 1 / 50 dilution) as the primary antibody. (5) The cells treated in (4) above were washed three times with PBS (5 minutes each time), and then immunostained for 30 minutes at room temperature using Alexa488-labeled goat anti-mouse IgG antibody ("A11001", 1 / 500 dilution) or Alexa488-labeled goat anti-rabbit IgG antibody ("A32731", 1 / 500 dilution) as the secondary antibody. (6) The cells treated in (4) above were washed three times with PBS (5 minutes each time), and then mounted in "Mounting Medium With DAPI (AB104139)."

[0056] Fluorescence microscopic images of cells immunostained by the above procedure are shown in Figure 2. It can be seen that the above procedure resulted in a sufficient number of skeletal muscle cells expressing Desmin and MYH1.

Claims

1. A medium composition for culturing mammalian pluripotent stem cells, which is a serum-free medium containing: (1) a CDK8 / 19 inhibitor; (2) a ROCK inhibitor; (3) a SMAD activator; and (4) a STAT3 activator.

2. The medium composition according to claim 1, wherein the serum-free medium further contains (5) an Src inhibitor.

3. The medium composition according to claim 2, wherein the serum-free medium further contains (6) a GSK3 inhibitor.

4. The medium composition according to claim 1, wherein the serum-free medium does not contain (7) a Wnt inhibitor.

5. The medium composition of claim 1, wherein the mammal is an artiodactyl.

6. The medium composition according to claim 5, wherein the ungulate is a bovine.

7. The medium composition of claim 1, wherein the pluripotent stem cells are stem cells derived from an embryo, including embryonic stem cells.

8. A culture method comprising a step of culturing mammalian pluripotent stem cells in a medium composition according to any one of claims 1 to 7.

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