JP2016093178A - Culture medium for differentiated cells derived from stem cells, production of differentiated cells from stem cells, and method for production of cell pharmaceutical composition containing differentiated cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique of preparing differentiated cells without the contamination with undifferentiated stem cells, in differentiated cells induced from stem cells; and to provide a pharmaceutical composition containing a stem cell survival inhibitor for inhibiting in vivo neoplastic transformation of a cell pharmaceutical composition containing differentiated cells derived from stem cells.SOLUTION: The invention provides a culture medium for differentiated cells derived from stem cells by which differentiated cells without contamination with undifferentiated stem cells can be obtained by compounding, into the undifferentiated stem cells, atrovastatin which has a specific survival inhibiting activity and inhibits the survival of undifferentiated stem cells without inducing the apoptosis of the differentiated cells.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a medium for differentiated cells derived from stem cells, production of differentiated cells from stem cells, and a method for producing a cell pharmaceutical composition containing the differentiated cells. More specifically, the present invention relates to a medium or the like that can selectively culture only differentiated cells by suppressing the survival of stem cells with atorvastatin.

  One of the challenges of regenerative medicine technology using pluripotent stem cells such as artificial pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) is to differentiate patients from pluripotent stem cells into desired types of cells. How to prevent the risk of pluripotent stem cells remaining in an undifferentiated state when transplanted into the body of the patient, transplanted into the patient's body together with the differentiated cells, and becoming a tumor and cancer in the patient's body (Refer nonpatent literature 1).

  As a test system for evaluating contamination of undifferentiated iPS cells or the like that may have tumorigenicity, a marker specific to undifferentiated pluripotent cells or a marker specific to cells with high differentiation potential (see Non-Patent Document 2) ) Expression as an index, and quantitative RT-PCR (qRT-PCR). However, in any case, contamination of undifferentiated pluripotent stem cells below a certain frequency cannot be detected, and it is confirmed that colonies such as iPS cells do not appear when the final product is cultured after returning to undifferentiated pluripotent stem cell culture conditions. Etc. are necessary.

  Atorvastatin (Monocalcum bis {(3R, 5R) -7- [2- (4-fluorophenyl) -5- (1-methylethyl) -3-phenyl-4- (phenylcarbamoyl) -1H-pyrol-1-yl] -3 , 5-dihydroxyheptanoate} trihydrate (see formula (I)) is a kind of drug called HMG-CoA reductase inhibitor (statin).

  Statin drugs are currently simvastatin, fluvastatin, lovastatin, atorvastatin, pitavastatin, pravastatin, pravastatin, and mevastatin (Mevastatin). Yes. Statin drugs are the most effective drugs for reducing blood low density lipoprotein (LDL) levels in patients at risk for cardiovascular disease. High levels of LDL in the blood are associated with the formation of coronary artery damage that promotes blockage of blood flow, rupture of blood vessels, and thrombosis (see Non-Patent Document 3).

  In relation to the present invention, Patent Document 1 discloses a technique for suppressing iPS cell tumorigenesis and differentiating into an objective differentiated cell. In the examples of this document, in an experiment in which iPS cells are induced into osteoblasts and transplanted into immunodeficient mice, differentiation induction is performed in a medium containing simvastatin, fluvastatin, or lovastatin, so that the tumor caused by the transplanted cells. It is described that the formation could be suppressed. However, Cited Document 1 does not specifically examine atorvastatin, and does not specifically disclose the induction of iPS cell differentiation into cardiomyocytes.

International Publication No. 2013/100080

J. et al. Cell Sci. 2010, 123, p. 643-651 PNAS, 2013, 110, 51, p. 20569-20574 The Pharmacological Basis of Therapeutics, 1996, 9, p. 879

  The main object of the present invention is to provide a technique for preparing differentiated cells free from contamination of undifferentiated stem cells in differentiated cells derived from stem cells.

[1] A medium for differentiated cells derived from stem cells, containing atorvastatin.
[2] The medium according to [1], wherein the stem cells are inducible pluripotent stem cells.
[3] The medium according to [1] or [2], wherein the differentiated cells are cardiomyocytes.
[4] The medium according to any one of [1] to [3], wherein the stem cells are derived from human.
[5] The medium according to any one of [1] to [4], containing atorvastatin at a concentration of 1 to 20 μM.
[6] The medium according to any one of [1] to [5], wherein the medium is a serum-free medium.

[7] A stem cell survival inhibitor comprising atorvastatin as an active ingredient.
[8] The stem cell survival inhibitor of [7], wherein the stem cells are inducible pluripotent stem cells.
[9] The stem cell survival inhibitor of [7] or [8], wherein the stem cells are derived from human.
[10] A pharmaceutical composition for suppressing tumor formation in vivo of a cell pharmaceutical composition containing differentiated cells derived from stem cells, comprising the stem cell survival inhibitor of any one of [7] to [9] object.

[11] A method for producing a differentiated cell from a stem cell, comprising a step of treating the cell after differentiation induction with atorvastatin.
[12] The method of [11], wherein the stem cell is an inducible pluripotent stem cell.
[13] The method according to [11] or [12], wherein the differentiated cells are cardiomyocytes [14] The method according to any one of [11] to [13], wherein the stem cells are derived from human.
[15] The method according to any one of [11] to [14], further comprising a step of culturing stem cells and a step of inducing differentiation of stem cells.

[16] A method for producing a cell pharmaceutical composition comprising differentiated cells derived from stem cells, the method comprising the step of inducing differentiation of the stem cells and the step of treating the cells after differentiation induction with atorvastatin.
[17] The method according to [16], wherein the stem cell is an inducible pluripotent stem cell.
[18] The method according to [16] or [17], wherein the differentiated cells are cardiomyocytes [19] The method according to any one of [16] to [18], wherein the stem cells are derived from human.
[20] The method according to any one of [16] to [19], further comprising a step of culturing stem cells.

[21] A method for separating only differentiated cells from a cell mixture containing stem cells and differentiated cells,
Treating the cell mixture with atorvastatin.
[22] The method of [21], wherein the stem cell is an inducible pluripotent stem cell.
[23] The method according to [21] or [22], wherein the differentiated cell is a cardiomyocyte. [24] The method according to any one of [21] to [23], wherein the stem cell and the differentiated cell are derived from human.
[25] The method according to any one of [21] to [24], further comprising a step of culturing a stem cell and a step of inducing differentiation of the stem cell.

[26] Use of atorvastatin for the production of the medium of [1], the stem cell survival inhibitor of [7], and the pharmaceutical composition of [10].
[27] Use of atorvastatin for the production of a cell pharmaceutical composition containing differentiated cells derived from stem cells.
[28] Use of atorvastatin for suppressing in vivo tumorigenesis of a cell pharmaceutical composition containing differentiated cells derived from stem cells.

  The present invention provides a technique for preparing differentiated cells free from contamination of undifferentiated stem cells in differentiated cells derived from stem cells.

It is a graph which shows the result of having evaluated the survival inhibitory activity of statins with respect to a cultured human iPS cell (Example 1). It is a graph which shows the result of having evaluated the survival inhibitory activity of atorvastatin with respect to a cultured human iPS cell (Example 1). It is a graph which shows the result of having evaluated the survival inhibitory activity of atorvastatin with respect to a cultured human iPS cell (Example 1). It is a graph which shows the result of having evaluated the survival inhibitory activity (cell death induction activity) of the atorvastatin with respect to a cultured cardiomyocyte (Example 2).

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

1. Medium for differentiated cells The medium for differentiated cells according to the present invention is used for inducing differentiated cells from stem cells, and is characterized by containing atorvastatin. In the present invention, it is apparent that atorvastatin has a survival inhibitory activity specific to undifferentiated stem cells and suppresses the survival of undifferentiated stem cells without inducing cell death of differentiated cells (particularly cardiomyocytes). became.

  In the medium according to the present invention, the concentration of atorvastatin is not particularly limited as long as it is a concentration that exhibits a survival inhibitory activity against undifferentiated stem cells and does not induce cell death of differentiated cells. Such a concentration can be appropriately set by those skilled in the art using the methods described in the Examples and conventionally known methods. The concentration of atorvastatin is, for example, 0.01 to 2000 μM, preferably 0.1 to 200 μM, more preferably 1 to 20 μM.

  The medium according to the present invention can be prepared by adding atorvastatin at the above concentration to a medium conventionally used for culturing stem cells or inducing differentiated cells from stem cells (basal medium). Examples of such a medium include the following.

[Basic medium]
RPMI-1640 medium, Eagle's MEM medium, Dulbecco's modified MEM medium, Glasgow's MEM medium, α-MEM medium, 199 medium, IMDM medium, DMEM medium Hybridoma Serum free medium, Chemically defined HybridSumFarm medium's medium F-12, Ham's Medium F-10, Ham's Medium F12K, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymout's MB752 / 1, CMRL-1 66, Williams' medium E, Brinster's BMOC-3 Medium, E8 Medium, E8 medium (above Thermo Fisher Scientific), ReproFF2 (Reprocell), EX-CELL 302 medium (SAFC) or EX-CELL-CD -CHO (SAFC) and mixtures thereof.

  The medium according to the present invention can be prepared by adding atorvastatin to these mediums in advance or during cell culture.

  In addition, physiologically active substances and nutrient factors necessary for cell survival or growth can be added to the medium as necessary. These additives may be added in advance to the medium, or may be added during cell culture. The method to be added during the culture may be in any form such as one solution or a mixed solution of two or more kinds, and may be continuous or intermittent addition.

A physiologically active substance, insulin, IGF-1, transferrin, and the like albumin or coenzyme Q _10.
Nutritional factors include sugars, amino acids, vitamins, hydrolysates or lipids.
Examples of the sugar include glucose, mannose, and fructose, and one kind or a combination of two or more kinds is used.
As amino acids, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, 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 the like, and one kind or a combination of two or more kinds is used.
Examples of vitamins include d-biotin, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyrodoxal, riboflavin, thiamine, cyanocobalamin or DL-α-tocopherol, and one or a combination of two or more Used.
Examples of the hydrolyzate include hydrolyzed soybeans, wheat, rice, peas, corn, cottonseed, yeast extract and the like.
Examples of lipids include cholesterol, linoleic acid, and linolenic acid.

  Further, an antibiotic such as kanamycin, streptomycin, penicillin or hygromycin may be added to the medium as necessary. When an acidic substance such as sialic acid is added to the medium, it is desirable to adjust the pH of the medium to pH 5-9, preferably pH 6-8, which is a neutral range suitable for cell growth.

  The medium according to the present invention may be a serum-containing medium or a serum-free medium. From the viewpoint of preventing contamination with heterogeneous animal-derived components, it is preferable not to contain serum or to use serum derived from the same species as the cultured stem cells. Here, the serum-free medium means a medium containing no unadjusted or unpurified serum. The serum-free medium may contain a purified blood-derived component or animal tissue-derived component (for example, a growth factor).

  The medium according to the present invention may or may not contain a serum substitute as well as serum. Serum substitutes include, for example, albumin substitutes such as albumin, lipid-rich albumin and recombinant albumin, plant starch, dextran, protein hydrolysates, transferrin or other iron transporters, fatty acids, insulin, collagen precursors, trace amounts An element, 2-mercaptoethanol, 3′-thioglycerol, or an equivalent thereof may be used. Specific examples of serum substitutes include, for example, those prepared by the method described in WO 98/30679, commercially available knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Life Technologies max) and Gluta Life Technologies).

[Stem cells]
The “stem cell” targeted by the present invention refers to an immature cell having self-replication ability and differentiation / proliferation ability, and depending on the differentiation ability, a pluripotent stem cell, a multipotent stem cell (multipotent stem cell). cell), unipotent stem cells, and the like. A “stem cell” is generally defined as an undifferentiated cell having “self-renewal ability” capable of proliferating while maintaining an undifferentiated state and “differentiation pluripotency” capable of differentiating into all three germ layers.
A pluripotent stem cell means a cell having an ability to differentiate into all tissues and cells constituting a living body.
A multipotent stem cell means a cell having the ability to differentiate into multiple types of tissues and cells, although not all types.
A unipotent stem cell means a cell having the ability to differentiate into a specific tissue or cell.

  The stem cell origin species is not particularly limited, and examples thereof include rodents such as rats, mice, hamsters, and guinea pigs, rabbit eyes such as rabbits, ungulates such as pigs, cows, goats, and sheep, and cats such as dogs and cats. The cells may be eyes, humans, monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees and other primates.

  Specific examples of stem cells include myoblasts, vascular endothelial cells, osteoblasts, adipocytes, myocytes, cardiomyocytes, mesenchymal stem cells that differentiate into chondrocytes, neural stem cells that differentiate into neurons and glial cells, leukocytes Hematopoietic stem cells or bone marrow stem cells that differentiate into red blood cells, platelets, mast cells, dendritic cells, etc., and differentiation / induction into various tissues through formation of pseudo embryos called embryoid bodies (EB bodies) from the spheroid state Embryonic stem cells (ES cells), inducible pluripotent cells (iPS cells), embryonic germ cells (EG) cells derived from primordial germ cells, known to proceed to the steps, Multipotent germline stem (mGS) isolated in the process of establishing GS cells from testis tissue Cells, such as multipotent adult progenitor cell (MAPC) pluripotent stem cells, such as isolated from the bone marrow and the like.

  Examples of pluripotent stem cells include the aforementioned ES cells or iPS cells. Stem cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei are also preferred as pluripotent stem cells (Nature, 1997, 385, p. 810, Science, 1998, 280, p. 1256, Nature Biotechnology, 1999, 17, p.456, Nature, 1998, 394, p.369, Nature Genetics, 1999, 22, p.127, Proc. Natl. Acad. Sci. USA, 1999, 96, p. 14984, Nature Genetics, 2000, 24, p. 109).

  Human ES cell lines are obtained from, for example, WA01 (H1) and WA09 (H9) from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 from the Institute of Regenerative Medicine, Kyoto University (Kyoto, Japan). Is possible.

  Examples of iPS cells include cells obtained by introducing a plurality of genes (reprogramming factors) into somatic cells such as skin cells and having acquired pluripotency similar to ES cells. For example, iPS cells obtained by introducing Oct3 / 4 gene, Klf4 gene, C-Myc gene and Sox2 gene, iPS cells obtained by introducing Oct3 / 4 gene, Klf4 gene and Sox2 gene (Nature Biotechnology, 2008) , 26, 101-106). Examples of genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, Eras, and ECAT15. -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1, etc. These initialization factors may be used alone or in combination. Combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 0775119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 045 626, WO 2010/056831, WO 2010/068955, WO 2010/098419, WO 2010/102267, WO 2010/111409, WO 2010/111422, WO 2010/115050, WO 2010/124290, WO 2010/147395, WO 2010/147612, Nat. Biotechnol. 2008, 26, p. 795-797, Cell Stem Cell, 2008, 2, p. 525-528, Stem Cells. 2008, 26, p. 2467-2474, Nat Biotechnol. 2008, 26, p. 1269-1275, Cell Stem Cell, 2008, 3, p. 568-574, Cell Stem Cell, 2008, 3, p. 475-479, Cell Stem Cell, 2008, 3, p. 132-135, Nat Cell Biol., 2009, 11, p. 197-203, Nat. Biotech., 2009, 27, p. 459-461, Proc Natl Acad Sci USA, 2009, 106, p. 8912-8917, Nature, 2009, 461, p. 643-649, Cell Stem Cell, 2009, 5, p. 491-503, Cell Stem Cell, 2010, 6, p. 167-74, Nature, 2010, 463, p. 1096-100, Stem Cells, 2010, 28, p. 713-720, Nature, 2011, 474, p. The combination of 225-9 is illustrated. iPS cells are available from predetermined institutions (RIKEN BioResource Center, Kyoto University).

  Examples of multipotent stem cells include somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and reproductive stem cells. The multipotent stem cell is preferably a mesenchymal stem cell, more preferably a bone marrow mesenchymal stem cell. Note that the mesenchymal stem cell broadly means a group of stem cells or progenitor cells that can differentiate into all or some mesenchymal cells such as osteoblasts, chondroblasts, and lipoblasts.

  The medium according to the present invention can be suitably used for culturing any stem cell, but is preferably used for culturing mesenchymal stem cells, ES cells or iPS cells, more preferably for culturing iPS cells. Can be used to culture human iPS cells. More specifically, as human iPS cells, 253G1 strain (RIKEN Cell Bank No. HPS0002), 201B7 strain (RIKEN Cell Bank No. HPS0063), 409B2 strain (RIKEN Cell Bank No. HPS0076), 454E2 strain (RIKEN Cell Bank No. HPS0077), HiPS -RIKEN-1A strain (RIKEN Cell Bank No. HPS0003), HiPS-RIKEN-2A strain (RIKEN Cell Bank No. HPS0009), HiPS-RIKEN-12A strain (RIKEN Cell Bank No. HPS0029), and Nippon-B2 strain (RIKEN Cell Bank No. HPS0223).

[Stem cell survival inhibitor, etc.]
As described above, atorvastatin has a survival inhibitory activity specific to undifferentiated stem cells, and suppresses the survival of undifferentiated stem cells without inducing cell death of differentiated cells (particularly cardiomyocytes). For this reason, atorvastatin can be used as a stem cell survival inhibitor in cell culture in vitro, and also in vivo as a stem cell survival inhibitor in vivo, a cell pharmaceutical composition containing stem cell-derived differentiated cells. May be used to suppress

2. Method for Producing Differentiated Cell The method for producing a differentiated cell according to the present invention includes a step of treating the cell after differentiation induction with atorvastatin. More specifically, the method for producing differentiated cells according to the present invention is characterized by including a procedure for culturing cells after differentiation induction in the above-described medium for differentiated cells according to the present invention. However, the method for producing differentiated cells according to the present invention is not intended to exclude that stem cells before differentiation induction are treated with atorvastatin. That is, in the method for producing differentiated cells according to the present invention, at least cells after differentiation induction (which may include stem cells that have maintained an undifferentiated state in addition to differentiated cells) are treated with atorvastatin, in addition to differentiation induction. Previous stem cells may also be treated with atorvastatin.

  Atorvastatin has a survival inhibitory activity specific to undifferentiated stem cells, and suppresses the survival of undifferentiated stem cells without inducing cell death of differentiated cells. For this reason, according to the method for producing a differentiated cell according to the present invention, it is possible to obtain a differentiated cell free from (or very little) of undifferentiated stem cells.

  In addition, if a cell mixture containing stem cells and differentiated cells is used in the atorvastatin treatment step similar to the method for producing differentiated cells according to the present invention, the survival of undifferentiated stem cells is suppressed without inducing cell death of the differentiated cells. Therefore, it is possible to separate only differentiated cells that are free from (or very little) undifferentiated stem cells from the cell mixture (method for separating differentiated cells).

  The method for producing differentiated cells according to the present invention is suitably used particularly for producing cardiomyocytes from stem cells. As will be described later in Examples, statins such as fluvastatin may exhibit cell death-inducing activity against cardiomyocytes differentiated from stem cells. For this reason, the method for producing differentiated cells according to the present invention is particularly suitable for producing cardiomyocytes from stem cells.

  The method for producing differentiated cells according to the present invention may further include a step of culturing stem cells and a step of inducing differentiation of stem cells, in addition to the treatment step with atorvastatin. In these steps, cell culture and stem cell differentiation induction may be performed according to a conventionally known technique, and can be performed in the presence or absence of atorvastatin.

  The incubator used for cell culture is not particularly limited, and examples thereof include flasks, dishes, petri dishes, microwell plates, microslides, chamber slides, tubes, trays, culture vessels such as culture bags or tanks. The substrate of these incubators is not particularly limited, and examples thereof include glass, various plastics such as polypropylene and polystyrene, metals such as stainless steel, or combinations thereof.

  The incubator may be cell-adhesive or non-cell-adhesive, and is appropriately selected according to the purpose. The cell-adhesive incubator can be coated with any cell-supporting substrate such as an extracellular matrix (ECM) for the purpose of improving the adhesion between the surface of the incubator and the cells. The cell support substrate can be any substance intended to adhere to stem cells or feeder cells (if used). Such cell support substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin (or a partial structure of laminin), and fibronectin and mixtures thereof such as matrigel and lysis Cell membrane preparations are mentioned (see Lancet, 2005, 365, 9471, p. 1636-1641).

  The cultured stem cells can be dispersed cells or non-dispersed cells. Dispersed cells refer to cells that have been treated to promote cell dispersion. Examples of the dispersed cells include cells forming a small cell mass composed of several cells (typically 2 to 50, 2 to 20, or 2 to 10 cells). The dispersed cells can be floating (suspension) cells or adherent cells.

The culture density of the cells is not particularly limited as long as the density can achieve the effect of promoting cell survival and proliferation. Preferably 1.0 × 10 ^{1 to} 1.0 × 10 ⁷ cells / ml, more preferably 1.0 × 10 ^{2 to} 1.0 × 10 ⁷ cells / ml, even more preferably 1.0 × 10 ³ to 1.0 × 10 ⁷ cells / ml, most preferably 3.0 × 10 ^{4 to} 1.0 × 10 ⁷ cells / ml.

Culture conditions such as temperature, CO ₂ concentration, oxygen concentration and pH can be appropriately set based on techniques conventionally used for culturing cells derived from animal tissues. For example, the culture temperature is not particularly limited, but may be 30 to 40 ° C, preferably 37 ° C. CO ₂ concentration is 1-10%, preferably be a 2-5%. The oxygen concentration can be 10-25%.

  When performing adherent culture of stem cells, the cells may be cultured in the presence of feeder cells. As the feeder cells, stromal cells such as fetal fibroblasts can be used (for example, Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Pt, 1994, Geskin A Laboratory Press, 1994). Oxford University Press, 1993; Proc. Natl. Acad. Sci. USA, 1981, 78, 12, p.7634-7638, Nature, 1981, 292, 5819, p.154-156, J. Virol., 1969, 4 , 5, p.549-553, Sc ence, 1996,272,5262, p.722-724, J. Cell. Physiol., 1982,112,1, p.89-95, International Publication No. WO 01/088100, see Nos. No. 2005/080554).

  Examples of the suspension culture of stem cells include suspension culture on a carrier (J. Biotechnology, 2007, 132, 2, p. 227-236) or suspension culture using a polymer such as methyl cellulose (Stem Cell Reports, 2014). 2, 5, p. 734-745). The term stem cell suspension culture refers to culturing stem cells in a medium under conditions that are non-adherent to the incubator or feeder cells (if used). Stem cell suspension culture includes stem cell dispersion culture and stem cell aggregation suspension culture. The term stem cell dispersion culture refers to culturing suspended stem cells, and examples include dispersion culture of small cell clusters composed of several (eg, 2 to 20) stem cells. When the dispersion culture is continued, the cultured dispersion cells form a larger stem cell mass, and then aggregated suspension culture can be performed. Examples of such agglutination suspension culture include embryoid body culture method (see Curr. Opin. Cell Biol., 1995, 7, 6, p. 862-869), SFEB method (Nature Neuroscience, 2005, 8, 3, p. 288-296, International Publication No. 2005/123902), a sphere culture method (Stem Cell Reports, 2014, 2, 5, p. 734-745) in which a cell line is passaged by mechanical treatment using a mesh filter. Can be mentioned.

  For stem cell differentiation induction, for example, in the cardiomyocyte differentiation induction process, 0.5 ng / ml BMP-4 is added to a medium (for example, STEMdiff APEL Medium, STEMCELL), and one day later, the medium is 10 ng / ml BMP-4. The medium was replaced with 10 ng / ml Activin A, 5 ng / ml bFGF, and after 4 days, the medium was replaced with 10 ng / ml VEGF, 150 ng / ml Dkk1, and after 8 days, the medium was replaced. Cardiomyocytes with autonomous pulsation can be confirmed by exchanging them with those added with 10 ng / ml VEGF, 150 ng / ml Dkk1, 10 ng / ml bFGF.

  Further, for example, in the chondrocyte differentiation induction process, mesenchymal stem cells are cultured in a medium (90% αMEM medium, 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 μM dexamethasone). Can be done by. Furthermore, for example, by adding a differentiation inducer such as retinoic acid to the medium, it becomes possible to differentiate the stem cells into nervous system cells. BMP inhibitors, Wnt inhibitors, Nodal inhibitors, retinoic acid and the like can also be used as differentiation inducers. For the formation of megakaryocytes required in the process of inducing platelet differentiation, thrombopoietin (TPO), interleukin 3 (IL3), interleukin 6 (through the embryoid body induced in a serum-containing medium (20% FCS) Factors such as IL6) and stem cell factor (SCF) may be used.

3. Method for Producing Cell Pharmaceutical Composition According to the present invention, a method for producing a cell pharmaceutical composition comprising a stem cell-derived differentiated cell comprises a step of inducing differentiation of a stem cell, and a step of treating the cell after differentiation induction with atorvastatin. It is characterized by including. This production method can further include a step of culturing stem cells.

  The differentiation induction step and the treatment step with atorvastatin can be performed by the same procedure as the above-described method for producing differentiated cells. In the method for producing a cell pharmaceutical composition according to the present invention, it is not excluded that stem cells before differentiation induction are treated with atorvastatin.

  Atorvastatin has a survival inhibitory activity specific to undifferentiated stem cells, and suppresses the survival of undifferentiated stem cells without inducing cell death of differentiated cells. Therefore, according to the method for producing a cell pharmaceutical composition according to the present invention, a cell pharmaceutical composition containing differentiated cells and free from (or very little) undifferentiated stem cells can be obtained.

  The cell pharmaceutical composition may be a dispersed differentiated cell, a differentiated cell population forming a cell cluster of a predetermined shape, or a differentiated cell population forming a tissue structure or organelle. The cell pharmaceutical composition can be utilized for cell sources for regenerative medicine. The cell pharmaceutical composition obtained by the production method according to the present invention has no (or very little) contamination of undifferentiated stem cells, so there is no risk of tumor formation or cancer within regeneration after transplantation, and excellent safety is expected. it can. For example, a cell pharmaceutical composition containing cardiomyocytes obtained by the cell pharmaceutical composition according to the present invention is formed into a sheet and can be suitably transplanted to the heart for the treatment of heart disease.

[Example 1: Inhibition of cell survival by atorvastatin in human iPS cell maintenance culture]
The inhibitory activity of atorvastatin on cultured human iPS cells was evaluated.

(Method)
For human iPS cells, human induced pluripotent stem cells (201B7) established by Professor Shinya Yamanaka of Kyoto University iPS Cell Laboratory were obtained from RIKEN Cell Bank (No. HPS0063) and used. Practical protocol for human pluripotent stem cell culture (2nd edition) (Created by RIKEN Center for Developmental and Regenerative Sciences, Stem Cell Research Support and Development Office: http://www.cdb.riken.jp/hsct/protocol.html) Then, undifferentiated human iPS cells were cultured on plastic culture dishes in which mouse fetal fibroblasts (inactivated by mitomycin treatment, MEF) were seeded as a feeder layer of the cells.

In the culture medium (maintenance medium), D-MEMF12 (Sigma D6421), final concentration 20% KSR (Life Technologies), final concentration 1% NON-ESSENTIAL AMINO ACID (× 100) (non-essential amino acid; SIGMA D7145), 2 mM What added L-glutamic acid and 80 micromol 2-mercaptoethanol was used. The culture was performed at 37 ° C. and 5% CO ₂ . Passaging was performed every 3-4 days. Dissociate the iPS cells from the feeder layer using a dissociation solution (phosphate buffer buffered physiological saline supplemented with 0.25% trypsin, 1 mg / ml collagenase IV solution, 1 mM CaCl ₂ ; all Life Technologies) After dispersing into small cell masses (a cell population of about 50-100 cells) by pipetting, they were seeded on the feeder layer formed by seeding MEF the day before.

The human iPS cells cultured as described above are dissociated from the feeder cells as small cell clusters and adsorbed to the bottom of a cell-adhesive culture plate (0.1% gelatin coat) in order to remove the contaminating feeder cells. The cells were cultured in a culture solution (assay medium) at 37 ° C. for 1 hour (iPS cell mass was not adsorbed on the plate, but contaminated feeder cells were strongly adsorbed). After crushing the iPS cell mass into small cell masses by pipetting, 1 × 10 ⁵ cells / 0.65 cm ² / medium volume on a Growth Factor Reduced BD Matrigel (BD) using a 24-well culture plate Seeded at 1.0 ml. After culturing for several days in an assay medium containing statins, the number of formed alkaline phosphatase staining positive (ALP +) colonies was counted as undifferentiated cells and compared with a control group containing no statins. The assay medium used was Essential 8 (Thermo Fisher Scientific, A151701).

(Result 1)
After culturing in an assay medium containing 20.0 μM statins for 48 hours, the number of colonies (ALP +) was measured. The number of colonies in the atorvastatin added group was significantly lower than that in the control group. For other statins (fluvastatin, lovastatin, mevastatin, simvastatin), no obvious survival inhibitory activity against undifferentiated stem cells was observed (FIG. 1).

(Result 2)
After culturing in an assay medium containing 10.0 μM atorvastatin for 24 hours, the number of colonies (ALP +) was measured. In the atorvastatin-added group, the number of colonies was significantly reduced to 60% or less compared to the control group, and suppression of survival of undifferentiated stem cells was observed (FIG. 2).

(Result 3)
After culturing in an assay medium containing 1 μM or 10 μM atorvastatin for 96 hours, the number of colonies (ALP +) was measured. In the atorvastatin addition group, the number of colonies was significantly reduced as compared to the control group, and the survival of undifferentiated stem cells was suppressed (FIG. 3).

[Example 2: Inhibition of cell survival by atorvastatin in differentiated cardiomyocytes]
The survival inhibitory activity (cell death inducing activity) of atorvastatin against differentiated cardiomyocytes differentiated from iPS cells was evaluated.

(Method)
Human iPS cells were dissociated from feeder cells as small cell clumps and adsorbed to the bottom of cell-adhesive culture plates (0.1% gelatin coated) to remove further contaminated feeder cells, and 37 ° C. in assay medium. The cells were cultured for 1 hour (iPS cell mass was not adsorbed on the plate, but contaminated feeder cells were strongly adsorbed). After crushing the iPS cell mass into small cell masses by pipetting, using a 48-well culture plate, the density is increased (1 × 10 ⁶ cells / 0.65 cm ² /0.6) on the Growth Factor Reduced BD Matrigel (BD). Seeded in a medium volume of 0.5 ml).

  Differentiation into the myocardium was performed using PSdif-Cardio Cardiomyocyte Differentiation Kit (Stem RD) according to the protocol attached to the kit. After culturing in a differentiation induction medium (PSdif-Cardio (registered trademark) A, B, and C) for 6 days, the medium was changed to a myocardial culture medium (CardioGro (registered trademark)). The pulsation of the cardiomyocytes induced to differentiate was confirmed under a microscope, and were set as a control group and an experimental group (adding atorvastatin 10.0 μM or fluvastatin 10.0 μM to the myocardial culture medium), and after 24 hours, Live / Dead Cell Staining The ratio of dead cells was measured using a fluorescence microscope using Kit II (PromoKine).

(result)
Fluvastatin showed a significant cell death-inducing activity against cardiomyocytes induced to differentiate from iPS cells (FIG. 4). On the other hand, in atorvastatin, the proportion of dead cells was the same as that in the control group, and differentiation suppression of differentiated cells and induction of cell death were not observed.

  From the above results, it was revealed that atorvastatin significantly suppresses only the survival of undifferentiated stem cells without inducing cell death of differentiated cells. These results indicate that atorvastatin has a survival inhibitory activity specific to undifferentiated stem cells.

Claims (28)

  A medium for differentiated cells derived from stem cells, containing atorvastatin.   The medium according to claim 1, wherein the stem cells are inducible pluripotent stem cells.   The medium according to claim 1 or 2, wherein the differentiated cells are cardiomyocytes.   The medium according to any one of claims 1 to 3, wherein the stem cell is derived from a human.   The medium according to any one of claims 1 to 4, comprising atorvastatin at a concentration of 1 to 20 µM.   The medium according to any one of claims 1 to 5, wherein the medium is a serum-free medium.   A stem cell survival inhibitor containing atorvastatin as an active ingredient.   The stem cell survival inhibitor according to claim 7, wherein the stem cells are inducible pluripotent stem cells.   The stem cell survival inhibitor according to claim 7 or 8, wherein the stem cell is derived from a human.   A pharmaceutical composition containing a stem cell survival inhibitor according to any one of claims 7 to 9, which is a pharmaceutical composition for suppressing tumor formation in vivo of a cellular pharmaceutical composition comprising differentiated cells derived from stem cells. .   A method for producing a differentiated cell from a stem cell, comprising a step of treating the cell after differentiation induction with atorvastatin.   The method according to claim 11, wherein the stem cell is an inducible pluripotent stem cell.   The method according to claim 11 or 12, wherein the differentiated cells are cardiomyocytes.   The method according to any one of claims 11 to 13, wherein the stem cell is derived from a human.   The method according to any one of claims 11 to 14, further comprising a step of culturing the stem cell and a step of inducing differentiation of the stem cell.   A method for producing a cell pharmaceutical composition comprising a stem cell-derived differentiated cell, the method comprising a step of inducing differentiation of the stem cell, and a step of treating the cell after differentiation induction with atorvastatin.   The method according to claim 16, wherein the stem cell is an inducible pluripotent stem cell.   The method according to claim 16 or 17, wherein the differentiated cells are cardiomyocytes.   The method according to any one of claims 16 to 18, wherein the stem cell is derived from a human.   The method according to any one of claims 16 to 19, further comprising culturing stem cells.   A method for separating only differentiated cells from a cell mixture containing stem cells and differentiated cells, comprising a step of treating the cell mixture with atorvastatin.   The method according to claim 21, wherein the stem cells are inducible pluripotent stem cells.   The method according to claim 21 or 22, wherein the differentiated cells are cardiomyocytes.   The method according to any one of claims 21 to 23, wherein the stem cells and the differentiated cells are derived from a human.   The method according to any one of claims 21 to 24, further comprising a step of culturing the stem cell and a step of inducing differentiation of the stem cell.   Use of atorvastatin for the production of the medium according to claim 1, the stem cell survival inhibitor according to claim 7, and the pharmaceutical composition according to claim 10.   Use of atorvastatin for the production of a cell pharmaceutical composition comprising differentiated cells derived from stem cells.   Use of atorvastatin for suppressing in vivo tumorigenesis of a cell pharmaceutical composition containing differentiated cells derived from stem cells.