JP6845500B2 - Method for producing skeletal muscle progenitor cells - Google Patents

Description

The present invention relates to a novel method for producing skeletal muscle progenitor cells, a reagent kit for inducing differentiation used in the method, and a therapeutic agent for myogenic diseases including skeletal muscle progenitor cells produced by the method.

Since the reports of pluripotent cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) obtained by introducing undifferentiated cell-specific genes into somatic cells, myogeny As a method for treating sexual diseases, particularly a method for treating muscular dystrophy, a treatment method for transplanting skeletal muscle progenitor cells induced to differentiate from these pluripotent stem cells has attracted attention.

So far, as a method for inducing differentiation of skeletal muscle precursor cells or skeletal muscle from pluripotent stem cells, (1) after adhering and culturing cells obtained by proliferating a single human ES cell by suspension culture in a serum-free culture medium. , CD73-positive cells are isolated and further cultured, then NCAM-positive cells are isolated and proliferated (Non-Patent Document 1), (2) Human ES cells treated with 5-Azacytidine (demethylating agent) are suspended-cultured. (Non-Patent Document 2), (3) Suspension culture of pluripotent stem cells, then adhesion culture, then dissociation and adhesion culture again (Patent Document 1) and the like have been developed.

However, these methods have a problem that the obtained skeletal muscle progenitor cells and the like do not sufficiently engraft at the transplant destination, and in order to perform muscle regenerative medicine, they are at an appropriate maturation stage suitable for transplantation. Skeletal muscle progenitor cells were needed, and methods for producing such cells needed to be developed.

Special table 2013-527746

Barberi T, et al. Nat Med. 13: 642-8, 2007 Zheng JK, et al. Cell Res. 16: 713-22, 2006

An object of the present invention is to provide a novel method for producing skeletal muscle precursors from pluripotent stem cells. An object of the present invention is also to provide skeletal muscle progenitor cells suitable for transplantation.

As a result of intensive research to solve the above problems, the present inventors have increased skeletal muscle progenitor cells from pluripotent stem cells by adopting a stepwise differentiation induction method using specific factors. We succeeded in guiding with efficiency. In addition, the present inventors have found that skeletal muscle progenitor cells obtained by such a method show an extremely high engraftment rate in transplantation, and have completed the present invention.

That is, the present invention is as follows.
[1] A method for producing skeletal muscle progenitor cells from pluripotent stem cells, which comprises the following steps (1) and (2A) or (2B).
(1) Steps of culturing pluripotent stem cells in a culture medium containing a TGF-β inhibitor and a GSK3β inhibitor (2A) Even if the cells obtained in the steps of (1) contain HGF and further contain IGF1. The cells obtained in the steps (2B) and (1) of culturing in a good culture medium are used.
(i) In a culture medium containing a TGF-β inhibitor, a GSK3β inhibitor, IGF1, HGF and bFGF,
(ii) In culture medium containing TGF-β inhibitor, GSK3β inhibitor and IGF1 and
(iii) The method according to [1], further comprising a step of sequentially culturing in a culture medium containing a TGF-β inhibitor, IGF1 and HGF [2] and the following step (3).
(3) A step of culturing the cells obtained in the steps (2A) or (2B) in a culture medium containing a TGF-β inhibitor, IGF1 and serum [3] The steps (1), (2B) and The method according to [1] or [2], wherein the TGF-β inhibitor of (3) is SB431542.
[4] The method according to any one of [1] to [3], wherein the GSK3β inhibitor in the step (1) is CHIR99021 and the concentration of the CHIR99021 in the medium is 5 μM or more.
[5] The method according to any one of [1] to [4], wherein the GSK3β inhibitor in the step (2B) is LiCl.
[6] The method according to any one of [2] to [5], wherein the serum in the step (3) is horse serum.
[7] The method according to any one of [2] to [6], further comprising the following step (4).
(4) The method according to [7], further comprising the step [8] of isolating the cells expressing Myf5 from the cells obtained in the steps (3), and the following step (5).
(5) A step of culturing the cells obtained in the steps (4) for at least 24 hours [9] The step (5) is a step of culturing in a culture medium containing SB431542, IGF1 and horse serum, [8]. ] The method described in.
[10] The method according to any one of [1] to [9], wherein the pluripotent stem cell is a human iPS cell or a human ES cell.
[11] A reagent kit for inducing differentiation of pluripotent stem cells into skeletal muscle progenitor cells, which comprises SB431542, CHIR99021, IGF1, HGF, and horse serum, and may optionally further contain LiCl and bFGF.
[12] The kit according to [11], wherein the pluripotent stem cells are human iPS cells or human ES cells.
[13] A therapeutic agent for myogenic diseases containing skeletal muscle progenitor cells produced by the method according to any one of [1] to [10].
[14] The agent according to [13], wherein the myogenic disease is muscular dystrophy.
[15] A method for treating a myogenic disease, which comprises administering an effective amount of skeletal muscle progenitor cells produced by the method according to any one of [1] to [10] to a subject.
[16] Skeletal muscle progenitor cells produced by the method according to any one of [1] to [10] for use in the treatment of myogenic diseases.

According to the method of the present invention, skeletal muscle progenitor cells can be efficiently produced from pluripotent stem cells. In addition, the obtained skeletal muscle progenitor cells show a high engraftment rate in a living body, and are therefore extremely useful in transplantation therapy.

The results of examining the differentiation state on the 7th day of induction of differentiation into skeletal muscle progenitor cells are shown. (A) shows the result of FACS analysis in which the cell group on the 7th day of differentiation induction was selected by CD271 (NGFR) and Pax3-GFP. (B) describes the cell groups in each compartment ((1) NGFR-Pax3 +, (2) NGFR + Pax3 +, (3) NGFR + Pax3- and (4) NGFR-Pax3-) for Pax3, T, Tbx6, Mesp2. , PDGFRa, KDR, CD56, Sox10, NGFR, Pax7 and Myf5 gene expression levels are shown by PCR. The value shows a comparative value with respect to the expression level in the teratoma on the 28th day. The results of FACS examination of the positive rate of Pax3 on the 14th day when passage was performed on the 7th day of induction of differentiation into skeletal muscle progenitor cells are shown. 75% in the figure shows the content of Pax3-positive cells. The results of PCR measurement of the expression levels of Tbx6, Wnt3a, Mesp2, PDGFRa, Pax3, and Meox1 in each day of the step of inducing differentiation into skeletal muscle progenitor cells are shown. The results of the fluorescence image of the cells on the 35th day of induction of differentiation into skeletal muscle progenitor cells are shown. (A) is an immunostained image of MHC (red) and DAPI (blue), (B) is a superposed image of Myogenin (red, DAPI (blue) immunostained image and bright field image, and (C) is. , Pax3-GFP (green) fluorescent image is shown. The results of PCR measurement of the expression levels of Myf5 and MyoD in each day of the step of inducing differentiation into skeletal muscle progenitor cells are shown. Induction of differentiation into skeletal muscle progenitor cells The results of fluorescence images of cells on days 57 (A) and 60 (B) are shown. (A) shows a fluorescence image of Pax3-GFP (green), and (B) shows an immunostaining image using an antibody of Embryonic MHC and Myogenin. Immunostaining images of Pax3-GFP positive cells (A) and negative cells (B) on day 84 after induction of differentiation into skeletal muscle progenitor cells are shown on day 7 after separation. The upper row shows immunostained images of MHC (red) and DAPI (blue), and the lower row shows a superposed image of Myogenin (red) and bright-field images. The immunostaining image using the Pax7 (green) and Myf5 (red) antibodies of the cell group on the 84th day of induction of differentiation into skeletal muscle progenitor cells is shown. Induction of differentiation into skeletal muscle progenitor cells Human nuclei and Spectrin (pink), eMYH (green), laminin (white) and mouse nuclei (blue) at the transplantation site 4 weeks after transplantation of cells on day 51 to NSG mice. ) Is shown. Induction of differentiation into skeletal muscle progenitor cells The results of an experiment in which Pax3-GFP-positive cells on day 84 were transplanted into NSG mice are shown. (A) shows a FACS analysis image of the cell group on the 84th day of differentiation induction. (B) shows immunostaining images of human nuclei (red) and eosin (pink) at the transplantation site 4 weeks after transplantation of Pax3-GFP-positive cells into NSG mice (left figure) and laminin (green) and mice. An immunostained image of the nucleus (blue) (right figure) is shown. Myf5-tdTomato positive cells (C3 +, shown in red in the figure) and negative cells (C3-, shown in blue in the figure) Myf5, MyoD, during each day of the step of inducing differentiation into skeletal muscle progenitor cells. The results of PCR measurement of the expression levels of Pax3 and Pax7 are shown. Induction of differentiation into skeletal muscle progenitor cells On day 44, Myf5-tdTomato positive cells (upper) and negative cells (lower) were separated on the 1st day (left figure), 7th day (center figure), and 14th day (center figure). The immunostained image of the cells in the right figure) is shown. On the 1st day (left figure) and 7th day (center figure), Myf5-tdTomato (red) and bright-field image are superimposed, and on the 14th day (right figure), MHC (red) and Myogenin (red). An immunostained image (green) is shown. Induction of differentiation into skeletal muscle progenitor cells On day 71, Myf5-tdTomato positive cells (left figure) or Myf5-tdTomato negative cells (right figure) were transplanted into NSG mice, and 4 weeks after transplantation to human spectrin at each transplantation site. An immunostained image is shown. Induction of differentiation into skeletal muscle progenitor cells On day 78, Myf5-tdTomato-positive cells were recultured for 24 hours, and then immunostained images of human spectrum (green) at the transplant site transplanted into NSG mice (left figure) and human nuclei (left figure). Immunostained images (red), laminin (white) and DAPI (blue) are shown (right figure). The results of FACS examination of the positive rate of Pax3 on the 14th day of induction of differentiation into skeletal muscle progenitor cells when the concentrations of TGF-β inhibitor and GSK3β inhibitor in step (1) were changed are shown. 95% and 92% in the figure indicate the content of Pax3-positive cells. In step (2A), the Myf5 positive rate on the 50th day of induction of differentiation into skeletal muscle progenitor cells when various combinations of TGF-β inhibitors, IGF-1, HGF, and bFGF were added or not was examined by FACS. The results are shown. The vertical axis of the figure is the ratio of the Myf5 positive rate under each condition when the Myf5 positive rate under the condition where all the additives are added is 1. Immunostaining of human Spectrin (green), human nucleus (red), laminin (white) and DAPI (blue) at the transplantation site where Myf5-tdTomato-positive cells on day 35 of induction of differentiation into skeletal muscle progenitor cells were transplanted into NSG mice. Show the image.

The present invention will be described in detail below.

As described above, the present invention comprises a method for producing skeletal muscle progenitor cells from pluripotent stem cells using a specific factor in a specific step, a differentiation-inducing reagent kit containing the specific factor used in the method, and the present invention. The present invention relates to a therapeutic agent for myogenic diseases containing skeletal muscle progenitor cells produced by the method.

In the present invention, "skeletal muscle" means mature muscle and includes muscle fibers, that is, muscle cells which are multinucleated cells. Further, in the present invention, the "skeletal muscle progenitor cell" means a cell that has not reached a mature muscle cell but is in a stage before the mature muscle cell and has an ability to selectively differentiate into a muscle cell. Here, skeletal muscle progenitor cells do not mean that they have no ability to differentiate into other mesophyll cells such as osteoblasts and fat cells, and in some cases, differentiation into cells other than muscle cells. Capable cells can also be included in the skeletal muscle progenitor cells of the present invention. Skeletal muscle progenitor cells are characterized by the expression of specific genes, such as marker genes such as MyoD, Myf5, Pax7, Myogenin, myosin heavy chain, NCAM, Desmin, SkMAct, MF20, M-Cadherin, Fgfr4 and VCAME1. It can be identified by detecting expression. The skeletal muscle progenitor cells in the present invention can preferably be Myf5-positive cells, more preferably Myf5 and Pax7-positive cells. Unless otherwise specified, the skeletal muscle progenitor cells in the present invention include skeletal muscle stem cells or satellite cells, and at least a part thereof can engraft as skeletal muscle stem cells or satellite cells after transplantation.

In the present invention, the differentiation-induced skeletal muscle progenitor cells may be provided as a cell population containing other cell types, or may be a purified cell population. Skeletal muscle progenitor cells need not be the same as naturally occurring skeletal muscle progenitor cells in humans, but may have characteristics that are considerably similar to those of skeletal muscle progenitor cells. Those having properties that are capable of substituting are particularly desirable.

The skeletal muscle progenitor cells produced by the method of the present invention may be any cell population containing at least skeletal muscle progenitor cells, and may include a cell population containing other types of cells together with skeletal muscle progenitor cells. The proportion of skeletal muscle progenitor cells is, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc., but is not limited thereto. Preferably, after producing the skeletal muscle progenitor cells, it is preferable to isolate the skeletal muscle progenitor cells from the cell population.

<Pluripotent stem cells>
The pluripotent stem cells that can be used in the present invention are pluripotent stem cells that can differentiate into all cells existing in the living body and also have proliferative ability. For example, but not limited to, embryonic stem (ES) cells, sperm stem (GS) cells, embryonic reproductive (EG) cells, induced pluripotent stem (iPS) cells, embryonicity derived from cloned embryos obtained by nuclear transplantation. Includes stem (ntES) cells, Muse cells, etc. Preferred pluripotent stem cells are ES cells, iPS cells and ntES cells, more preferably human ES cells, human iPS cells, and even more preferably human iPS cells from the viewpoint of being used for the treatment of myogenic diseases. Is.

(A) Embryonic stem cells
ES cells are pluripotent and self-renewing proliferative stem cells established from the inner cell mass of early mammalian embryos (eg, blastocysts) such as humans and mice.

ES cells are embryo-derived stem cells derived from the inner cell mass of the blastocyst, which is the embryo after the morula at the 8-cell stage of the fertilized egg, and have the ability to differentiate into all the cells that make up the adult, so-called multi-differentiation. It has the ability and the ability to proliferate by self-replication. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and ES cell lines were subsequently established in primates such as humans and monkeys (JA Thomson et. al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod ., 55: 254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).

ES cells can be established by removing the inner cell mass from the blastocyst of the fertilized egg of the target animal and culturing the inner cell mass on a fibroblast feeder. For cell maintenance by subculture, use a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). It can be carried out. For methods of establishing and maintaining ES cells in humans and monkeys, see, for example, USP 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. US A. 92: 7844-7848; Thomson JA, et. al. (1998), Science. 282: 1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932; M. Ueno et al. (2006), Proc . Natl. Acad. Sci. USA, 103: 9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222: 273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99: 1580-1585; Klimanskaya I, et al. (2006), Nature. 444: 481-485, etc.

As the culture medium for ES cell production, for example, DMEM / F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng / ml bFGF was used. , 37 ° C, 2% CO ₂ / 98% Human ES cells can be maintained in a moist atmosphere of air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26: 215-224). Also, ES cells should be passaged every 3-4 days, where passage is 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing _{, for example, 1 mM CaCl 2 and 20% KSR.} Can be done using.

ES cells can generally be selected by the Real-Time PCR method using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, and Nanog as an index. In particular, in the selection of human ES cells, the expression of genetic markers such as OCT-3 / 4, NANOG, and ECAD can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443). -452).

Human ES cell lines, for example, WA01 (H1) and WA09 (H9) are obtained from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan). It is possible.

(B) Sperm stem cells Sperm stem cells are testis-derived pluripotent stem cells, which are the origin cells for spermatogenesis. Similar to ES cells, these cells can induce differentiation into cells of various lineages, and have properties such as the ability to produce chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. (M. Kanatsu-Shinohara et al.). 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). It is self-renewable in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and sperm by repeating passage under the same culture conditions as ES cells. Stem cells can be obtained (Masanori Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Edition), pp. 41-46, Yodosha (Tokyo, Japan)).

(C) Embryonic germ cells Embryonic germ cells are cells that are established from primordial germ cells in the embryonic period and have pluripotency similar to ES cells, such as LIF, bFGF, and stem cell factor. It can be established by culturing primordial germ cells in the presence of the substance of (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550. -551).

(D) Induced pluripotent stem cells Induced pluripotent stem (iPS) cells are similar to ES cells, which can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); International Published WO 2007/069666). Reprogramming factors are genes that are specifically expressed in ES cells, their gene products or non-cording RNAs, or genes that play an important role in maintaining undifferentiated ES cells, their gene products or non-cording RNAs, or It may be composed of low molecular weight compounds. Genes contained in reprogramming factors include, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15. -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 and the like are exemplified, and these reprogramming factors may be used alone or in combination. The combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, 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 / 050626, WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010/102267, WO2010 / 111409, WO 2010/111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008) , Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467-2474 , Huangfu D, et al. (2008), Nat Biotechnol. 26: 1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11: 197-203, RL Judson et al., (2009), Nat. Biotech., 27: 459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106: 8912-8917 , Kim JB, et al. (2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5: 491-503, Heng JC, et al. (2010), Cell The combinations described in Stem Cell. 6: 167-74, Han J, et al. (2010), Nature. 463: 1096-100, Mali P, et al. (2010), Stem Cells. 28: 713-720 Illustrated.

The reprogramming factors include histone deacetylase (HDAC) inhibitors [eg, small molecule inhibitors such as valproic acid (VPA), tricostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA for HDAC (eg). , HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.), MEK inhibitors (eg PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen synthase kinase- 3 Against small molecule inhibitors such as Bio and CHIR99021, DNA methyltransferase inhibitors (eg 5-azacytidine), histone methyltransferase inhibitors (eg BIX-01294), Suv39hl, Suv39h2, SetDBl and G9a Nucleic acid expression inhibitors such as siRNA and shRNA), L-channel calcium agonist (eg Bayk8644), butyric acid, TGFβ inhibitor or ALK5 inhibitor (eg LY364947, SB431542, 616453 and A-83-01), p53 inhibition Agents (eg siRNA and shRNA for p53), ARID3A inhibitors (eg siRNA and shRNA for ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295 and mir-302, Wnt Signaling (eg soluble Wnt3a) ), Neuropeptide Y, prostaglandins (eg, prostaglandins E2 and prostaglandins J2), hTERT, SV40LT, UTF1, IRX6, GLISL, PITX2, DMRTBl, etc. In this specification, the factors used for the purpose of improving the establishment efficiency are not particularly distinguished from the reprogramming factors.

In the case of protein form, the reprogramming factor may be introduced into somatic cells by techniques such as lipofection, fusion with cell membrane penetrating peptides (eg, HIV-derived TAT and polyarginine), and microinjection.
On the other hand, in the case of DNA morphology, it can be introduced into somatic cells by, for example, a vector such as a virus, a plasmid, or an artificial chromosome, lipofection, liposome, or microinjection. Viral vectors include retroviral vectors and lentiviral vectors (Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007. ), Adenovirus vector (Science, 322, 945-949, 2008), adeno-associated virus vector, Sendai virus vector (WO 2010/008054) and the like. Further, as the artificial chromosome vector, for example, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC, PAC) and the like are included. As the plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). The vector can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc. so that nuclear reprogramming substances can be expressed, and, if desired, drug resistance genes ( For example, canamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidin kinase gene, diphtheriatoxin gene, and other selectable marker sequences, green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG, and other reporter gene sequences. Can include. In addition, the above vector has LoxP sequences before and after the gene encoding the reprogramming factor or the promoter and the gene encoding the reprogramming factor that binds to the promoter after introduction into somatic cells. You may.

In the case of RNA form, it may be introduced into somatic cells by a method such as lipofection or microinjection, and in order to suppress degradation, RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) is used. May be (Warren L, (2010) Cell Stem Cell. 7: 618-630).

Cultures for iPS cell induction include, for example, DMEM, DMEM / F12 or DME cultures containing 10-15% FBS (these cultures also include LIF, penicillin / streptomycin, puromycin, L-glutamine). , Non-essential amino acids, β-mercaptoethanol, etc.) or commercially available culture medium [for example, mouse ES cell culture medium (TX-WES culture medium, Thrombo X), primate ES cells Culture medium for culture (culture solution for primate ES / iPS cells, Reprocell), serum-free medium (mTeSR, Stemcell Technology)] and the like are included.

As an example of the culture method, for example, in the _{presence of 5% CO 2} at 37 ° C., the somatic cells are brought into contact with the reprogramming factor on DMEM or DMEM / F12 culture medium containing 10% FBS and cultured for about 4 to 7 days. Then, the cells are re-sown on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.), and about 10 days after contact between the somatic cells and the reprogramming factor, the cells are used in a culture medium for primate ES cell culture containing bFGF. It can be cultured to give rise to iPS-like colonies about 30-about 45 days or more after the contact.

Alternatively, in the _{presence of 5% CO 2} at 37 ° C., DMEM culture medium containing 10% FBS (for example, LIF, penicillin / streptomycin, etc.) on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.). (Puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc. can be appropriately contained), and ES-like colonies can be generated after about 25 to about 30 days or more. .. Desirably, instead of feeder cells, the reprogrammed somatic cells themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or extracellular matrix (eg, Laminin-). 5 (WO2009 / 123349) and Matrigel (BD)) are exemplified.
In addition to this, a method of culturing using a medium containing no serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci US A. 106: 15720-15725). Furthermore, in order to increase the establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237. -241 or WO2010 / 013845).

During the above culture, fresh culture solution and culture solution are exchanged once a day from the second day after the start of culture. The number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 × 10 ³ to about 5 × 10 ⁶ cells ^{per 100 cm 2 culture dish.}

iPS cells can be selected according to the shape of the formed colonies. On the other hand, when a drug resistance gene expressed in conjunction with a gene expressed when somatic cells are reprogrammed (for example, Oct3 / 4, Nanog) is introduced as a marker gene, a culture medium containing the corresponding drug (selection). Established iPS cells can be selected by culturing in (culture solution). In addition, iPS cells are selected by observing with a fluorescence microscope when the marker gene is a fluorescent protein gene, by adding a luminescent substrate when it is a luciferase gene, and by adding a chromogenic substrate when it is a luciferase gene. can do.

As used herein, the term "somatic cell" refers to any animal cell (preferably a mammalian cell, including human) except germline cells such as eggs, oocytes, ES cells or totipotent cells. Say. Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells, as well as primary cultured cells. , Passed cells, and established cells are all included. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue precursor cells, (3) lymphocytes, and epithelium. Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lung cells, renal cells And differentiated cells such as fat cells are exemplified.

In the present invention, the individual mammal from which the somatic cells are collected is not particularly limited, but is preferably human. When the obtained iPS cells are used for human regenerative medicine, somatic cells should be collected from the patient or another person with the same or substantially the same HLA type from the viewpoint of not causing rejection. Is particularly preferable. Here, the HLA type is "substantially the same" when the transplanted cells are transplanted into a patient by inducing differentiation from the iPS cells derived from the somatic cells by using an immunosuppressant or the like. It means that the HLA types match to the extent that they can survive. For example, the main HLA (for example, 3 loci of HLA-A, HLA-B and HLA-DR, or 4 loci including HLA-C) may be the same (the same applies hereinafter).

(E) ES cells derived from cloned embryos obtained by nuclear transplantation
nt ES cells are ES cells derived from cloned embryos produced by nuclear transplantation technology and have almost the same characteristics as ES cells derived from fertilized eggs (T. Wakayama et al. (2001), Science, 292). : 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. (2007), Nature, 450: 497-502). That is, ES cells established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nuclei of unfertilized eggs with the nuclei of somatic cells are nt ES (nuclear transfer ES) cells. For the production of nt ES cells, a combination of nuclear transplantation technology (JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (above) is used (Wakayama). Seika et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Edition), pp. 47-52). In nuclear transplantation, somatic cell nuclei can be injected into unfertilized unfertilized eggs of mammals and cultured for several hours to reprogram them.

(F) Multilineage-differentiating Stress Enduring cells (Muse cells)
Muse cells are pluripotent stem cells produced by the method described in WO 2011/007900, specifically, fibroblasts or bone marrow stromal cells treated with long-term trypsin, preferably 8 or 16 hours of trypsin. It is a pluripotent cell obtained by suspension culture after treatment, and is positive for SSEA-3 and CD105.

<Method of producing skeletal muscle progenitor cells from pluripotent stem cells>
According to the present invention, in producing skeletal muscle progenitor cells from pluripotent cells, a method including the following steps (1) and (2A) or (2B) can be used.
(1) Steps of culturing pluripotent stem cells in a culture medium containing a TGF-β inhibitor and a GSK3β inhibitor (2A) Even if the cells obtained in the steps of (1) contain HGF and further contain IGF1. The cells obtained in the steps (2B) and (1) of culturing in a good culture medium are used.
(i) In a culture medium containing a TGF-β inhibitor, a GSK3β inhibitor, IGF1, HGF and bFGF,
(ii) In culture medium containing TGF-β inhibitor, GSK3β inhibitor and IGF1 and
(iii) Step of sequentially culturing in a culture medium containing a TGF-β inhibitor, IGF1 and HGF.

The present invention also uses TGF-β inhibitors, IGF1 and cells obtained in steps (3) (2A) or (2B) in addition to steps (1) to (2A) or (2B). A method comprising the step of culturing in a culture medium containing serum can be used.

Further, the present invention comprises, in addition to the above steps (1) to (3), a step of isolating cells expressing Myf5 from the cell population obtained in the steps (4) and (3). Can be used.

Further, in the present invention, in addition to the steps (1) to (4), a method including a step of reculturing the cells obtained in the steps (5) and (4) can be used.

The method for inducing differentiation of skeletal muscle progenitor cells in the present invention will be described in detail below.

Preparation for Differentiation Induction This step may include culturing pluripotent stem cells prior to inducing differentiation of pluripotent stem cells into skeletal muscle progenitor cells. Here, pluripotent stem cells may be separated by an arbitrary method and cultured by suspension culture, or may be cultured by adhesive culture using a coated culture dish. Adhesive culture is preferably used in this step. Here, as a method for separating pluripotent stem cells, for example, only a method for mechanically separating, a separation solution having protease activity and collagenase activity (for example, Accutase (TM) and Accumax (TM)) or collagenase activity is used. Examples thereof include a separation method using a separation solution having a substance, a dissociation method using Trypsin / EDTA, and the like. Preferably, a method of dissociating cells with Trypsin / EDTA is used.

In the present invention, the suspension culture is to form an embryo-like body by culturing cells in a non-adherent state to a culture dish, and is not particularly limited, but is artificial for the purpose of improving adhesion to cells. Culture dishes that have not been specifically treated (for example, coated with extracellular matrix) or that have been artificially suppressed (for example, coated with poly-hydroxyethyl methacrylate (poly-HEMA)). Can be done using.

In the present invention, adhesive culture can be performed by culturing on feeder cells or using a culture vessel coated with extracellular matrix. The coating treatment can be performed by putting a solution containing the extracellular matrix in a culture vessel and then appropriately removing the solution.

In the present invention, the feeder cell means another cell that plays an auxiliary role used for adjusting the culture conditions of the target cell. In the present invention, the extracellular matrix is a supramolecular structure existing outside the cell, and may be naturally derived or an artificial product (recombinant). Examples include substances such as collagen, proteoglycans, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, laminin or fragments thereof. These extracellular matrices may be used in combination and may be preparations from cells such as BD Matrigel (TM). Examples of artificial objects include fragments of laminin. In the present invention, laminin is a protein having a heterotrimer structure having one α chain, one β chain, and one γ chain, and is not particularly limited. For example, the α chain is α1, α2, α3, and so on. It is α4 or α5, the β chain is β1, β2 or β3, and the γ chain is exemplified by γ1, γ2 or γ3. In the present invention, the laminin fragment is not particularly limited as long as it is a laminin fragment having an integrin-binding activity, and examples thereof include an E8 fragment which is a fragment obtained by digestion with elastase.

Prior to inducing differentiation of pluripotent stem cells into skeletal muscle progenitor cells, the step of culturing the pluripotent stem cells is preferably adherent culture, and the adherent culture is a culture vessel coated with Matrigel (TM). It can be an adhesive culture using. In the step of culturing pluripotent stem cells, the culture medium used can be prepared using the medium used for culturing animal cells as the basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and StemPro34 (invitrogen). , RPMI-base medium, mTeSR1 and a mixed medium thereof, etc. are included. In this step, mTeSR1 is preferably used. The medium may contain serum or may be serum-free. If desired, the medium may be, for example, albumin, transferase, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen. It may contain one or more serum substitutes such as precursors, trace elements, 2-mercaptoethanol (2ME), thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, low molecular weight compounds, antibiotics, antioxidants, serum, buffers, inorganic salts and the like. The preferred medium is mTeSR1.

The culture period of the step of culturing pluripotent stem cells is exemplified as 1 day or more and 10 days or less, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days. It can be a day, 10 days, etc., preferably 4 days.

When separating pluripotent stem cells in the step of culturing pluripotent stem cells, it is preferable that the medium contains a ROCK inhibitor. The ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK), but for example, Y-27632 can be preferably used in the present invention.

The concentration of Y-27632 in the medium is not particularly limited, but is preferably 1 μM to 50 μM, for example, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM. , 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, but not limited to these. More preferably, it is 10 μM.

The period for adding the ROCK inhibitor to the medium may be the culture period of the step of culturing the pluripotent stem cells, and may be a period for suppressing cell death at the time of monodisperse, for example, at least 1. It's a day.

In the step of culturing pluripotent stem cells, the culturing temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culturing is performed in an atmosphere of _{CO 2 containing air.} The CO ₂ concentration is about 2-5%, preferably 5%.

In the step of culturing pluripotent stem cells, the medium can be exchanged in the middle of the culturing period. The medium used for the medium exchange may be a medium having the same components as the medium before the medium exchange, or a medium having different components. Preferably, a medium having the same components is used. The time of medium replacement is not particularly limited, but is carried out, for example, every 1 day, every 2 days, every 3 days, every 4 days, and every 5 days after the start of culturing in a fresh medium. In this step, the medium change is preferably carried out every two days.

Step (1): A step of culturing pluripotent stem cells in a culture medium containing a TGF-β inhibitor and a GSK3β inhibitor In this step (1), pluripotent stem cells are subjected to a TGF-β inhibitor and a GSK3β inhibitor. It is a step of culturing in the containing culture solution. The culture medium used in this step (1) can be prepared using the medium used for culturing animal cells as the basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and StemPro34 (invitrogen). , RPMI-base medium, and mixed media thereof and the like are included. In this step (1), a mixed medium of IMDM medium and Ham's F12 medium is preferable.

The basal medium may contain serum or may be serum-free. If desired, for example, albumin, transferase, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, selenium (subcerene) It may contain one or more serum supplements such as (sodium acid), collagen precursors, trace elements, 2-mercaptoethanol (2ME), thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non- It may also contain one or more substances such as essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, serums, buffers, inorganic salts. In this step (1), the preferred basal medium is a mixed medium of IMDM medium and Ham's F12 medium supplemented with albumin, transferrin, fatty acid, insulin, selenium and thiolglycerol.

In the present invention, the TGFβ inhibitor is not particularly limited as long as it is a substance that inhibits the signal transduction from the binding of TGFβ to the receptor to SMAD, but for example, it inhibits the binding of TGFβ to the ALK family, which is a receptor. And substances that inhibit the phosphorylation of SMAD by the ALK family. In the present invention, the TGFβ inhibitor is, for example, Lefty-1 (for example, NCBI Accession No., mouse: NM_010094, human: NM_020997), SB431542, SB202190 (above, RKLindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A-83-01 (WO 2009146408) and derivatives thereof. Will be done.

The TGFβ inhibitor used in this step (1) can preferably be SB431542.

The concentration of SB431542 in the medium is not particularly limited, but is preferably 1 μM to 50 μM, for example, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM. , 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, but not limited to these. More preferably, it is 2 μM to 10 μM, and particularly preferably 5 μM.

In the present invention, the GSK-3β inhibitor is defined as a substance that inhibits the kinase activity of the GSK-3β protein (for example, the ability to phosphorylate β-catenin), and many are already known, for example, GSK. Lithium chloride (LiCl), which was first discovered as a -3β inhibitor, BIO (also known as GSK-3β inhibitor IX; 6-bromoinsilvin 3'-oxym), which is an indylvin derivative, and SB216763 (3), which is a maleimide derivative. -(2,4-dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione), GSK-3β inhibitor VII, which is a phenylα bromomethylketone compound (4-Dibromoacetophenone), L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2), a cell membrane-permeable phosphorylated peptide, and CHIR99021 (6- [2- [2- [2-] [4- (2,4-Dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) pyrimidin-2-ylamino] ethylamino] pyridine-3-carbonitrile) can be mentioned. These compounds are commercially available from, for example, Calbiochem, Biomol, etc. and can be easily used, but they may be obtained from other sources or may be prepared by themselves.

The GSK-3β inhibitor used in this step (1) can preferably be CHIR99021.

The concentration of CHIR99021 in the medium is not particularly limited, but is preferably higher than the concentration used in GSK-3β inhibition of 1 μM, eg, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, etc., but are not limited thereto. More preferably, it is 5 μM or more (for example, 5 μM to 50 μM, preferably 5 μM to 10 μM).

In this step (1), the culture period is exemplified by 10 days or more and 30 days or less, for example, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, It can be 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, etc., preferably 16th to 21st. Between, more preferably 16 days.

In this step (1), the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of _{CO 2 containing air.} The CO ₂ concentration is about 2-5%, preferably 5%.

In this step (1), from the viewpoint of increasing the content of desired cells, passage is preferably included in the step. In the present invention, passage is an operation of dissociating cells in culture from a container and reseeding them. In the step of dissociating cells, cells adhering to each other to form a group are dissociated (separated) into individual cells. As a method for dissociating cells, for example, a method for mechanically dissociating, a dissociation solution having protease activity and collagenase activity (for example, Accutase (TM) and Accumax (TM)), and a dissociation solution having only collagenase activity are used. Examples include the dissociation method used, and the dissociation method using Trypsin / EDTA. Preferably, a method of dissociating cells with Trypsin / EDTA is used.

In this step (1), the subculture is not particularly limited, but every 4 to 10 days from the start of this step, for example, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, preferably. Is held every 7 days.

Immediately after passage, the culture medium may contain a ROCK inhibitor for the purpose of preventing cell death of reseeded cells. The ROCK inhibitor can be used under the same conditions as described above.

In this step (1), it is desirable to replace the medium as appropriate. The time of medium replacement is not particularly limited, but is carried out, for example, every 1 day, every 2 days, every 3 days, every 4 days, and every 5 days after the start of culturing in a fresh medium. In this step, the medium change is preferably carried out every two days.

Step (2A): A step of culturing in a culture medium containing HGF and may further contain IGF1 In this step (2A), the cells obtained in the above step (1) may contain HGF and may further contain IGF1. It is a step of culturing in a good culture medium. The culture medium used in this step (2A) can be prepared using the medium used for culturing animal cells as the basal medium. The basal medium includes, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen). , RPMI-base medium, SF-O3 medium (Adia Co., Ltd.) and a mixed medium thereof. In this step (2A), SF-O3 medium is preferably used.

The basal medium may contain serum or may be serum-free. If desired, for example, albumin, transferase, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, selenium (subcerene) It may contain one or more serum supplements such as (sodium acid), collagen precursors, trace elements, 2-mercaptoethanol (2ME), thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non- It may also contain one or more substances such as essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, serums, buffers, inorganic salts. In this step (2), the preferred basal medium is SF-O3 medium supplemented with albumin and 2-mercaptoethanol.

The concentration of HGF in the medium used in this step (2A) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng. / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / ml , 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but not limited to these. More preferably, it is 10 ng / ml.

In this step (2A), it is preferable to carry out the culture in a culture solution containing IGF1 in addition to HGF. The concentration of IGF1 in the medium used in this step (2A) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng. / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / ml , 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but not limited to these. More preferably, it is 10 ng / ml.

In this step (2A), the culture period is exemplified as 1 day or more and 40 days or less, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th , 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, etc., preferably , 20 days.

In this step (2A), the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of _{CO 2 containing air.} The CO ₂ concentration is about 2-5%, preferably 5%.

Step (2B): (i) in a culture medium containing TGF-β inhibitor, GSK3β inhibitor, IGF1, HGF and bFGF, (ii) in a culture medium containing TGF-β inhibitor, GSK3β inhibitor and IGF1, and (iii) Step of sequentially culturing in a culture medium containing a TGF-β inhibitor, IGF1 and HGF In this step (2B), the cells obtained in the above step (1) are subjected to.
(i) In a culture medium containing a TGF-β inhibitor, a GSK3β inhibitor, IGF1, HGF and bFGF,
(ii) In culture medium containing TGF-β inhibitor, GSK3β inhibitor and IGF1 and
(iii) This is a step of sequentially culturing in a culture medium containing a TGF-β inhibitor, IGF1 and HGF. This step is a step performed in place of the previous step step (2A). Hereinafter, the sub-steps (i) to (iii) will be described in order.

Sub-step (i): A step of culturing in a culture medium containing a TGF-β inhibitor, GSK3β inhibitor, IGF1, HGF and bFGF The culture medium used in this sub-step (i) is a medium used for culturing animal cells. Can be prepared as a basal medium. The basal medium includes, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen). , RPMI-base medium, SF-O3 medium (Adia Co., Ltd.) and a mixed medium thereof. In this sub-step (i), SF-O3 medium is preferably used.

The basal medium may contain serum or may be serum-free. If desired, for example, albumin, transferase, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, selenium (subcerene) It may contain one or more serum supplements such as (sodium acid), collagen precursors, trace elements, 2-mercaptoethanol (2ME), thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non- It may also contain one or more substances such as essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, serums, buffers, inorganic salts. In this sub-step (i), the preferred basal medium is SF-O3 medium supplemented with albumin and 2-mercaptoethanol.

As the TGFβ inhibitor used in this sub-step (i), the same ones as described above can be used, but SB431542 may be preferable. The concentration of SB431542 in the medium used in this sub-step (i) is not particularly limited, but is preferably 1 μM to 50 μM, for example, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, but not limited to these. More preferably, it is 10 μM.

As the GSK-3β inhibitor used in this sub-step (i), the same ones as described above can be used, but LiCl can be preferable. The concentration of LiCl in the medium used in this sub-step (i) is not particularly limited, but is preferably 1 mM to 50 mM, for example, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15mM, 20mM, 25mM, 30mM, 40mM, 50mM, but not limited to these. More preferably, it is 5 mM.

The concentration of IGF1 in the medium used in this sub-step (i) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / It is, but is not limited to, ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, and 100 ng / ml. More preferably, it is 10 ng / ml.

The concentration of HGF in the medium used in this sub-step (i) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / It is, but is not limited to, ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, and 100 ng / ml. More preferably, it is 10 ng / ml.

The concentration of bFGF in the medium used in this sub-step (i) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / It is, but is not limited to, ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, and 100 ng / ml. More preferably, it is 10 ng / ml.

In this sub-step (i), the culture period is exemplified by 1 day or more and 10 days or less, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days. , 10 days, etc., preferably 4 days.

In this sub-step (i), the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of _{CO 2-containing air.} The CO ₂ concentration is about 2-5%, preferably 5%.

Sub-step (ii): A step of culturing in a culture medium containing a TGF-β inhibitor, a GSK3β inhibitor and IGF1 In this sub-step (ii), the cells obtained in the sub-step (i) are subjected to TGF-β. This is a step of culturing in a culture medium containing an inhibitor, a GSK3β inhibitor and IGF1. The culture medium used in this sub-step (ii) can be prepared using the medium used for culturing animal cells as the basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and StemPro34 (invitrogen). , RPMI-base medium, SF-O3 medium and mixed media thereof and the like are included. In this sub-step (ii), SF-O3 medium is preferably used.

The basal medium may contain serum or may be serum-free. If desired, for example, albumin, transferase, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, selenium (subcerene) It may contain one or more serum supplements such as (sodium acid), collagen precursors, trace elements, 2-mercaptoethanol (2ME), thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non- It may also contain one or more substances such as essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, serums, buffers, inorganic salts. In this sub-step (ii), the preferred basal medium is SF-O3 medium supplemented with albumin and 2-mercaptoethanol.

As the TGFβ inhibitor used in this sub-step (ii), the same ones as described above can be used, but SB431542 may be preferable. The concentration of SB431542 in the medium used in this sub-step (ii) is not particularly limited, but is preferably 1 μM to 50 μM, for example, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, but not limited to these. More preferably, it is 10 μM.

As the GSK-3β inhibitor used in this sub-step (ii), the same ones as described above can be used, but LiCl can be preferable. The concentration of LiCl in the medium used in this sub-step (ii) is not particularly limited, but is preferably 1 mM to 50 mM, for example, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15mM, 20mM, 25mM, 30mM, 40mM, 50mM, but not limited to these. More preferably, it is 5 mM.

The concentration of IGF1 in the medium used in this sub-step (ii) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml. , 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but not limited to these. More preferably, it is 10 ng / ml.

In this sub-step (ii), the culture period is exemplified by 1 day or more and 10 days or less, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days. , 10 days, etc., preferably 3 days.

In this sub-step (ii), the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of _{CO 2-containing air.} The CO ₂ concentration is about 2-5%, preferably 5%.

Sub-step (iii): A step of culturing in a culture medium containing a TGF-β inhibitor, IGF1 and HGF In this sub-step (iii), the cells obtained in the sub-step (ii) are subjected to a TGF-β inhibitor. , IGF1 and HGF in a culture medium containing IGF1 and HGF. The culture medium used in the sub-step (iii) can be prepared using the medium used for culturing animal cells as the basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and StemPro34 (invitrogen). , RPMI-base medium, SF-O3 medium and mixed media thereof and the like are included. In this sub-step (iii), SF-O3 medium is preferably used.

The basal medium may contain serum or may be serum-free. If desired, for example, albumin, transferase, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, selenium (subcerene) It may contain one or more serum supplements such as (sodium acid), collagen precursors, trace elements, 2-mercaptoethanol (2ME), thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non- It may also contain one or more substances such as essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, serums, buffers, inorganic salts. In this sub-step (iii), the preferred basal medium is SF-O3 medium supplemented with albumin and 2-mercaptoethanol.

As the TGFβ inhibitor used in this sub-step (iii), the same ones as described above can be used, but SB431542 may be preferable. The concentration of SB431542 in the medium used in this sub-step (iii) is not particularly limited, but is preferably 1 μM to 50 μM, for example, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, but not limited to these. More preferably, it is 10 μM. The TGFβ inhibitor used in is preferably SB431542.

The concentration of IGF1 in the medium used in this sub-step (iii) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml. , 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but not limited to these. More preferably, it is 10 ng / ml.

The concentration of HGF in the medium used in this sub-step (iii) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml. , 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but not limited to these. More preferably, it is 10 ng / ml.

In this sub-step (iii), the culture period is exemplified as 7 days or more and 20 days or less, for example, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days. , 16th, 17th, 18th, 19th, 20th, etc., preferably 14th.

In this sub-step (iii), the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of _{CO 2-containing air.} The CO ₂ concentration is about 2-5%, preferably 5%.

In this sub-step (iii), it is desirable to replace the medium as appropriate. The time of medium replacement is not particularly limited, but is carried out, for example, every 1 day, every 2 days, every 3 days, every 4 days, and every 5 days after the start of culturing in a fresh medium. Preferably, it is done every two days. Alternatively, from another point of view, the medium exchange may be carried out, for example, once a week, twice a week, three times a week, four times a week after starting the culture in fresh medium. Preferably, it is done twice a week.

Step (3): Culturing in a culture medium containing a TGF-β inhibitor, IGF1 and serum In this step (3), the cells obtained in the above step (2A) or (2B) are inhibited from TGF-β. This is a step of culturing in a culture medium containing an agent, IGF1 and serum. The culture medium used in this step (3) can be prepared using the medium used for culturing animal cells as the basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and StemPro34 (invitrogen). , RPMI-base medium and mixed media thereof and the like are included. In this step (3), DMEM medium is preferably used.

The basal medium may contain serum or may be serum-free. If desired, for example, albumin, transferase, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, selenium (subcerene) It may contain one or more serum supplements such as (sodium acid), collagen precursors, trace elements, 2-mercaptoethanol (2ME), thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non- It may also contain one or more substances such as essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, serums, buffers, inorganic salts. In this step (3), the preferred basal medium is DMEM medium supplemented with serum, L-glutamine and 2-mercaptoethanol. The serum used in this step (3) is horse serum, and the concentration in the basal medium is 1 to 10%, more preferably 2%.

As the TGFβ inhibitor used in this step (3), the same ones as described above can be used, but SB431542 may be preferable. The concentration of SB431542 in the medium used in this step (3) is not particularly limited, but is preferably 500 nM to 50 μM, for example, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, but not limited to these. More preferably, it is 5 μM.

The concentration of IGF1 in the medium used in this step (3) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / It is, but is not limited to, ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, and 100 ng / ml. More preferably, it is 10 ng / ml.

In this step (3), the culture period is exemplified by 9 days or more and 50 days or less, preferably 20 days or more and 40 days or less, and 30 days or more and 40 days or less. For example, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st , 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, etc.

In this step (3), the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of _{CO 2 containing air.} The CO ₂ concentration is about 2-5%, preferably 5%.

In this step (3), it is desirable to replace the medium as appropriate. The time of medium replacement is not particularly limited, but is carried out, for example, every 1 day, every 2 days, every 3 days, every 4 days, and every 5 days after the start of culturing in a fresh medium. Preferably, it is done every two days. Alternatively, from another point of view, the medium exchange may be carried out, for example, once a week, twice a week, three times a week, four times a week after starting the culture in fresh medium. Preferably, it is done twice a week.

Step (4): Isolation of cells expressing Myf5 In this step (4), cells expressing Myf5 are isolated from the cell population containing the skeletal muscle progenitor cells obtained in step (3). It is a process. In this step (4), the isolation of cells expressing Myf5 is a method capable of detecting the expression of Myf5 in the cells and selectively obtaining cells expressing Myf5 based on the expression or expressing Myf5. The method is not particularly limited as long as it is a method capable of detecting non-expressing cells and selectively removing cells not expressing Myf5 based on the detection. Therefore, it may be isolated using a gene that substitutes for Myf5. As a gene that substitutes for Myf5, a gene existing on the cell surface is preferable, and for example, CD34 or M-cadherin is exemplified.

Isolation of cells expressing Myf5 can be performed, for example, using an antibody that specifically binds to Myf5 or a gene that substitutes for Myf5. Alternatively, it can be done by detecting a marker gene that is functionally linked to the Myf5 promoter. In the present invention, the "marker gene" is a gene encoding a protein that can be translated intracellularly and function as an index, and the protein assists, for example, a fluorescent protein, a photoprotein, fluorescence, luminescence, or coloring. Proteins, including, but not limited to, proteins encoded by drug resistance genes.

In the present invention, the fluorescent proteins include blue fluorescent proteins such as Sirius, BFP and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan and CFP; TurboGFP, AcGFP, TagGFP and Azami-Green (for example, hmAG1). ), ZsGreen, EmGFP, EGFP, GFP2, HyPer and other green fluorescent proteins; TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana and other yellow fluorescent proteins; Orange fluorescent protein such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry; Red fluorescent protein such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (eg hdKeimaRed), mrAsberry Examples include, but are not limited to, near-infrared fluorescent proteins such as mPlum.

In the present invention, the photoprotein can be exemplified by aequorin, but is not limited thereto. In addition, examples of proteins that assist fluorescence, luminescence, or color development include, but are not limited to, enzymes that decompose fluorescence, luminescence, or color development precursors such as luciferase, phosphatase, peroxidase, and β-lactamase.

In the present invention, the protein encoded by the drug resistance gene may be any protein that has resistance to the corresponding drug. The drug resistance genes in the present invention include, but are not limited to, for example, antibiotic resistance genes. Examples of the antibiotic resistance gene include a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, a blastsaidin resistance gene, a gentamicin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, and a chloramphenicol resistance gene. ..

In the present invention, the marker gene can be detected, for example, by introducing a vector in which the sequence of the promoter region of the Myf5 gene is linked upstream of the marker gene into the cell. In this case, cells expressing Myf5 can be identified with cells expressing the marker gene, so it is possible to isolate cells expressing Myf5 depending on whether the marker gene is positive or not. it can.

In the present invention, cell isolation is not particularly limited, but can be performed by, for example, a flow cytometry method. In the flow cytometry method, cell particles are flowed at high speed in a very thin running fluid and irradiated with laser light to emit light such as fluorescence generated by the particles (when the cells are fluorescently labeled in advance) and scattered light. It is for measurement, and if a cell sorter is provided, the target cells can be isolated.

Step (5) Step of re-culturing the isolated cells This step (5) is a step of re-culturing the skeletal muscle progenitor cells isolated in the step (4) in the culture medium. The culture medium used in this step (5) can be prepared using the medium used for culturing animal cells as the basal medium, and for example, the same medium as in step (3) is used, but the culture medium is not limited thereto.

In this step (5), the culture period may be any period as long as the engraftment rate at the transplant destination increases when the skeletal muscle progenitor cells are transplanted, but 6 hours or more and 60 hours or less are exemplified, for example, 6 It can be hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, etc., preferably 24 hours.

<Reagent kit for inducing differentiation of skeletal muscle progenitor cells from pluripotent stem cells>
In one embodiment of the present invention, a reagent kit for inducing differentiation of skeletal muscle progenitor cells from pluripotent stem cells is provided. The kit includes skeletal muscle progenitor cell inducers (eg, lyophilized, frozen solution dissolved in a suitable buffer, etc.), cells, reagents and the above-mentioned skeletal muscle progenitor cell inducers containing specific factors for the induction of skeletal muscle progenitor cells. It may contain a culture solution. The kit may further include a document or instructions describing the procedure for inducing differentiation.

<Use of pluripotent stem cell-derived skeletal muscle progenitor cells>
The pluripotent stem cell-derived skeletal muscle progenitor cells produced by the method of the present invention can be used for various purposes. For example, autologous or allogeneic allogeneic transplantation of skeletal muscle progenitor cells differentiated from iPS cells induced using somatic cells collected from a subject or another person of the same or substantially the same HLA type. Stem cell therapy by transplantation becomes possible. The target disease in the present invention is exemplified by a myopathic disease (myopathy). Myogenic diseases include, for example, muscular dystrophy (eg, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, congenital muscular dystrophy, muscular dystrophy, myotonic muscular dystrophy, etc.), congenital muscular dystrophy, distal muscular dystrophy, mitochondrial dystrophy. Examples thereof include hereditary myopathy such as, polymyopathy, dermatitis, non-hereditary muscular dystrophy such as severe muscular dystrophy, glycogenosis, and periodic limb palsy. A more preferred subject is muscular dystrophy.

As another aspect of the present invention, the skeletal muscle progenitor cells differentiated from the iPS cells derived from the subject are considered to better reflect the state of the muscle cells in the body of the subject. It can also be suitably used for an in vitro evaluation system for toxicity. Furthermore, it can be preferably used as a tool for pathological research of muscle diseases whose cause is unknown.

Therapeutic agent for myogenic disease The present invention provides a therapeutic agent for myogenic disease containing skeletal muscle progenitor cells produced from pluripotent stem cells by the above method. As shown in Examples below, when the skeletal muscle progenitor cells of the present invention were transplanted into NSG mice that had muscle damage by treating with Cardiotoxin (CTx), engraftment of the transplanted skeletal muscle progenitor cells was observed. .. Therefore, the skeletal muscle progenitor cells provided in the present invention are useful for treating myogenic diseases.

As skeletal muscle progenitor cells for the treatment of hereditary muscle diseases such as muscular dystrophy, skeletal muscle progenitor cells induced to differentiate from pluripotent stem cells derived from other people who have the same or substantially the same HLA type as the patient. Cells are preferably used. In human regenerative medicine, it is not easy to obtain human ES cells with the same or substantially the same HLA type, so human iPS cells are used as pluripotent stem cells for inducing skeletal muscle progenitor cells. It is preferable to use it.

In another embodiment, it is also possible to use skeletal muscle progenitor cells differentiated from iPS cells derived from the patient's own body cells as the skeletal muscle progenitor cells for the treatment of hereditary muscle disease. For example, iPS cells derived from somatic cells of DMD patients are deficient in the dystrophin gene, so a normal dystrophin gene is introduced into the iPS cells. Since the dystrophin cDNA has a total length of 14 kb and the adeno-associated virus (AAV) vector, which is optimal for gene transfer into muscle cells, has a capacity of only about 4.5 kb, the current gene therapy strategy is to shorten the functional dystrophin gene. (Microdistrophin gene (3.7 kb)) is introduced with an AAV vector, or a 6.4 kb mini dystrophin gene is introduced with a retrovirus / lentiviral vector capable of inserting larger DNA, or the full-length dystrophin gene is naked. Alternatively, a method of introduction using a Gutted adenovirus vector has been attempted. In the case of iPS cells, the highest transfer efficiency can be obtained with retrovirus / lentivirus, and full-length cDNA can be introduced with an artificial chromosome, which has the advantage of expanding gene therapy options. In the case of limb-girdle muscular dystrophy, since the cause is a sarcoglycan gene abnormality, the gene may be introduced into iPS cells. Alternatively, the mutation site of the causative gene can be repaired by utilizing the endogenous DNA repair mechanism or homologous recombination of iPS cells. That is, a chimeric RNA / DNA oligonucleotide (chimeraplast) having a sequence having a normal mutation site is introduced and bound to a target sequence to form a mismatch, which activates an endogenous DNA repair mechanism to induce gene repair. Alternatively, gene repair can be performed by introducing a homologous single-stranded DNA of 400-800 bases into the mutation site and causing homologous recombination. By inducing differentiation of the iPS cells thus obtained in which the disease-causing gene has been repaired into skeletal muscle progenitor cells through the above steps, normal skeletal muscle progenitor cells derived from the patient can be produced.

Since patients with hereditary muscle disease do not naturally have a normal gene product, even the patient's own skeletal muscle progenitor cells may have an immune response to the normal gene product (eg, dystrophin). Therefore, in any case, it is preferable to use an immunosuppressive agent in combination when transplanting skeletal muscle progenitor cells. Alternatively, for the purpose of avoiding this immune response, in the case of DMD, the dystrophin gene, which is a dystrophin homolog that is also expressed in patient skeletal muscle, may be introduced to replace the dystrophin function.

The skeletal muscle progenitor cells produced by the method of the present invention can be formulated in the form of cells, or can be formulated as they are without sorting.

In the present invention, the desirable skeletal muscle progenitor cell for transplantation is a cell produced through the production steps (1) to (5) of the present invention. By going through the step (5) in the manufacturing process of the present invention, the engraftment rate of the transplanted cells at the transplant destination can be increased. The culture period in the step (5) is preferably 24 hours, but is not limited to this, and may be any period as long as the cell engraftment rate at the transplant destination increases.

The skeletal muscle progenitor cells (including a cell population containing skeletal muscle progenitor cells; the same shall apply hereinafter) of the present invention may be mixed with a pharmaceutically acceptable carrier according to conventional means to inject, suspend, drip, etc. Manufactured as a parenteral preparation of. Pharmaceutically acceptable carriers that can be included in the parenteral preparation include, for example, isotonic solutions containing saline, glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). An aqueous solution for injection can be mentioned. The agents of the present invention include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, lidocaine hydrochloride, prokine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol). , Etc.), preservatives (eg, sodium benzoate, benzalkonium chloride, etc.), antioxidants (eg, ascorbic acid, sodium edetate, etc.) and the like.

When the agent of the present invention is formulated as an aqueous suspension, skeletal muscle progenitor cells may be suspended in the ^{aqueous solution so as to have a concentration of about 1.0 × 10 5} − about 1.0 × 10 ^{7 cells / mL.} Since the preparation thus obtained is stable and has low toxicity, it can be safely administered to mammals such as humans. The administration method is not particularly limited, but is preferably injection or infusion, and examples thereof include intravenous administration, intraarterial administration, and intramuscular administration (local administration to the affected area). The dose of the agent of the present invention varies depending on the administration target, treatment target site, symptom, administration method, etc., but is usually used in DMD patients (assuming a body weight of 60 kg), for example, in the case of intravenous injection. ^{It is convenient to administer about 1.0 × 10 5} − about 1 × 10 ⁷ cells as the amount of skeletal muscle progenitor cells at intervals of about 1 to about 2 weeks, about 4 to about 8 times.

Screening Method for Therapeutic or Preventive Agents for Myogenic Diseases The present invention provides a screening method for candidate agents that are useful agents for the treatment or prevention of myogenic diseases.

In the present invention, a method for screening a therapeutic or prophylactic agent for a myogenic disease may include the following steps:
(1) A step of contacting a candidate substance with skeletal muscle progenitor cells induced to differentiate from iPS cells derived from a patient with myogenic disease.
(2) A step of selecting as a therapeutic or prophylactic agent for myogenic diseases when the pathological condition of the skeletal muscle progenitor cells is alleviated as compared with the case where the cells are not brought into contact with the candidate substance.

When the myogenic disease is muscular dystrophy, the pathology of skeletal muscle progenitor cells can be observed as a deficiency or mutation in the dystrophin protein in the skeletal muscle progenitor cells, or as positive for an inflammatory marker. Here, as an inflammation marker, the activity of prostaglandin D2 or NFkB is exemplified. Alleviation of the condition can be confirmed, for example, by expression of short dystrophin protein or reduction of inflammatory markers by dystrophin protein or exon skipping.

In the present invention, the candidate substance is, for example, a cell extract, a cell culture supernatant, a microbial fermentation product, an extract derived from a marine organism, a plant extract, a purified protein or a crude protein, a peptide, a non-peptide compound, or a synthetic low molecular weight compound. , And natural compounds are exemplified.

In the present invention, candidate substances are also (1) biological libraries, (2) synthetic library methods using deconvolution, and (3) "one-bead one-compound" libraries. It can be obtained using any of the many approaches in combinatorial library methods known in the art, including methods and (4) synthetic library methods using affinity chromatography sorting. Biological library methods using affinity chromatography sorting are limited to peptide libraries, but the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers, or compounds (Lam (1997). ) Anticancer Drug Des. 12: 145-67). Examples of methods for synthesizing molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994) J. Med. Chem . 37: 1233-51). Compound libraries include solutions (see Houghten (1992) Bio / Techniques 13: 412-21) or beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555- 6), bacteria (US Pat. No. 5,223,409), spores (US Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-9) or Phage (Scott and Smith (1990) Science 249: 386-90; Devlin (1990) Science 249: 404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87 : 6378-82; Felici (1991) J. Mol. Biol. 222: 301-10; U.S. Patent Application No. 2002103360).

Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited thereto.

Pluripotent stem cells Human iPS cells (201B7) were donated by Professor Yamanaka of Kyoto University and cultured by a conventional method (Takahashi K, et al. Cell. 131: 861-72, 2007).

The EGFP sequence was ligated to the 3'side of the start codon of the Pax3 locus by homologous recombination of 201B7 according to a conventional method, and an iPS cell line (Pax3-GFP iPSCs) expressing EGFP was prepared in cooperation with Pax3 expression.

Preparation for differentiation induction Prior to differentiation induction into skeletal muscle progenitor cells, human iPS cells (Pax3-GFP iPSCs or Myf5-tdTomato C3 iPSCs) were subjected to low-density culture on a culture vessel coated with MatriGel. Briefly, one 6 cm dish of human iPS cells was treated in a manner similar to normal passage. After dissociation from the MSNL feeder with CTK solution and washed twice with PBS, 1 mL 0.25% Trypsin / 1 mM EDTA was added and incubated at 37 ° C. for 5 minutes. Subsequently, 5 ml of medium was added, pipetting was performed about 15 times, and cells were collected in a 15 ml tube. After centrifugation at 900 rpm for 5 minutes, the supernatant was removed and resuspended in 2 ml mTeSR1 supplemented with 10 μM Y-27632. Then, the number of cells was counted. Separately, dilute MatriGel 50-fold (when left standing for 2 hours) or 100-fold (when left standing for 24 hours) with human iPS cell maintenance medium (without bFGF added), add to the culture vessel, let stand and coat with MatriGel. Was done. Excess MatriGel was removed by suction from the culture vessel, and mTeSR1 supplemented with 10 μM Y-27632 was added in an amount of 2 ml / well at a time. Human Pax3-GFP iPSC single cells, coated 6 cm 1 sheet per 3x10 ⁴ cells dishes in MATRIGEL, and seeded by 1 × 10 ⁴ per 6well plates 1well coated with MATRIGEL. Each culture vessel was shaken well to mix so that the cells spread evenly, and the cells were incubated at 37 ° C. The medium was replaced with mTeSR1 on the 2nd day (Day-2), and the culture was continued until the 4th day (Day 0).

Induction of differentiation into skeletal muscle progenitor cells The iPS cells obtained above were induced to differentiate into skeletal muscle progenitor cells as follows.

<Process (1) (Day 0-20)>
Medium was CDMi basal medium supplemented with 10 μM SB431542 and 10 μM CHIR99021 (1% Albumin from bovine serum (SIGMA), 1% Penicillin-Streptomycin Mixed Solution (Nacalai Tesque), 1% CD Lipid Concentrate (Invitrogen), 1% Insulin- IMDM (1X) Iscove's Medified Dullbecco's Medium (+) L-Glutamine (+) 25 mM HEPES (Invitrogen) and F-12 (1X) Nutrient Mixture (Ham) with Transferrin-Selenium (Invitrogen), 450 μM 1-Thioglycerol (SIGMA) ) (+) L-Glutamine (Invitrogen) mixed 1: 1) at 2 ml / well. Then, the medium was exchanged on the 2nd day (Day 2) and the 5th day (Day 5).

On the 7th day (Day 7), the succession was carried out by the following method. The medium was aspirated, washed twice with PBS, then 500 μL 0.25% Trypsin / 1 mM EDTA was added and incubated at 37 ° C. for 5 minutes. Subsequently, 2.5 ml of CDMi basal medium was added and pipetted to dissociate the cells into single cells. After centrifugation at 900 rpm for 5 minutes at 4 ° C., the supernatant was removed and resuspended in CDMi basal medium. Then, the number of cells was counted. To a culture vessel coated with MatriGel, add CDMi basal medium supplemented with 10 μM SB431542, 5 μM CHIR99021 and 10 μM Y-27632, and add the above cells to ^{1x10 6} cells per 6 cm dish or 4 × 10 ⁵ cells per 6 well plate. Seeded. Medium exchange was performed with CDMi basal medium supplemented with 10 μM SB431542 and 5 μM CHIR99021 on Day 8 (Day 8), Day 10 (Day 10) and Day 12.

On the 14th day (Day 14), the succession was carried out in the same way as Day 7. However, the number of cells to be seeded, 6 × 10 ⁵ cells per one case of 10cm dish, 2 × 10 ⁵ cells per one case of 6cm dish, for 6well plate 1 × 10 ⁵ per one well, or 24well In the case of plates, there were 3 x 10 ⁴ pieces per well.

On the 15th day (Day15), 17th day (Day17) and 19th day (Day19), medium exchange was performed with CDMi basal medium supplemented with 10 μM SB431542 and 5 μM CHIR99021.

<Process (2B) (i) (Day 21-24)>
The cell medium obtained in step (1) was mixed with 0.2% BSA, 200 μM 2-ME (2-Mercaptoethanol), 5 mM LiCl (Nacarai), 10 μM SB431542, 10 ng / ml IGF-1 (Peprotech), 10 ng / ml HGF. The cells were replaced with SF-O3 (Sanko Pure Drug) supplemented with (Peprotech) and 10 ng / ml bFGF (Oriental yeast), and the culture was continued.

<Process (2B) (ii) (Day 25-27)>
The cell medium obtained in steps (2B) and (i) was replaced with SF-O3 supplemented with 0.2% BSA, 200 μM 2-ME, 5 mM LiCl, 10 μM SB431542 and 10 ng / ml IGF-1, and cultured for 3 days. did.

<Process (2B) (iii) (Day 28-41)>
The cell medium obtained in steps (2B) and (ii) was replaced with SF-O3 supplemented with 0.2% BSA, 200 μM 2-ME, 10 μM SB431542, 10 ng / ml IGF-1 and 10 ng / ml HGF, and 2 Incubated for a week. At this time, the medium was exchanged twice a week with the same fresh medium.

<Process (3) (Day42 ~)>
At this stage, MYH3-positive fetal thin myotube cells were scattered. Therefore, in order to promote further maturation, the cell medium obtained in steps (2B) and (iii) was added to DMEM basal medium supplemented with 2% Horse Serum (HS), 5 μM SB431542 and 10 ng / ml IGF-1. I exchanged it. After that, observation was continued as appropriate, and the cells were cultured until at least Day 80. During the culture, the medium was changed twice a week.

Differentiation state on the 7th day of differentiation induction
In order to investigate the state of induction of differentiation into skeletal muscle progenitor cells on Day 7, FACS analysis and comparison of marker gene expression levels were performed. Briefly, for FACS analysis, the cells on Day 7 of the above step (1) were collected, and CD271 negative Pax-GFP positive cells were focused on using CD271 (NGFR) and Pax3-GFP as indicators. The expression level of the marker gene was measured by RT-PCR by measuring the expression level of each gene of Pax3, T, Tbx6, Mesp2, PDGFRa, KDR, CD56, Sox10, NGFR, Pax7 and Myf5.

As a result, it was found that the expression level of marker genes (T, Tbx6 and Mesp2) of cells in the Paraxial mesoderm-somite stage was increased in the Pax3-GFP positive CD271 negative cell group (Fig. 1). At this time, when differentiation induction was performed using a low dose of 1 μM CHIR99021, CD271 positive cells accounted for the majority. Therefore, by using CHIR99021 of 5 μM or more, cells in the Paraxial mesoderm-somite stage were efficiently induced. It was suggested that

Effect of passage on the 7th day of differentiation induction
FACS analysis was performed to investigate the effect of passage on Day 7. Briefly, as shown in the above step (1), the cells were passaged on Day 7 and then cultured until Day 14, and the cells were collected and sorted by FACS using Pax3-GFP as an index. It was.

As a result, it was found that the ratio of Pax3-GFP-positive cells increased to 75% by passage on Day 7 (Fig. 2). It was suggested that Pax3-GFP positive cells were induced more efficiently than when no passage was performed.

Effects of CHiR99021 and SB431542 on day 14 of induction of differentiation FACS analysis was performed to investigate the effects of CHIR99021 and SB431542 on Day 14. Briefly, in step (1), cells cultured until Day 14 were collected in a medium in which CHIR99021 and SB431542 were added in several amounts under the same conditions, and the cells were collected by FACS using Pax3-GFP as an index. It was done by sorting. The results are shown in FIG. In the figure, CH and SB on the graph indicate CHIR99021 and SB431542, respectively, and the number on the right side indicates their concentration (μM).

As a result, it was found that the ratio of Pax3-GFP positive cells increased to 95% in 10 μM CHIR99021 and 5 μM SB431542.

Gene expression state in the Paraxial mesoderm induction process In order to investigate the expression state of the marker gene over time up to the 14th day of induction of differentiation, analysis by RT-PCR was performed. Briefly, the expression status of each gene of Tbx6, Wnt3a, Mesp2, PDGFRa, Pax3, and Meox1 was examined by RT-PCR.

As a result, it was found that the expression of the paraxial mesoderm marker increased until the 7th day of differentiation induction, and after that, the expression of the Somite marker increased (Fig. 3).

Cell observation on day 35 of differentiation induction
When the differentiation status of cells cultured until Day 35 was examined by antibody staining of MHC and Myogenin and fluorescent staining of GFP (Pax3-GFP), Pax3-GFP-positive fetal myotube cell differentiation was induced. Was found (Fig. 4). It was suggested that fetal myotube cells were efficiently induced.

Gene expression state in the induction process up to the 42nd day of differentiation induction In order to investigate the temporal expression state of the marker gene up to the 42nd day of differentiation induction, analysis by RT-PCR was performed. Briefly, the expression status of each gene of Myf5 and MyoD was examined by RT-PCR.

As a result, it was found that the expression of the skeletal muscle differentiation regulator was increased on the 42nd day of the induction of differentiation (Fig. 5).

Maturation of muscle stem cells
In order to confirm the maturation of muscle stem cells after Day 42, observation by antibody staining and fluorescent staining was performed. Briefly, cells cultured up to days 57 and 60 were examined for their differentiation status by antibody staining of Embryonic MHC and Myogenin and fluorescent staining of GFP (Pax3-GFP).

As a result, it was found that Pax3-GFP-negative mature muscle stem cells appeared (Fig. 6A). In addition, Embryonic MHC or Myogenin-positive myotube cells were confirmed (Fig. 6B). This suggests that mature muscle stem cells can be efficiently induced by the existing differentiation induction after the 42nd day.

Effect of long-term culture
The cell group (Pax3-GFP positive cells and negative cells) on the 84th day (Day 84) was stained with an antibody to examine the myotube differentiation potential. Briefly, after sorting by FACS using Pax3-GFP as an index on Day 84, antibody staining of MHC and Myogenin was performed on each cell group cultured under the same conditions for 7 days.

As a result, it was confirmed that Pax3-GFP-negative cells on day 84 of induction of differentiation contained cells strongly stained with MHC and Myogenin (Fig. 7). This suggests that there are Pax3-GFP-negative in vitro myotube-differentiated cells on the 84th day of differentiation induction.

Furthermore, antibody staining was performed to examine whether satellite cells were present in the cell group on Day 84. Briefly, antibody staining of Pax7 and Myf5 was performed on the cell population on Day 84.

As a result, it was found that in the cell group on the 84th day of differentiation induction, there were cells expressing a marker gene similar to that of satellite cells, Pax7 positive and Myf5 negative (Fig. 8).

Transplant experiment (1)
The effect of skeletal muscle progenitor cells on day 51 of differentiation induction in transplantation into mice was investigated. Briefly, the cell group on day 51 of the above step (3) was intramuscularly transplanted into the tibialis anterior muscle of NSG mice one day after CTx treatment. Four weeks after transplantation, the area around the transplantation site was excised, sections were prepared according to a conventional method, and antibody staining of human nuclei, spectrin, eMYH and laminin was performed to evaluate the engraftment and muscle regeneration of the transplanted cells.

As a result, when the cell group on the 51st day of differentiation induction was transplanted, engraftment of the transplanted cells and formation of muscle fibers were confirmed (Fig. 9). It was suggested that there are cells capable of forming phase muscle fibers.

Transplant experiment (2)
The effect of skeletal muscle progenitor cells on day 84 of differentiation induction in transplantation into mice was investigated. Briefly, Pax3-GFP positive cells were selected by FACS from the cell group on day 84 in the above step (3). Subsequently, 2.6 × 10 ⁵ Pax3-GFP-positive cells were intramuscularly transplanted into the tibialis anterior muscle of NSG mice 1 day after CTx treatment. Four weeks after transplantation, antibody staining of human nuclei, eosin and laminin was performed to evaluate transplanted cell engraftment and muscle regeneration.

As a result, when Pax3-GFP-positive cells on day 84 of differentiation induction were transplanted, fusion with host muscle fibers was confirmed (Fig. 10). Therefore, the Pax3-GFP-positive cell group on day 84 of differentiation induction was confirmed. It was suggested that there are cells that contribute to muscle regeneration.

Pluripotent stem cells
By homologous recombination of 201B7 according to a conventional method, the tdTomato sequence is ligated to the 5'side of the start codon of the Myf5 locus, and an iPS cell line (Myf5-tdTomato C3 iPSCs) expressing tdTomato in cooperation with Myf5 expression is prepared. did.

Induction of differentiation into skeletal muscle progenitor cells The iPS cells obtained above were prepared for differentiation induction as in Example 1, and differentiation induction of skeletal muscle progenitor cells was performed as follows.

<Process (1') (Days 0 to 17)>
Culturing was carried out for 18 days under the same conditions as in step (1) of Example 1. On the 7th day (Day7) and the 14th day (Day14), the subculture was performed in the same manner as described above.

<Process (2B) (i) (Day 18-21)>
The cells obtained in step (1') were cultured for 4 days under the same conditions as in steps (2B) and (i) of Example 1.

<Process (2B') (ii) (Day 22-24)>
The cells obtained in steps (2B) and (i) were cultured for 3 days under the same conditions as in steps (2B) and (ii) of Example 1.

<Process (2B') (iii) (Day 25-39)>
The cells obtained in steps (2B') and (ii) were cultured for 14 days under the same conditions as in steps (2B) and (iii) of Example 1.

<Process (3') (Day40 ~)>
The cells obtained in steps (2B') and (iii) were continuously cultured under the same conditions as in step (3) of Example 1, and the cells obtained after an appropriate process were used.

Gene expression status during differentiation induction process
Using Myf5-tdTomato C3 iPSCs, differentiation was induced into muscle progenitor cells by the method described in Example 1. The cell groups (Myf5-tdTomato positive cells and negative cells) at this time were analyzed by RT-PCR in order to investigate the expression state of the marker gene over time from Day 34 to Day 85. Briefly, Myf5-tdTomato positive or negative cells were selected by FACS for the Day 84 cell group in the above step (3'), and each gene of Myf5, MyoD, Pax3, and Pax7 was expressed by RT-PCR, respectively. I checked the condition.

As a result, it was found that cells having high expression of Pax7 as well as Myf5 were included in the Myf5-tdTomato-positive cell group (Fig. 11). That is, it was suggested that a group of cells likely to be satellite cells was included.

Evaluation of Myf5-positive cells
The cell group (Myf5-tdTomato positive cells and negative cells) on the 44th day (Day 44) was stained with an antibody to examine myotube differentiation ability. Briefly, antibody staining of MHC and Myogenin was performed on each cell group cultured under the condition of step (3') for 1, 7 or 14 days after sorting by FACS using Myf5-tdTomato as an index on Day44. Was done.

As a result, it was confirmed that the Myf5-tdTomato-positive cells on the 44th day of differentiation induction contained many cells that changed into cells strongly stained with MHC and Myogenin (Fig. 12). This suggests that Myf5-tdTomato-positive cells include cells capable of muscle cell differentiation.

Transplant experiment (1')
A transplant experiment was conducted on the skeletal muscle progenitor cells (Myf5-positive cells on day 71) induced by the differentiation induction method of this example in order to investigate the effect on transplantation. Briefly, Myf5-tdTomato-positive cells were selected by FACS from the cell group on day 71 in the above step (3'). 7.5 × 10 ⁵ Myf5-tdTomato positive cells immediately after selection or 6.1 × 10 ⁵ Myf5-tdTomato negative cells immediately after selection were intramuscularly transplanted into the tibialis anterior muscle of NSG mice 1 day after CTx treatment. .. Four weeks after transplantation, human spectrin antibody staining was performed to evaluate muscle regeneration.

As a result, muscle regeneration was confirmed when Myf5-positive cells on the 71st day of differentiation induction were transplanted (Fig. 13). Therefore, the Myf5-positive cells on the 71st day of differentiation induction were a cell population that contributed to muscle regeneration. Was suggested to exist.

Transplant experiment (2')
A transplant experiment was conducted on the skeletal muscle progenitor cells (Myf5-positive cells on day 78) induced by the differentiation induction method of this example in order to investigate the effect on transplantation. Briefly, Myf5-positive cells were selected by FACS from the cell group on day 78 in the above step (3'). Subsequently, 5 × 10 ⁵ Myf5-positive cells were recultured for 24 hours and then intramuscularly transplanted into the tibialis anterior muscle of NSG mice 1 day after CTx treatment. Four weeks after transplantation, human spectrin antibody staining was performed to evaluate muscle regeneration.

As a result, muscle regeneration was confirmed when Myf5-positive cells on day 78 of differentiation induction were recultured for 24 hours and then transplanted (Fig. 14).

Pluripotent stem cells
By homologous recombination of 201B7 according to a conventional method, the tdTomato sequence is linked to the 5'side of the start codon of the Myf5 locus, and iPS cell lines expressing tdTomato in cooperation with Myf5 expression (Myf5-tdTomato C3 iPSCs and Myf5-) tdTomato E16 iPSCs) was prepared.

Induction of differentiation into skeletal muscle progenitor cells The iPS cells obtained above were prepared for differentiation induction as in Example 1, and differentiation induction of skeletal muscle progenitor cells was performed as follows.

<Process (1 ") (Days 0 to 17)>
Culturing was carried out for 18 days under the same conditions as in step (1) of Example 1. On the 7th day (Day7) and the 14th day (Day14), the subculture was performed in the same manner as described above.

<Process (2A) (Day 18-39)>
The cell medium obtained in step (1 ") was added to SF-O3 (Sanko Pure Drug) supplemented with 0.2% BSA and 200 μM 2-ME (2-Mercaptoethanol) to 10 μM SB431542 and / or 10 ng / ml IGF-. The medium was replaced with a medium supplemented with 1 (Peprotech) and / or 10 ng / ml HGF (Peprotech) and / or 10 ng / ml bFGF (Oriental yeast), or a medium without any addition, and the cells were cultured for 21 days.

<Process (3 ") (Day40 ~)>
The cells obtained in the step (2A) were continuously cultured under the same conditions as in the step (3) of Example 1, and the cells obtained after an appropriate process were used.

Gene expression status during differentiation induction process
Using Myf5-tdTomato C3 iPSCs and Myf5-tdTomato E16 iPSCs, differentiation was induced into muscle progenitor cells by the above steps (1 "), (2A) and (3"). Regarding the cell groups (Myf5-tdTomato positive cells and negative cells) at this time, the ratio of the Myf5-tdTomato positive cell group was evaluated. As a result, in step 2A, Myf5-tdTomato-positive cells were present, and after culturing in a medium containing HGF and no TGFβ inhibitor and bFGF, particularly a medium containing only HGF and IGF1, Myf5 It was found that it contained a large amount of -tdTomato-positive cells (Fig. 16).

Transplant experiment (1 ")
A transplant experiment was conducted on the skeletal muscle progenitor cells (Myf5-positive cells on day 35) induced by the differentiation induction method of this example in order to investigate the effect on transplantation. Briefly, Myf5-tdTomato-positive cells were selected by FACS from the cell group on day 35 in the above step (2A). 3.7 × 10 ⁵ Myf5-tdTomato positive cells immediately after selection or 3.7 × 10 ⁶ Myf5-tdTomato negative cells immediately after selection were intramuscularly transplanted into the tibialis anterior muscle of NSG mice 1 day after CTx treatment. .. Four weeks after transplantation, human Spectrin antibody staining was performed to evaluate muscle regeneration.

As a result, muscle regeneration was confirmed when Myf5-positive cells were transplanted on the 35th day of differentiation induction (Fig. 17). Therefore, the Myf5-positive cells on the 35th day of differentiation induction were a cell population that contributed to muscle regeneration. Was suggested to exist.

Although the present invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that preferred embodiments can be modified. The present invention is intended that the invention can be practiced in ways other than those described in detail herein. Accordingly, the present invention includes all modifications contained within the spirit and scope of the appended claims.
The contents of all publications, including the patents and patent application specifications mentioned herein, are incorporated herein by reference in their entirety to the same extent as expressly stated. ..

This application is based on Japanese Patent Application No. 2014-267085 filed in Japan on December 29, 2014, the contents of which are incorporated herein by reference in its entirety.

According to the method of the present invention, it is possible to provide an efficient method for inducing differentiation of pluripotent stem cells into skeletal muscle precursors. In addition, the skeletal muscle precursors obtained by the method of the present invention show a high engraftment rate in a living body, and are therefore extremely useful in transplantation therapy.

Claims (15)

A method for producing human skeletal muscle progenitor cells from human pluripotent stem cells, which comprises the following steps (1) and (2A) or (2B).
(1) The cell population obtained in the steps (2A) and (1) of culturing a population of human pluripotent stem cells in a culture medium containing a TGF-β inhibitor and a GSK3β inhibitor for 14 days or more and 21 days or less. , The cell population obtained in the steps (2B) and (1) of culturing in a culture medium containing HGF and may further contain IGF1 for 14 days or more and 21 days or less.
(i) 1 day or more and 10 days or less in a culture medium containing a TGF-β inhibitor, GSK3β inhibitor, IGF1, HGF and bFGF,
(ii) 1 day or more and 10 days or less in a culture medium containing a TGF-β inhibitor, GSK3β inhibitor and IGF1 and
(iii) Step of sequentially culturing in a culture medium containing a TGF-β inhibitor, IGF1 and HGF for 7 days or more and 20 days or less. The method according to claim 1, further comprising the following step (3).
(3) A step of culturing the cell population obtained in the step (2A) or (2B) in a culture medium containing a TGF-β inhibitor, IGF1 and serum for 9 to 50 days. The method according to claim 1 or 2, wherein the TGF-β inhibitor in steps (1) and (2B) is SB431542. The method according to any one of claims 1 to 3, wherein the GSK3β inhibitor in the step (1) is CHIR99021, and the concentration of the CHIR99021 in the medium is 5 μM or more. The method according to any one of claims 1 to 4, wherein the GSK3β inhibitor in the step (2B) is LiCl. The method according to claim 2, wherein the serum in the step (3) is horse serum. The method according to claim 2 or 6, further comprising the following step (4).
(4) A step of isolating cells expressing Myf5 from the cells obtained in the steps of (3). The method according to claim 7, further comprising the following step (5).
(5) A step of culturing the cells obtained in the steps of (4) for at least 24 hours. The method according to claim 8, wherein the step (5) is a step of culturing in a culture medium containing SB431542, IGF1 and horse serum. The method according to any one of claims 1 to 9, wherein the human pluripotent stem cell is a human iPS cell or a human ES cell. The method according to any one of claims 1 to 10, wherein the proportion of PAX3 positive cells in the entire cell population obtained in the step (1) is 90% or more. 11. The method of claim 11, comprising the step (2B). The method according to any one of claims 1 to 11, wherein the step (2A) is included, and the step is performed in a culture medium containing HGF and IGF1 and not containing a TGF-β inhibitor and bFGF. A reagent kit for inducing differentiation of human pluripotent stem cells into human skeletal muscle progenitor cells, which comprises SB431542, CHIR99021, IGF1, HGF and horse serum, and may optionally further contain LiCl and bFGF. The kit according to claim 14, wherein the human pluripotent stem cells are human iPS cells or human ES cells.