JP2018082639A - Method for producing induced pluripotent stem cell from somatic cell, and medium for culturing induced pluripotent stem cell in undifferentiated state - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing an induced pluripotent stem cell from a somatic cell, conveniently and inexpensively, without using a feeder cell.SOLUTION: A method for producing an induced pluripotent stem cell from a somatic cell has a culture step in which: a nucleus initialization factor is introduced into a somatic cell, and cultured in a low oxygen environment without using a feeder cell. In the culture step, used is a medium containing at least one inorganic salt, at least one amino acid, at least one vitamin, D-glucose, sodium phenol red, sodium pyruvate, antibiotic, and serum.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing induced pluripotent stem cells from somatic cells and a medium for culturing induced pluripotent stem cells in an undifferentiated state.

  By introducing a very small number of factors into a somatic cell and culturing it, it is transformed into a pluripotent stem cell having the ability to differentiate into cells of various tissues and organs and the ability to proliferate almost infinitely. This cell is referred to as an “induced pluripotent stem cell” and is expressed as “induced pluripotent stem cell” in English. For this reason, the acronym is called “iPS cell”.

  In 2006, Professor Yamanaka of Kyoto University found four genes (Oct3 / 4, Sox2, Klf4, and c-Myc) that are characteristically working in ES cells, and using retroviral vectors, By introducing the gene into mouse skin cells (fibroblasts) and culturing for several weeks, mouse iPS cells were successfully produced (see, for example, Non-Patent Document 1). Furthermore, in 2007, it was announced that the above four genes were similarly introduced into human skin cells and that human iPS cells were successfully produced.

Yamanaka S., et al., “Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors”, Cell, vol.126, p663-676, 2006.

  In the method of Prof. Yamanaka et al., IPS cells are prepared by seeding the cells into which the above four genes have been introduced on feeder cells and using a dedicated medium usually used for preparing stem cells. Since feeder cells are used, there is a risk of contamination and the like, and it is difficult to use the prepared iPS cells as they are for clinical use such as regenerative medicine. In addition, a dedicated medium for producing stem cells is expensive, and it is difficult to produce a large amount of iPS cells.

  The present invention has been made in view of the above circumstances, and without using feeder cells, a method for producing induced pluripotent stem cells from simple and inexpensive somatic cells and culturing induced pluripotent stem cells in an undifferentiated manner A culture medium is provided.

  As a result of intensive studies to achieve the above object, the present inventors have found that iPS cells can be easily produced without using feeder cells by using a medium containing general components. It came to complete.

That is, the present invention includes the following aspects.
[1] A method for producing induced pluripotent stem cells from somatic cells, comprising a culture step of introducing a nuclear reprogramming factor into somatic cells and culturing in a hypoxic environment without using feeder cells, In the culturing step, at least one inorganic salt selected from the group consisting of calcium chloride, ferric sulfate hydrate, anhydrous magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and anhydrous monosodium phosphate, arginine, At least one amino acid selected from the group consisting of cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine, choline chloride, folic acid, myo-inositol, niacin Amides, hemi-calcium D-pantothenate A medium containing at least one vitamin selected from the group consisting of pyridoxine, riboflavin, and thiamine, D-glucose, phenol red sodium, sodium pyruvate, antibiotics, and serum is used. Production method.
[2] The production method according to [1], wherein the nuclear reprogramming factor is an Oct family gene, a Klf family gene, and a Sox family gene.
[3] The production method according to [1] or [2], wherein the somatic cells are fibroblasts.
[4] The production method according to any one of [1] to [3], wherein the oxygen concentration is 10% in the culturing step.
[5] The production method according to any one of [1] to [4], wherein the D-glucose concentration in the medium is 0.5 g / L or more and 2 g / L or less.
[6] A medium for culturing artificial pluripotent stem cells in an undifferentiated state, comprising calcium chloride, ferric sulfate hydrate, anhydrous magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and anhydrous phosphorus At least one inorganic salt selected from the group consisting of monosodium acid, L-arginine, L-cysteine, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L- At least one amino acid selected from the group consisting of methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; choline chloride, folic acid, myo-inositol, niacinamide, D- from hemi-calcium pantothenate, pyridoxine, riboflavin, and thiamine At least one vitamin selected from the group, and D- glucose medium and phenol red, sodium pyruvate and, comprising an antibiotic, a serum, a.
[7] The medium according to [6], wherein the D-glucose concentration in the medium is 0.5 g / L or more and 2 g / L or less.

  According to the present invention, a method for producing induced pluripotent stem cells from simple and inexpensive somatic cells without using feeder cells and a medium for culturing induced pluripotent stem cells undifferentiated can be provided. .

(A) A mouse tail fibroblast into which a nuclear reprogramming factor (Oct3 / 4, Sox2, Klf4, and c-Myc) was introduced using the lipofection method in Example 1 was cultured and formed on the 21st day after the introduction. It is an image which shows a colony. (B) Mouse tail fibroblasts into which nuclear reprogramming factors (Oct3 / 4, Sox2, Klf4, and c-Myc) were introduced using the electroporation method in Example 1 were cultured, and 6 days after the introduction It is an image which shows the formed colony. Mouse tail fibroblasts into which nuclear reprogramming factors (Oct3 / 4, Sox2, Klf4, and c-Myc) have been introduced using the electroporation method in Example 1 are cultured, and changes in the number of colonies over time are shown. It is a graph. Mouse tail fibroblasts into which nuclear reprogramming factors (Oct3 / 4, Sox2, Klf4, and c-Myc) were introduced using the lipofection method or electroporation method in Example 1 were cultured, and 136-190 after introduction, respectively. It is the image which colony was collect | recovered on the day or the 46th-56th day after introduction | transduction, DNA was extracted, and insertion of the exogenous gene (Klf4 and c-Myc) was confirmed by PCR. (A) Mouse tail fibroblasts into which nuclear reprogramming factors (Oct3 / 4, Sox2, Klf4, and c-Myc) were introduced using the lipofection method in Example 1 were cultured, and 162 to 163 days after introduction It is the image which collect | recovered colonies, extracted RNA, and confirmed the expression of the initialization marker gene by RT-PCR method. (B) Mouse tail fibroblasts into which nuclear reprogramming factors (Oct3 / 4, Sox2, Klf4, and c-Myc) have been introduced using the lipofection method or electroporation method in Example 1, and after each introduction It is an image in which colonies were collected on days 162 to 163 or 14 to 57 days after introduction, RNA was extracted, and expression of the initialization marker gene was confirmed by RT-PCR. (C) Mouse tail fibroblasts into which nuclear reprogramming factors (Oct3 / 4, Sox2, Klf4, and c-Myc) have been introduced using the electroporation method in Example 1, and cultured for 14 to 57 days after introduction It is the image which collect | recovered colonies to eyes, extracted RNA, and confirmed the expression of the initialization marker gene by RT-PCR method. Mouse tail fibroblasts into which nuclear reprogramming factors (Oct3 / 4, Sox2, Klf4, and c-Myc) were introduced using the electroporation method in Example 2 were either LIF-free or LIF-containing DMEM (serum and antibiotics) It is an image which shows the result of having cultivated using (contained) and staining the colony with alkaline phosphatase 33 days after introduction or 43 days after introduction.

<Method for producing induced pluripotent stem cells from somatic cells>
In one embodiment, the present invention is a method for producing induced pluripotent stem cells from somatic cells, wherein a nuclear reprogramming factor is introduced into somatic cells and cultured in a hypoxic environment without using feeder cells. A culturing step, wherein in the culturing step, at least one selected from the group consisting of calcium chloride, ferric sulfate hydrate, anhydrous magnesium sulfate, potassium chloride, sodium hydrogen carbonate, sodium chloride, and anhydrous monosodium phosphate Inorganic salts and L-arginine, L-cysteine, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L- At least one amino acid selected from the group consisting of threonine, L-tryptophan, L-tyrosine, and L-valine; At least one vitamin selected from the group consisting of phosphorus, folic acid, myo-inositol, niacinamide, hemi-calcium D-pantothenate, pyridoxine, riboflavin, and thiamine, D-glucose, phenol red sodium, sodium pyruvate, A production method using a medium containing antibiotics and serum is provided.

  According to the production method of the present embodiment, it is possible to easily produce induced pluripotent stem cells without using feeder cells. Moreover, since no feeder cells are used, there is little contamination and it is suitable for clinical use.

In the present specification, the term “artificial pluripotent stem cell” refers to the ability to differentiate into cells of various tissues and organs by artificially introducing several types of genes into adult-derived cells such as skin and culturing. It means a pluripotent stem cell that has the ability to proliferate almost infinitely. In English, it is expressed as “induced prime stem cell”. Hereinafter, “artificial pluripotent stem cells” may be referred to as “iPS cells”.
The manufacturing method of this embodiment is demonstrated in detail below.

[Culture process]
First, a nuclear reprogramming factor is introduced into somatic cells that have been cultured in advance to the required number of cells.

In the present specification, “somatic cells” means all cells except germline cells such as eggs and oocytes, or totipotent cells such as ES cells, and the type thereof is not particularly limited. For example, fetal somatic cells or mature somatic cells may be used. Somatic cells derived from mammals including humans are preferable, somatic cells derived from mice or primates are more preferable, and somatic cells derived from humans are particularly preferable. More specifically, as somatic cells, for example, tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells; tissue progenitor cells; lymphocytes, epithelial cells, muscle cells, fibroblasts Examples thereof include differentiated cells such as cells (skin cells and the like), hair cells, liver cells, and gastric mucosa cells. Among them, the somatic cell is preferably a fibroblast.
When using induced pluripotent stem cells for treatment of diseases, it is desirable to use somatic cells isolated from the patient itself or somatic cells isolated from others who have the same type of HLA antigen. Somatic cells and somatic cells involved in disease treatment can be used.

  As used herein, “mammal” includes all mammals. Among them, as mammals, primates (eg, humans, monkeys, chimpanzees, etc.), rodents (eg, mice, rats, guinea pigs, hamsters, etc.), ungulates (eg, cows, horses, sheep, goats, pigs, etc.) And pet animals (eg, dogs, cats, rabbits, etc.) are preferred.

(Nuclear reprogramming factor)
The nuclear reprogramming factor used in the present embodiment mainly includes three types of genes: Oct family gene, Klf family gene, and Sox family gene.
As for the Oct family gene, the Klf family gene, and the Sox family gene, specific examples of the family gene are described in International Publication No. WO2007 / 69666. The Oct family gene is preferably Oct3 / 4, the Klf family gene is preferably Klf4, and the Sox family gene is preferably Sox2. These genes may be wild-type genes, and some (for example, 1 to 30, preferably 1 to 20, more preferably 1 to 10, further preferably 1 to 5, particularly preferably). Is a mutant gene in which one or two bases are substituted, inserted, and / or deleted, or a chimeric gene obtained by an appropriate combination of family genes, and has the same function or improved as a wild-type gene. It may be a gene having a different function.

  In addition to the above three genes, at least one gene selected from the group consisting of Myc family gene, Lin family gene, Sall1, Sall4, and Nanog may be used. A gene selected from the group consisting of the Myc family gene, Sall1, and Sall4 is a nuclear reprogramming factor that has an effect of increasing the efficiency of nuclear reprogramming, and is a nuclear reprogramming factor that is essential for nuclear reprogramming (for example, the Oct family By using in combination with a combination of a gene and a Sox family gene, or a combination of an Oct family gene and a Klf family gene, the efficiency of nuclear reprogramming can be significantly improved.

C-Myc, L-Myc, and N-Myc are known as Myc family genes. c-Myc is a cancer-related transcription factor. There are two related genes in c-Myc (N-Myc and L-Myc) (Adhikari et al., Nat. Rev. Mol. Cell Biol., vol. 6, p635-645, 2005). L-Myc is described in Table 1 of International Publication No. WO2007 / 69666.
Lin28 and Lin28b are known as the Lin family gene, and Lin28 is preferable.
Sall4 is described in Tables 4 and 5 of International Publication No. WO2007 / 69666.
Sall1 is an ES cell-specific expression gene and is known as one of the zinc finger proteins, and is thought to be involved in kidney development. The NCBI accession numbers for Sall1 are NM_021390 (mouse) and NM_002968 (human). The NC28 accession numbers of Lin28 are NM_145833 (mouse) and NM_024647 (human).
Nanog is an ES cell-specific expression gene and is an undifferentiated marker gene identified by Professor Yamanaka of Kyoto University (Yamanaka S., et al., Cell. Vol. 113, No. 5, p631-642, 2003). ).

  The five types of genes may be wild-type genes, and several (for example, 1 to 30, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, Particularly preferably, it is a mutant gene obtained by substitution, insertion and / or deletion of one or two bases, or an appropriate combination of family genes, and has the same function as a wild-type gene, or It may be a gene having an improved function.

Among the genes exemplified above, a preferable combination as a nuclear reprogramming factor to be introduced is a known combination described in International Publication WO2007 / 069666, International Publication WO2009 / 057831, Japanese Translation of PCT International Publication No. 2012-507258, or the like. Just choose. For example, four genes of Oct3 / 4, Klf4, Sox2, and c-Myc may be introduced as nuclear reprogramming factors.
In addition, each amino acid and nucleotide sequence of the gene exemplified above can be obtained by accessing GenBank (NCBI, USA).

  Moreover, when reprogramming somatic cells derived from a specific mammal, it is preferable to use the same mammal-derived reprogramming factor. For example, when an iPS cell is derived from a human somatic cell, a human-derived reprogramming factor is used.

A method for introducing the above-exemplified gene into a somatic cell is not particularly limited. For example, a vector capable of expressing the gene is generally used. Specific means using a retroviral vector as a vector are disclosed in “International Publication WO2007 / 69666”, “Cell, 126, pp. 663-676, 2006”, and “Cell, 131, pp. 861-872, 2007”. Has been. For the case of using a lentiviral vector as a vector, Science, 318, pp. 1917-1920, 2007. In addition, the case of using a plasmid which is a non-viral vector is described in Okita et al. (Science, Published online: October 9, 2008; 10.1126 / science. 1164270). Appropriate means can be selected and employed.
When using a vector, two or more types of genes selected from the above genes may be incorporated into the vector, or several types of vectors incorporating one type of gene may be used. If one or two of the above genes are already expressed in the somatic cell to be initialized, remove the one or two genes expressed from the combination of the above genes to be introduced You can also.

When using a viral vector, for example, a retroviral vector, as a means for introducing the gene into somatic cells, first prepare a viral vector containing each gene to be introduced, and then transfer each viral vector to a separate packaging cell. Culture supernatants containing each virus are prepared independently by infecting and culturing the cells. Subsequently, gene transfer may be performed by preparing a mixture of these culture supernatants and infecting a virus containing the gene. By adopting this means, the gene transfer efficiency may be remarkably increased, and may be preferable particularly when performing nuclear reprogramming without using c-Myc.
Alternatively, a plurality of viral vectors may be transfected into a single packaging cell, and a culture supernatant containing the plurality of viral vectors may be prepared and used for gene transfer.

In addition, when a non-viral vector such as a plased vector is used as a means for introducing the gene into a somatic cell, the vector can be introduced into the somatic cell by any method known in the art. Examples of the method include, but are not limited to, a calcium phosphate method, an electroporation method, a lipofection method, an aggregation method, a microinjection method, a particle gun method, a DEAE-dextran method, and the like.
In the lipofection method, for example, the vector is encapsulated in a cationic liposome such as lipofectamine, and the obtained liposome is fused with a somatic cell, thereby introducing the vector into the somatic cell. Cells usually attract cationic liposomes because their surfaces are anionic. Examples of liposomes include liposomes modified with molecules such as sugar and polyethylene glycol (PEG).
In the electroporation method, for example, a micropore is formed in a cell membrane by applying an electric pulse to the suspension containing the vector and the somatic cell, and the vector is introduced into the cell.
In the micronucleus cell fusion method, for example, mammalian cells (for example, mouse A9 cells (ATCC VA20110-2209)) containing the above vector and having the ability to form micronuclei are treated with a polyploid inducer (eg, colcemid, colchicine, etc.). To form a micronucleated multinucleated cell, and then form a micronucleated body by cytochalasin treatment, collect it by centrifugation, etc., and fuse the obtained micronucleated body with a somatic cell. Should be introduced.

Methods for introducing reprogramming factors into cultured mammalian somatic cells in the form of proteins include conventional methods such as methods using protein delivery reagents, protein transport domains (PTDs) or cell penetrating peptides (CPPs). Examples include a method using a fusion protein, a microinjection method, a liposome method, and a lipofection method.
Protein delivery agents include, for example, cationic lipid-based BioPOTER Protein Delivery Reagent (manufactured by Gene Therapy Systems), Pro-JectTM Protein Transfection Reagent (manufactured by PIERCE), ProVectin (IMGENEX Prot, et. GenomeONE (Ishihara Sangyo, Japan) using a HVJ envelope (derived from an inactivated Sendai virus), and a permeabilized peptide-based Penetrain Peptide (from Q biogene), Chariot Kit (from Active Motif) ) Are commercially available.
The protein or peptide can be introduced into the cell according to the instruction manual attached to each reagent, and the general procedure is as follows. That is, the reprogramming factor is diluted with a suitable solvent (for example, a buffer such as PBS or HEPES), and then a delivery reagent is added thereto, and the mixture is incubated at room temperature for 5 to 15 minutes. To form a complex with This complex is added to the cells in pre-exchanged serum-free medium, after which the cells are incubated at 37 ° C. for 1 hour to several hours. Thereafter, the medium is removed and replaced with a serum-containing medium.

  Disclosed as PTDs used are Drosophila-derived AntP; HIV-derived TAT (Frankel, A. et al., Cell 55: 1189-1193 (1988)); Green, M. et al. And Loewstein, P.A. M.M. Cell 55: 1179-1188 (1988)); Penetratin (Derossei, D. et al., J. Biol. Chem. 269: 10444-10450 (1994)); Buforin II (Park, C. B. et al., Proc. Natl. Acad. Sci. USA 97: 8245-8250 (2000)); Transportan (Pouga, M. et al., FASEB J. 12: 67-77 (1998); MAP (model amphipathic epoxide) J. et al., Biochim.Biophys. Acta.1414: 127-139 (1998)), K-FGF (Lin, Ze). al., J. Biol. Chem. 270: 14255-14258 (1995)); Ku70 (Sawada, M. et al., Nature Cell Biol. 5: 352-357 (2003)); Prion (Lundberg, P. et. al., Biochem. Biophys.Res Commun. 299: 85-90 (2002)); pVEC (Elmquist, A. et al., Exp. Cell Res. 269: 237-244 (2001)); Morris, MC et al., Nature Biotechnol. 19: 1173-1176 (2001)); Pep-7 (Gao, C. et al., Bioorg. M). Chem. 10: 4057-4065 (2002)); SynB1 (Rousselle, C. et al., Mol. Pharmacol. 57: 679-686 (2000)); HN-1 (Hong, FD & Clayman) , G.L., Cancer Res. 60: 6551-6556 (2000)); CPP derived from PTD includes polyarginine such as 11R (Cell Stem Cell 4: 381-384 (2009)) and 9R (Cell Stem Cell 4: 472-476 (2009)).

  A vector is produced that expresses the fusion protein and that incorporates the cDNA encoding the reprogramming factor and the PTD or CPP sequence to express the DNA encoding the fusion protein, and then recovering the fusion protein Introduce into cells. The introduction can be performed in the same manner as described above except that no protein delivery reagent is added.

  Microinjection is a method in which a protein solution is introduced into a cell by placing the protein solution in a glass needle having a tip diameter of about 1 μm, and the needle is inserted into the cell, whereby the protein can be reliably introduced into the cell.

  The operation of introducing the protein can be performed once or more, for example, 1 to 10 times, 1 to 5 times, etc., preferably 2 times or more. The time interval between repeated operations is, for example, 6 to 48 hours, preferably 12 to 24 hours.

Next, the somatic cells after introduction of the nuclear reprogramming factor are cultured. The culture density of the cells after introduction of the nuclear reprogramming factor is preferably set lower than in the case of normal animal cell culture. For example, it is preferable to continue the culture at a cell density of 1.0 × 10 ⁴ cells / cm ² or more and 1.0 × 10 ⁵ cells / cm ² or less. In the production method of the present embodiment, feeder cells are not used in the culturing step.
As culture | cultivation temperature, 25 to 40 degreeC is preferable, 30 to 39 degreeC is more preferable, and 35 to 39 degreeC is further more preferable.
The culture environment may be arbitrarily set as long as the environment suitable for the cell culture used can be maintained for an arbitrary period without directly affecting the maintenance or proliferation of the cells. If necessary, a hydrodynamic addition such as reflux may be added as long as the culture environment is not significantly changed.
The culture environment is preferably a hypoxic environment, specifically, the oxygen concentration is preferably 1% or more and 10% or less, more preferably 5% or more and 10% or less, and more preferably 10%. It is particularly preferred that
In the conventional iPS cell culture, the oxygen concentration is 20%, whereas in the culturing process of the present embodiment, the oxygen concentration is 10% or less, so that the iPS cells are efficiently proliferated in an undifferentiated state. be able to.
Further, the carbon dioxide concentration in the culture environment may be about 5%, for example.

(Culture medium)
The culture medium used for the culture process is a basic medium containing components (inorganic salts, carbohydrates, amino acids, vitamins) necessary for the survival and growth of cells plus phenol red sodium, sodium pyruvate, antibiotics, and serum. If it is.
The inorganic salt contained in the medium is to help maintain the osmotic balance of the cells and to help regulate the membrane potential.
There is no special limitation as an inorganic salt, For example, salts, such as calcium, copper, iron, magnesium, potassium, sodium, zinc, are mentioned. Salts are usually used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates.
Among them, the inorganic salt includes at least one selected from the group consisting of calcium chloride, ferric sulfate hydrate, anhydrous magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and anhydrous monosodium phosphate. It is more preferable that it contains all of calcium chloride, ferric sulfate hydrate, anhydrous magnesium sulfate, potassium chloride, sodium hydrogen carbonate, sodium chloride, and anhydrous monosodium phosphate.

  The osmolality of the medium may be, for example, 200 mOsm / kg or more and 400 mOsm / kg or less, for example, 290 mOsm / kg or more and 350 mOsm / kg or less, for example, 280 mOsm / kg or more and 310 mOsm / kg or less, for example, 280 mOsm / kg or more. It may be less than 300 mOsm / kg (specifically, 280 mOsm / kg).

The carbohydrate is not particularly limited, and examples thereof include glucose, galactose, maltose, and fructose. Among them, the carbohydrate is preferably glucose, and more preferably D-glucose.
The concentration of carbohydrate (preferably D-glucose) in the medium is preferably 0.5 g / L or more and 2 g / L.
The concentration of D-glucose is about 4 g / L or more and about 10 g / L or less in the medium used for culturing conventional iPS cells, whereas the concentration of D-glucose is the above in the medium used in the culturing process of this embodiment. By being in the range and having a low glucose concentration, iPS cells can be efficiently propagated in an undifferentiated state.

The amino acid is not particularly limited. For example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L -Histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof Etc. Among them, L-arginine, L-cysteine, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, It preferably contains at least one selected from the group consisting of L-tryptophan, L-tyrosine, and L-valine, L-arginine, L-cysteine, L-glutamine, L-glycine, L-histidine, L-isoleucine , L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine are more preferable.
In general, the concentration of glutamine contained in the medium is 0.05 g / L or more and 1 g / L or less (usually 0.1 g / L or more and 0.75 g / L or less). Each amino acid other than glutamine contained in the medium is 0.001 g / L or more and 1 g / L (usually 0.01 g / L or more and 0.15 g / L or less). Amino acids may be synthetically derived.

  The vitamin is not particularly limited. For example, thiamine (vitamin B1), riboflavin (vitamin B2), niacinamide (vitamin B3), hemi-calcium D-pantothenate, (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-α tocopherol (vitamin E), biotin (vitamin H), menadione (vitamin K), Examples thereof include choline chloride and myo-inositol. Among them, the vitamin preferably contains at least one selected from the group consisting of choline chloride, folic acid, myo-inositol, niacinamide, hemi-calcium D-pantothenate, pyridoxine, riboflavin, and thiamine, choline chloride, folic acid, More preferably, all of myo-inositol, niacinamide, hemi-calcium D-pantothenate, pyridoxine, riboflavin, and thiamine are included.

Phenol red sodium is an acid and base titration indicator and is used to know the liquidity of the medium.
Sodium pyruvate is a glycolytic metabolite and is the first metabolite in which glucose is catabolized as an organic acid in the Emden-Meyerhof pathway (EM pathway). Therefore, it can become a source of energy for cells.

  Antibiotics include, for example, gentamicin, amphotericin, ampicillin, minomycin, kanamycin, penicillin, streptomycin, gentacin, tylosin, aureomycin, and the like used for normal animal cell culture. These antibiotics may be contained alone or in combination of two or more. The concentration of the antibiotic contained in the cell culture medium is not particularly limited, and may be, for example, from 0.1 μg / mL to 100 μg / mL.

Examples of serum include, but are not limited to, FBS / FCS (Fetal Bovine / Calf Serum), NCS (Newborn Calf Serum), CS (Calf Serum), HS (Horse Serum), and the like.
The concentration of serum contained in the medium may be, for example, 2% by mass or more and 10% by mass or less.

  The medium used in the present embodiment may be a known basic medium supplemented with phenol red sodium, sodium pyruvate, the above antibiotics, and the above serum, and more specifically, for example, DMEM, Minimum Essential. Medium (MEM), RPMI-1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM / F-12), Glasgow Min E However, it is not limited to these.

The medium may further contain a growth factor, leukemia inhibiting factor (LIF), or hormone.
Examples of the growth factor include, but are not limited to, a cell growth factor and a cell adhesion factor.
More specifically, examples of the growth factor include epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (basic fibroblast growth factor). bFGF), Insulin-like growth factor-1 (IGF-1), Macrophage-derived growth factor (MDGF), Platelet-derived growth factor (PDGF) Tumor angiogenesis factor (TAF), etc. Is mentioned. These growth factors may be included singly or in combination.
The concentration of the growth factor contained in the medium is not particularly limited, and may be, for example, 1 ng / mL or more and 10 μg / mL or less.

LIF is a cytokine discovered as a factor that inhibits proliferation of leukemia cells and induces differentiation into macrophages, belongs to the IL-6 family, and activates JAK1 and JAK2 via LIFR / gp130 receptors. In addition, LIF has a function of maintaining the undifferentiated nature of mouse ES / iPS cells, and in recent years, human LIF has attracted attention as one of the factors necessary for naïve human iPS / ES cells.
The concentration of LIF contained in the medium is not particularly limited, and may be, for example, from 1 ng / mL to 10 μg / mL.

Examples of hormones include insulin, glucagon, triiodothyronine, and adrenocortical hormone. These hormones may be included singly or in combination.
The concentration of the hormone contained in the medium is not particularly limited, and may be, for example, 1 ng / mL or more and 10 μg / mL or less.

The cells can then be allowed to generate iPS colonies after 20 days or more and 30 days or less, with appropriate media changes.
During the culture, the medium is replaced with a fresh medium once every 2-3 days from the second day after the start of the culture.

  By culturing the obtained iPS colony in the same culture environment, iPS cells can be passaged.

  iPS cells can be identified by tests based on the characteristics of pluripotent cells such as ES cells. Specifically, when cells are tested for characteristics such as expression of ES cell-specific marker gene, semi-permanent cell proliferation ability, differentiation pluripotency (formation of three germ layers), and the cells have these characteristics, The cells are identified as iPS cells (Takahashi, K. et al., Cell 131: 861-872 (2007)).

Examples of ES cell-specific marker genes include Oct3 / 4, Nanog, Lin28, PH34, Dnmt3b, Noda1, SSEA3, SSEA4, Tra-1-60, Tra-1-81, Tra2-49 / 6F (alkaline phosphatase), etc. Is mentioned.
Expression of the marker gene in the cell can be detected by RT-PCR method using primers specific for amplification of these genes.

As for the semi-permanent cell growth ability, it is confirmed that the cells grow exponentially in a cell culture test over about 4 months to 6 months or more. The colony doubling time (or population doubling time) of human iPS cells is, for example, about 46.9 ± 12.4 hours, 47.8 ± 6.6 hours, or 43.2 ± 11.5 hours. Therefore, this value can be used as an index of proliferation ability (Takahashi, K. et al., Cell 131: 861-872 (2007)).
In addition, since iPS cells have high telomerase activity, the activity may be detected by, for example, the TRAP (telomeric repeat amplification protocol) method.

Pluripotency (formation of 3 germ layers) can be confirmed by, for example, formation of teratoma and identification of each tissue (or cell) of 3 germ layers (endoderm, mesoderm, and ectoderm) in teratoma tissue. . Specifically, in the case of mouse iPS cells, cells are injected subcutaneously into nude mice, and in the case of human iPS cells, cells are injected into the testes of Scid mice to confirm tumor formation. (Or cell), neural tube tissue (or cell), muscle tissue (or cell), adipose tissue (or cell), intestinal tract-like tissue (or cell), etc. are confirmed.
Based on each of the above tests, it is possible to confirm that the cell is an iPS cell and select an iPS cell colony.

[Application of induced pluripotent stem (iPS) cells]
The iPS cells obtained by the production method of the present embodiment have pluripotency and have characteristics very similar to those of ES cells. For this reason, iPS cells can be used as substitute cells for ES cells.

  iPS cells can be induced into a variety of differentiated cells, progenitor cells and tissues because of their pluripotency. Specifically, inducing various differentiated cells such as nerve cells and cardiomyocytes from iPS cells in the presence of factors such as activin A / BMP4 (bone morphogenetic protein 4) and VEGF (basic endothelial growth factor). Can do. The differentiated cells obtained in this way can be used for treatment of patients (so-called regenerative medicine) by transplanting them into patients, for example, into defective tissues of patients.

  In addition, by introducing iPS cells into blastocysts of mammals (excluding humans) and transplanting the embryos into the uterus of a foster parent of the same animal species, part of the genotypes and traits of iPS cells A chimeric animal inherited can be produced (see International Publication No. WO2007 / 069666). Modification of a specific gene in iPS cells, elucidation of gene function by knockout (KO) or knockin (KI), preparation of a disease model, production of a substance (protein, etc.), etc. are possible. At this time, iPS cells derived from previously genetically modified somatic cells can also be used.

<Medium for culturing induced pluripotent stem cells undifferentiated>
In one embodiment, the present invention is a medium for culturing induced pluripotent stem cells in an undifferentiated state, comprising calcium chloride, ferric sulfate hydrate, anhydrous magnesium sulfate, potassium chloride, sodium bicarbonate, At least one inorganic salt selected from the group consisting of sodium chloride and anhydrous monosodium phosphate; and L-arginine, L-cysteine, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, At least one amino acid selected from the group consisting of L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; choline chloride, folic acid, myo -Inositol, niacinamide, D-hemicalcium pantothenate, pyridoxine, ribofura Emissions, and to provide at least a one vitamin selected from the group consisting of thiamine, and D- glucose, and phenol red, sodium, and sodium pyruvate, and antibiotics, serum, a medium containing.

  According to the culture medium of this embodiment, artificial pluripotent stem cells can be cultured undifferentiated without using feeder cells.

Examples of the medium of the present embodiment include the same media as those exemplified above in <Method for producing induced pluripotent stem cells from somatic cells>.
The inorganic salt contained in the medium of this embodiment is selected from the group consisting of calcium chloride, ferric sulfate hydrate, anhydrous magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and anhydrous monosodium phosphate. It is preferable to include at least one, and it is more preferable to include all of calcium chloride, ferric sulfate hydrate, anhydrous magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and anhydrous monosodium phosphate.

  The osmolality of the medium of the present embodiment may be, for example, 200 mOsm / kg or more and 400 mOsm / kg or less, for example, 290 mOsm / kg or more and 350 mOsm / kg or less, for example, 280 mOsm / kg or more and 310 mOsm / kg or less. It may be 280 mOsm / kg or more and less than 300 mOsm / kg (specifically, 280 mOsm / kg).

The carbohydrate contained in the medium of the present embodiment is preferably D-glucose.
The concentration of carbohydrate (preferably D-glucose) in the medium is preferably 0.5 g / L or more and 2 g / L.
In a general medium, the concentration of D-glucose is 1 g / L or more and 10 g / L or less, whereas in the medium used in the culture process of the present embodiment, the concentration of D-glucose is in the above range, and the low glucose concentration Therefore, iPS cells can be efficiently proliferated in an undifferentiated state.

As amino acids contained in the medium of the present embodiment, L-arginine, L-cysteine, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L- It preferably contains at least one selected from the group consisting of phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, L-arginine, L-cysteine, L-glutamine, L -All of glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine More preferably.
In general, the concentration of glutamine contained in the medium is 0.05 g / L or more and 1 g / L or less (usually 0.1 g / L or more and 0.75 g / L or less). Each amino acid other than glutamine contained in the medium is 0.001 g / L or more and 1 g / L (usually 0.01 g / L or more and 0.15 g / L or less). Amino acids may be synthetically derived.

  The vitamin contained in the medium of the present embodiment preferably contains at least one selected from the group consisting of choline chloride, folic acid, myo-inositol, niacinamide, hemi-calcium D-pantothenate, pyridoxine, riboflavin, and thiamine. More preferably, all of choline chloride, folic acid, myo-inositol, niacinamide, hemi-calcium D-pantothenate, pyridoxine, riboflavin, and thiamine.

Phenol red sodium is an acid and base titration indicator and is used to know the liquidity of the medium.
Sodium pyruvate is a glycolytic metabolite and is the first metabolite in which glucose is catabolized as an organic acid in the Emden-Meyerhof pathway (EM pathway). Therefore, it can become a source of energy for cells.

  Antibiotics include, for example, gentamicin, amphotericin, ampicillin, minomycin, kanamycin, penicillin, streptomycin, gentacin, tylosin, aureomycin, and the like used for normal animal cell culture. These antibiotics may be contained alone or in combination of two or more. The concentration of the antibiotic contained in the cell culture medium is not particularly limited, and may be, for example, from 0.1 μg / mL to 100 μg / mL.

Examples of serum include, but are not limited to, FBS / FCS (Fetal Bovine / Calf Serum), NCS (Newborn Calf Serum), CS (Calf Serum), HS (Horse Serum), and the like.
The concentration of serum contained in the medium may be, for example, 2% by mass or more and 10% by mass or less.

  The medium used in the present embodiment may be a known basic medium supplemented with phenol red sodium, sodium pyruvate, the above antibiotics, and the above serum, and more specifically, for example, DMEM, Minimum Essential. Medium (MEM), RPMI-1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM / F-12), Glasgow Min E However, it is not limited to these.

The medium may further contain a growth factor, leukemia inhibiting factor (LIF), or hormone.
Examples of the growth factor include, but are not limited to, a cell growth factor and a cell adhesion factor.
More specifically, examples of the growth factor include epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (basic fibroblast growth factor). bFGF), Insulin-like growth factor-1 (IGF-1), Macrophage-derived growth factor (MDGF), Platelet-derived growth factor (PDGF) Tumor angiogenesis factor (TAF), etc. Is mentioned. These growth factors may be included singly or in combination.
The concentration of the growth factor contained in the medium is not particularly limited, and may be, for example, 1 ng / mL or more and 10 μg / mL or less.

LIF is a cytokine discovered as a factor that inhibits proliferation of leukemia cells and induces differentiation into macrophages, belongs to the IL-6 family, and activates JAK1 and JAK2 via LIFR / gp130 receptors. In addition, LIF has a function of maintaining the undifferentiated nature of mouse ES / iPS cells, and in recent years, human LIF has attracted attention as one of the factors necessary for naïve human iPS / ES cells.
The concentration of LIF contained in the medium is not particularly limited, and may be, for example, from 1 ng / mL to 10 μg / mL.

Examples of hormones include insulin, glucagon, triiodothyronine, and adrenocortical hormone. These hormones may be included singly or in combination.
The concentration of the hormone contained in the medium is not particularly limited, and may be, for example, 1 ng / mL or more and 10 μg / mL or less.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to a following example.

[Example 1]
(1) Collection of mouse tail fibroblasts Fibroblasts were collected from the tail of BALB / c mice (8-week-old male, 13-week-old male, and 45-week-old female), and 10% fetal bovine serum ( FBS) and antibiotics (penicillin and streptomycin) -containing Dulbecco's modified Egourdium (hereinafter sometimes referred to as “DMEM”) were used for pre-culture.

(2) Introduction of nuclear reprogramming factor Next, Oct3 / 4, Sox2, Klf4, and c-Myc are mounted on mouse tail fibroblasts cultured and seeded to 30 × 10 ⁴ cells / cm ^2. The two plasmid vectors (pCX-OKS-2A 4 μg and pCX-cMyc 2 μg) (manufactured by addgene) were introduced by a lipofection method using FuGENE (registered trademark) 6 (manufactured by Promega).
Alternatively, two plasmid vectors (pCX-OKS-2A 0.5 μg and pCX-cMyc 0.5 μg or pCX-OKS-2A 1.0 μg and pCX loaded with Oct3 / 4, Sox2, Klf4, and c-Myc -CMyc 1.0 μg) (manufactured by addgene) was introduced by an electroporation method using Nucleofector (manufactured by Lonza).

(3) Cell Culture Next, the mouse tail fibroblasts after introduction were cultured as they were. As a medium, Dulbecco's modified Egourdium (hereinafter sometimes referred to as “DMEM”) containing 10% fetal bovine serum (hereinafter sometimes referred to as “FBS”) and antibiotics (penicillin / streptomycin) are included. Using. The culture conditions were 37 ° C., 10% oxygen concentration, and 5% carbon dioxide concentration.

(4) Formation of colonies In mouse tail fibroblasts into which reprogramming factors were introduced by lipofection, colony formation was observed on the 21st day after introduction (see FIG. 1 (A)). On the other hand, in the mouse tail fibroblasts into which the reprogramming factor was introduced by electroporation, colony formation was observed on the 5th day after the introduction (see FIG. 1B). 6th day image).

(5) Confirmation of undifferentiated state by alkaline phosphatase staining method Next, it was confirmed by the alkaline phosphatase method that the undifferentiated state was maintained for each formed colony.
Specifically, Alkaline Phosphatase Substrate Kit III <VECTOR Blue> (manufactured by Vector Laboratories) was used.
First, cells were fixed with 1 × PBS containing 4% paraformaldehyde for 10 minutes at room temperature for immunohistochemical analysis. After washing with 1 × PBS, a substrate solution (precipitate (reaction product) colored intense blue) was added and allowed to react for 20 minutes at room temperature. It was then washed with 1 × PBS and observed (not shown).
Alternatively, it was performed using AP Staining Kit (manufactured by System Biosciences).
First, for immunohistochemical analysis, cells were fixed with Fixation Solution at room temperature for 5 minutes. After washing with 1 × PBS, a substrate solution (precipitate (reaction product) colored intense red) was added and allowed to react for 20 minutes at room temperature. Subsequently, it was washed with 1 × PBS and observed.
As a result, it was confirmed that the mouse tail fibroblasts into which the reprogramming factor was introduced by any of the lipofection method and the electroporation method were maintained in an undifferentiated state.

(6) Confirmation of insertion of exogenous gene Next, in order to confirm whether or not the vector of the nuclear reprogramming factor was inserted, DNA was extracted from the cells and the insertion was confirmed by the PCR method.
Specifically, first, using a NucleoSpin Tissue kit (manufactured by TaKaRa) from a colony of mouse tail fibroblasts formed by each introduction method 136-190 days after introduction or 46-56 days after introduction, DNA was extracted according to the method recommended by the manufacturer. The mouse Klf4 and c-Myc genes were then amplified by PCR. PCR was performed with KOD-Plus- (manufactured by TOYOBO). The primer sequences used are shown in Table 1 below.

  Subsequently, each of the obtained PCR products was confirmed by agarose electrophoresis. The results are shown in FIG. In FIG. 3, colonies 1, 2 and 7 show the results of using mouse tail fibroblast colonies into which phasing factors have been introduced by the lipofection method, and colonies 3 to 6 and 8 are phased by the electroporation method. The result using the colony of the mouse | mouth tail fibroblast which introduce | transduced the factor is shown. Further, “Fibroblasts DNA” subjected to agarose electrophoresis together as a positive control means total DNA extracted from mouse tail fibroblasts, and “pCX” means pCX-OKS-2A or pCX-cMyc, respectively. To do.

  From FIG. 1, the mouse Klf4 and c-Myc genes, which are exogenous genes, are integrated and integrated in mouse tail fibroblasts into which the reprogramming factor has been introduced by either the lipofection method or the electroporation method. Confirmed that there is a case not to.

(7) Confirmation of initialization marker gene expression Subsequently, in order to confirm the expression of the initialization marker gene, RNA was extracted from the cells, and the expression was confirmed by RT-PCR.
Specifically, first, a NucleoSpin RNA kit (manufactured by TaKaRa), or Maxwell (from the mouse tail fibroblasts colony formed by each of the introduction methods 136 to 190 days after introduction or 46 to 56 days after introduction, or Maxwell ( RNA was extracted according to the method recommended by the manufacturer using 16 LEV simple RNA Cells and Tissue Kit (manufactured by Promega). Subsequently, the extracted RNA was subjected to reverse transcription reaction using PrimeScript (registered trademark) RT-PCR Kit (manufactured by TaKaRa). Subsequently, PCR was performed with KOD-Plus- (manufactured by TOYOBO). As initialization marker genes, five genes, c-Myc, Oct3 / 4, Sox2, Nanog, and Rex1, were detected. The primer sequences used are shown in Table 2 below.

  Each PCR product was then confirmed by agarose electrophoresis. The results are shown in FIGS. 4 (A) to (C), colonies 1 to 12 and 13 to 20 show results using colonies of mouse tail fibroblasts into which a nuclear reprogramming factor has been introduced by the lipofection method, and colonies 21 to 24, And 25-35 and 8 show the result using the colony of the mouse | mouth tail fibroblast which introduce | transduced the staging factor by the electroporation method. “Fibroblasts cDNA” means complementary DNA extracted from mouse tail fibroblasts, “NC” means sterilized water subjected to agarose electrophoresis as a negative control, and “P "C." refers to pCX-cMyc (cMyc), pCX-OKS-2A (OCT3 / 4 and Sox2), which were subjected to agarose electrophoresis together as a positive control, and DNA extracted from mouse tail fibroblasts (Nanog and Rex1).

4 (A) to 4 (C), in mouse tail fibroblasts into which reprogramming factor was introduced by lipofection method, at least any one reprogramming marker among the above four reprogramming marker genes in all colonies. Was expressed. Further, in mouse tail fibroblasts into which an initialization factor was introduced by electroporation, colony 25-35 expressed at least one of the four types of initialization marker genes. .
Furthermore, in the mouse tail fibroblasts into which the reprogramming factor was introduced by the lipofection method, in colony 2, and in the mouse tail fibroblasts into which the reprogramming factor was introduced by the electroporation method, in colony 27, the above four types of reprogramming were performed. Expression of all of the marker genes was confirmed.
From the above, it was confirmed that iPS cells can be easily produced without using feeder cells in culture using DMEM containing 10% FBS and antibiotics (penicillin and streptomycin).

[Example 2]
(1) Collection of mouse tail fibroblasts Using the same method as in (1) of Example 1, mouse tail fibroblasts were collected and DMEM containing 10% FBS and antibiotics (penicillin and streptomycin) was used. Cultured in advance.

(2) Introduction of nuclear reprogramming factor Next, using the same method as (2) of Example 1, two plasmid vectors (pCX-OKS-) loaded with Oct3 / 4, Sox2, Klf4, and c-Myc were used. 2A 1.0 μg and pCX-cMyc 1.0 μg) (manufactured by addgene) were introduced by an electroporation method using Nucleofector (manufactured by Lonza).

(3) Cell Culture Next, the mouse tail fibroblasts after introduction were seeded in a 150 mm petri dish at 50 × 10 ⁴ cells without using feeder cells. As the medium, leukemia inhibitory factor (LIF) -free (-) or contained (+) DMEM (containing 10% FBS and antibiotics (penicillin / streptomycin)) was used. The culture conditions were 37 ° C., 10% oxygen concentration, and 5% carbon dioxide concentration. The cells using LIF (−) DMEM were cultured from the introduction of reprogramming factor to day 33, and the cells using LIF (+) DMEM were cultured from the introduction of reprogramming factor to day 43. It was.

(4) Confirmation of undifferentiated state by alkaline phosphatase staining method Next, cells cultured using DMEM of LIF (−) on day 33 after introduction of reprogramming factor, and LIF on day 43 after introduction of reprogramming factor The cells cultured using (+) DMEM were subjected to alkaline phosphatase staining using the same method as in Example 1, (5). The results are shown in FIG.

  FIG. 5 confirmed that the undifferentiated state was maintained in mouse tail fibroblasts cultured using either LIF (−) or LIF (+) DMEM and any medium.

  According to the present invention, artificial pluripotent stem cells can be easily produced without using feeder cells. Moreover, since no feeder cells are used, there is little contamination and it is suitable for clinical use.

Claims (7)

A method for producing induced pluripotent stem cells from somatic cells,
Introducing a nuclear reprogramming factor into somatic cells, without using feeder cells, and culturing in a hypoxic environment,
In the culturing step,
At least one inorganic salt selected from the group consisting of calcium chloride, ferric sulfate hydrate, anhydrous magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and anhydrous monosodium phosphate;
L-arginine, L-cysteine, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L- At least one amino acid selected from the group consisting of tryptophan, L-tyrosine, and L-valine;
At least one vitamin selected from the group consisting of choline chloride, folic acid, myo-inositol, niacinamide, hemi-calcium D-pantothenate, pyridoxine, riboflavin, and thiamine;
D-glucose,
Phenol red sodium,
Sodium pyruvate,
Antibiotics,
A production method comprising using a medium containing serum.   The production method according to claim 1, wherein the nuclear reprogramming factor is an Oct family gene, a Klf family gene, and a Sox family gene.   The production method according to claim 1 or 2, wherein the somatic cells are fibroblasts.   The manufacturing method according to any one of claims 1 to 3, wherein in the culturing step, the oxygen concentration is 10%.   The production method according to any one of claims 1 to 4, wherein the D-glucose concentration in the medium is 0.5 g / L or more and 2 g / L or less. A medium for culturing induced pluripotent stem cells in an undifferentiated state,
At least one inorganic salt selected from the group consisting of calcium chloride, ferric sulfate hydrate, anhydrous magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and anhydrous monosodium phosphate;
At least one amino acid selected from the group consisting of arginine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine;
At least one vitamin selected from the group consisting of choline chloride, folic acid, myo-inositol, niacinamide, hemi-calcium D-pantothenate, pyridoxine, riboflavin, and thiamine;
D-glucose,
Phenol red sodium,
Sodium pyruvate,
Antibiotics,
And a serum.   The medium according to claim 6, wherein the D-glucose concentration in the medium is 0.5 g / L or more and 2 g / L or less.