# Patent

## Title
Method for inducing differentiation of pluripotent stem cells into skeletal muscle or skeletal muscle progenitor cells

## Abstract

A method for inducing the differentiation of pluripotent stem cells into skeletal muscle or skeletal muscle progenitor cells is provided. Specifically, a method for producing artificial skeletal muscle or skeletal muscle progenitor cells from human pluripotent stem cells is provided, comprising the following steps of: (1) culturing human pluripotent stem cells by suspension culture; (2) culturing a cell population after suspension culture by adhesion culture; (3) dissociating cells after adhesion culture; and (4) culturing the dissociated cells by adhesion culture. Artificial skeletal muscle or induced skeletal muscle progenitor cells prepared by the method are also provided.

## Background
TECHNICAL FIELD
The present invention relates to a method for inducing differentiation of pluripotent stem cells into skeletal muscle or skeletal muscle progenitor cells.
The present invention also relates to artificial skeletal muscle or skeletal muscle progenitor cells prepared by the above method.
BACKGROUND ART
Cells having pluripotency such as embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) obtained via introduction of undifferentiated cell-specific genes into somatic cells have been reported (U.S. Pat. No. 5,843,780 or WO 2007/069666). Hence, as a method for treating myogenic diseases, and particularly, a method for treating muscular dystrophy, a therapeutic method involving transplanting skeletal muscle progenitor cells resulting from induction of the differentiation of pluripotent stem cells has recently received attention. Similarly, development of a therapeutic agent using homogenous skeletal muscle is also under consideration.
Here, methods for inducing the differentiation of ES cells into skeletal muscle progenitor cells or skeletal muscle have been developed as follows: (1) a method that involves proliferating a single human ES cell by suspension culture, culturing the resulting cells by adhesion culture in a serum free culture solution, isolating CD73 positive cells, further culturing the cells, isolating NCAM positive cells, and proliferating the cells (Barberi T, et al. Nat Med. 13: 642-8, 2007); and (2) a method that involves treating human ES cells with 5-Azacytidine (demethylating agent), culturing the treated human ES cells by suspension culture to form embryoid bodies, and then further carrying out adhesion culture (Zheng JK, et al. Cell Res. 16:713-22, 2006), for example.
However, these methods are problematic in that: it is necessary to isolate cells several times and although engraftment of induced skeletal muscle can be confirmed by transplantation into model mice, the resulting amount of the skeletal muscle is extremely low.
DISCLOSURE OF THE INVENTION
Problem to be Solved by the Invention
One object of the present invention is to provide a method for inducing the differentiation of pluripotent stem cells into skeletal muscle or skeletal muscle progenitor cells by the steps of suspension culture, adhesion culture, and dissociation-adhesion culture.
Means for Solving the Problem
The present invention has the following characteristics.

    [1] A method for producing artificial skeletal muscle or skeletal muscle progenitor cells from human pluripotent stem cells, comprising the following steps of:
    (1) culturing human pluripotent stem cells by suspension culture,
    (2) culturing a cell population after suspension culture by adhesion culture,
    (3) dissociating cells after adhesion culture; and
    (4) culturing dissociated cells by adhesion culture.
    [2] The method according to [1], wherein in step (1) above, human pluripotent stem cells are cells of colonies formed by adhesion culture and the colonies are directly subjected to suspension culture.
    [3] The method according to [1] or [2], wherein in step (2) above, cells are cultured in a serum free medium containing insulin, transferrin, and sodium selenite.
    [4] The method according to any one of [1] to [3], wherein in step (4) above, dissociated cells are cultured at an initial density of 1,000-3,000 cells/cm2.
    [5] The method according to any one of [1] to [4], wherein in step (4) above, cells are cultured in a medium containing fetal calf serum and horse serum.
    [6] The method according to any one of [1] to [5], wherein in step (1) above, the culture period is 7 days.
    [7] The method according to any one of [1] to [6], wherein in step (2) above, the culture period is 14 days.
    [8] The method according to any one of [1] to [7], wherein in step (4) above, the culture period is at least 21 days.
    [9] The method according to [5], wherein in step (4) above, after the above culture in a medium containing the fetal calf serum and horse serum, the medium is exchanged with a serum free medium containing insulin, transferrin, and sodium selenite, and then cells are further cultured.
    [10] The method according to any one of [1] to [9], wherein the human pluripotent stem cells are human embryonic stem (ES) cells or human induced pluripotent stem (iPS) cells.
    [11] A kit for producing skeletal muscle or skeletal muscle progenitor cells from human pluripotent stem cells, containing with instructions the following substances:
    (1) a serum free medium containing insulin, transferrin, and sodium selenite;
    (2) a medium containing fetal calf serum and horse serum; and
    (3) a cell dissociation solution.
    [12] Artificial skeletal muscle or skeletal muscle progenitor cells, which are prepared from human pluripotent stem cells by the method according to any one of [1] to [10].
    [13] The artificial skeletal muscle or skeletal muscle progenitor cells according to [12], containing DNA encoding normal human dystrophin in a form that can be expressed.
    [14] Use of the artificial skeletal muscle or skeletal muscle progenitor cells prepared from human pluripotent stem cells by the method according to any one of [1] to [10] in preparation of a medicament for treating a myogenic disease.
    [15] Composition comprising the artificial skeletal muscle or skeletal muscle progenitor cells prepared from human pluripotent stem cells by the method according to any one of [1] to [10].


The contents as disclosed in the description and/or drawings of U.S. Provisional Patent Application No. 61/326,929, to which the present application claims priority, are incorporated herein.
Effect of the Invention
Artificial skeletal muscle or skeletal muscle progenitor cells can be prepared by the method according to the present invention.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows a scheme for culturing, by which skeletal muscle or skeletal muscle progenitor cells are prepared from human pluripotent stem cells.
FIG. 2 shows the following immunostaining images of cells on day 70 after induction of differentiation: (a) DAPI (4′,6-diamidino-2-phenylindole); (b) myogenin (Clone: F5D); (c) myosin (skeletal muscle); and (d) an image obtained by merging these images. In FIG. 2, open arrowheads indicate myogenin positive cell nuclei and white arrowheads indicate myosin-positive cells.
FIG. 3 shows the images of cells on day 70 after induction of differentiation, specifically: an immunostaining images (A) and a phase-contrast images (B) of (a) DAPI (4′,6-diamidino-2-phenylindole); (b) myogenin; and (c) myosin (skeletal muscle).
FIG. 4 shows the immunostaining images of muscle tissue on 4 weeks after transplantation of differentiated cells: (a) human Laminin (merosin); (b) murine/human Laminin; (c) merged image of (a) and (b); (d) DAPI (4′,6-diamidino-2-phenylindole); (e) human Laminin A/C; (f) murine/human Laminin; (g) merged image of (d) and (e); (h) merged image of (e) and (f); and (i) merged image of (d), (e) and (f).



EMBODIMENTS FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
The present invention relates to a method for inducing the differentiation of skeletal muscle or skeletal muscle progenitor cells, comprising culturing human pluripotent stem cells by suspension culture, adhesion culture, and then adhesion culture again after dissociation.
<Pluripotent Stem Cells>
Pluripotent stem cells that can be used in the present invention are stem cells having both pluripotency, by which the cells are capable of differentiating into all cells existing in vivo, and proliferation potency. Examples of these pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, embryonic stem (nuclear transfer ES: ntES) cells from clone embryos obtained by nuclear transplantation, spermatogonial stem cells (“germline stem cells: GS cells”), embryonic germ cells (“EG cells”), and induced pluripotent stem (iPS) cells. Examples of preferable pluripotent stem cells include ES cells, ntES cells, and iPS cells.
(A) Embryonic Stem Cells
ES cells are stem cells having pluripotency and proliferation potency via self-replication, which are established from inner cell mass of early embryos (e.g., blastocysts) of a mammal such as a human or a mouse.
ES cells are stem cells from embryos originated from inner cell mass of blastocysts that are embryos after the morula stage of fertilized eggs. ES cells have so-called pluripotency, by which they are capable of differentiating into all cells composing an adult, and proliferation potency via self-replication. ES cells were discovered in mice in 1981 (M. J. Evans and M. H. Kaufman (1981), Nature 292: 154-156). Thereafter, ES cell lines were also established in primates including humans, monkeys, and the like (J. A. Thomson et al. (1999), Science 282:1145-1147; J. A. Thomson et al. (1995), Proc. Natl. Acad. Sci. U.S.A., 92:7844-7848; J. A. Thomson et al. (1996), Biol. Reprod., 55: 254-259; J. A. Thomson and V. S. Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).
ES cells can be established by removing inner cell mass from blastocysts of fertilized eggs of a subject animal and then culturing the inner cell mass on fibroblasts as feeders. Also, cell maintenance by subculture can be carried out using a medium supplemented with substances such as a leukemia inhibitory factor (LIF) and a basic fibroblast growth factor (bFGF). Methods for establishment and maintenance of human and monkey ES cells are described in H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. U.S.A., 103: 9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222: 273-279; and H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. U.S.A., 99: 1580-1585, for example.
As a medium for preparation of ES cells, a DMEM/F-12 medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM nonessential amino acid, 2 mM L-glutamic acid, 20% KSR, and 4 ng/ml 13-FGF is used, for example. Human ES cells can be maintained under wet atmosphere of 2% CO2/98% air at 37° C. (O. Fumitaka et al. (2008), Nat. Biotechnol., 26: 215-224). Also, it is necessary for ES cells to subculture every 3 to 4 days. At this time, subculture can be carried out using 0.25% trypsin and 0.1 mg/ml collagenase IV in PBS containing 1 mM CaCl2 and 20% KSR, for example.
ES cells can be generally selected by using the expression of a gene marker such as alkaline phosphatase, Oct-3/4 and Nanog as an index. The markers can be detected by Real-time PCR, western-blotting, immunostaining and so on. In particular, for selection of human ES cells, the expression of a gene marker 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, such as KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).
(B) Spermatogonial Stem Cells
Spermatogonial stem cells are testis-derived pluripotent stem cells, serving as an origin for spermatogenesis. Spermatogonial stem cells can also be induced to differentiate into cells of various lines in a manner similar to that in ES cells. For example, the cells have properties such that a chimeric mouse can be produced when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). Spermatogonial stem cells are self-replicable in a medium containing a glial cell line-derived neurotrophic factor (GDNF) or spermatogonial stem cells can be obtained by repeated subculture of the cells under culture conditions similar to those for ES cells (Masanori Takebayashi et al., (2008), Experimental Medicine, Vol. 26, No. 5 (Extra Number), pp. 41-46, YODOSHA (Tokyo, Japan)).
(C) Embryonic Germ Cells
Embryonic germ cells are cells established from primordial germ cells at the prenatal period and have pluripotency similar to that of ES cells. Embryonic germ cells can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and a stem cell factor (Y. Matsui et al. (1992), Cell, 70: 841-847; J.L. Resnick et al. (1992), Nature, 359: 550-551).
(D) Induced Pluripotent Stem Cells
Induced pluripotent stem (iPS) cells can be prepared by introducing a specific nuclear reprogramming substance in the form of DNA or protein into somatic cells. iPS cells are somatic cell-derived artificial stem cells having properties almost equivalent to those of ES cells, such as pluripotency and proliferation potency via self-replication (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; M. Nakagawa et al. (2008) Nat. Biotechnol., 26: 101-106; international publication WO 2007/069666). A nuclear reprogramming substance may be a gene specifically expressed in ES cells, a gene playing an important role in maintenance of undifferentiation of ES cells, or a gene product thereof. Examples of such nuclear reprogramming substance include, but are not particularly limited to, Oct3/4, Klf4, Klfl, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Esrrb, and Esrrg. These reprogramming substances may be used in combination upon establishment of iPS cells. Such combination may contain at least one, two, or three reprogramming substances above and preferably contains four reprogramming substances above.
The nucleotide sequence information of the mouse or human cDNA of each of the above nuclear reprogramming substances and the amino acid sequence information of a protein encoded by the cDNA can be obtained by referring to NCBI accession numbers described in WO 2007/069666. Also, the mouse and human cDNA sequence and amino acid sequence information of L-Myc, Lin28, Lin28b, Esrrb, and Esrrg can be each obtained by referring to the following NCBI accession numbers. A person skilled in the art can prepare desired nuclear reprogramming substances by a conventional technique based on the cDNA sequence or amino acid sequence information.














Gene 






Name
Mouse

Human







L-Myc
NM_008506 
(SEQ ID NOS: 1 and 2)
NM_001033081
(SEQ ID NOS: 3 and 4)


Lin28
NM_145833
(SEQ ID NOS: 5 and 6)
NM_024674
(SEQ ID NOS: 7 and 8)


Lin28b
NM_001031772
(SEQ ID NOS: 9 and 10)
NM_001004317
(SEQ ID NOS: 11 and 12)


Esrrb
NM_011934
(SEQ ID NOS: 13 and 14)
NM_004452
(SEQ ID NOS: 15 and 16)


Esrrg
NM_011935
(SEQ ID NOS: 17 and 18)
NM_001438
(SEQ ID NOS: 19 and 20)









These nuclear reprogramming substances may be introduced in the form of protein into somatic cells by a technique such as lipofection, binding with a cell membrane-permeable peptide, or microinjection. Alternatively, they can also be introduced in the form of DNA or RNA into somatic cells by a technique such as a technique using a vector (such as a virus, a plasmid and an artificial chromosome), lipofection, a liposome, or microinjection. Examples of a viral vector include a retrovirus vector, a lentivirus vector (these are according to Cell, 126, pp. 663-676, 2006; Cell, 131, pp. 861-872, 2007; and Science, 318, pp. 1917-1920, 2007), an adenovirus vector (Science, 322, 945-949, 2008), an adeno-associated virus vector, and a Sendai virus vector (Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-62, 2009). Also, examples of an artificial chromosome vector include a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), and a bacterial artificial chromosome (BAC and PAC). As a plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). A vector can contain regulatory sequences such as a promoter, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site, so that a nuclear reprogramming substance can be expressed. Examples of a promoter to be used herein include an EF 1 a promoter, a CAG promoter, an SRα promoter, an SV40 promoter, an LTR promoter, a CMV (cytomegalovirus) promoter, an RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, and an HSV-TK (herpes simplex virus thymidine kinase) promoter. Particularly preferable examples of such a promoter include an EF1α promoter, a CAG promoter, MoMuLV LTR, a CMV promoter, and an SRα promoter. The above vector may further contain, if necessary, a selection marker sequence such as a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, and a puromycin resistance gene), a thymidine kinase gene, and a diphtheria toxin gene, and a reporter gene sequence such as a green fluorescent protein (GFP), β glucuronidase (GUS), and FLAG. Also, in order to cleave a gene encoding a nuclear reprogramming substance, or a promoter and a gene encoding a nuclear reprogramming substance binding thereto together after introduction into somatic cells, the above vector may have LoxP sequences located before and after the relevant portion. In another preferable embodiment, a method that involves incorporating a transgene into a chromosome using a transposon, causing transferase to act on cells using a plasmid vector or an adenovirus vector, and then completely removing the transgene from the chromosome can be used. An example of a preferable transposon is piggyBac that is a lepidopteran insect-derived transposon (Kaji, K. et al., Nature, 458: 771-775 (2009), Woltjen et al., Nature, 458: 766-770 (2009), WO 2010/012077). Furthermore, a vector may also contain sequences of replication origins for lymphotrophic herpes virus, BK virus, and Bovine papilloma virus and sequences of replication factor thereof, so that the vector is replicated without incorporation into a chromosome so as to be present episomally. For example, EBNA-1 and oriP, or Large T and SV40ori sequences may be contained (WO 2009/115295, WO 2009/157201, and WO 2009/149233). Also, for simultaneous introduction of a plurality of nuclear reprogramming substances, an expression vector that enables polycistronic expression may be used. For polycistronic expression, sequences encoding nuclear reprogramming substances may be linked via an IRES or a foot and mouth disease virus (FMDV) 2A coding region (Science, 322: 949-953, 2008, WO 2009/092042 and WO 2009/152529).
Upon nuclear reprogramming, to improve the efficiency for inducing iPS cells, in addition to the above factors, histone deacetylase (HDAC) inhibitors [e.g., low-molecular-weight inhibitors such as valproic acid (VPA) (Nat. Biotechnol., 26(7): 795-797 (2008)), trichostatin A, sodium butyrate, MC 1293, and M344, and nucleic acid expression inhibitors such as siRNA and shRNA against HDAC (e.g., HDAC1 siRNA Smartpool® (Millipore) and HuSH 29mer shRNA Constructs against HDAC1 (OriGene))], DNA methyltransferase inhibitors (e.g., 5′-azacytidine) (Nat. Biotechnol., 26(7): 795-797 (2008)), G9a histone methyltransferase inhibitors [e.g., low-molecular-weight inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)) and nucleic acid expression inhibitors such as siRNA and shRNA against G9a (e.g., G9a siRNA (human) (Santa Cruz Biotechnology))], L-channel calcium agonists (e.g., Bayk8644) (Cell Stem Cell, 3, 568-574 (2008)), p53 inhibitors (e.g., siRNA and shRNA against p53) (Cell Stem Cell, 3, 475-479 (2008)), Wnt Signaling activator (e.g., soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), cytokines such as LIF or bFGF, ALK5 inhibitors (e.g., SB431542) (Nat Methods, 6: 805-8 (2009)), mitogen-activated protein kinase signalling inhibitors, glycogen synthase kinase-3 inhibitors (PloS Biology, 6(10), 2237-2247 (2008)), miRNA such as miR-291-3p, miR-294, and miR-295 (R. L. Judson et al., Nat. Biotech., 27: 459-461 (2009)), for example, can be used.
Examples of a culture medium for inducing iPS cells include (1) DMEM, DMEM/F12, or a DME medium containing 10-15% FBS (these media may further appropriately contain LIF, penicillin/streptomycin, L-glutamine, nonessential amino acids, β-mercaptoethanol, and the like), (2) a medium for ES cell culture containing bFGF or SCF, such as a medium for mouse ES cell culture (e.g., TX-WES medium (Thromb-X)), and a medium for primate ES cell culture (e.g., a medium for primate (human & monkey) ES cells, ReproCELL, Kyoto, Japan).
An example of culture methods is as follows. Somatic cells are brought into contact with nuclear reprogramming substances (DNA, RNA or protein) on a DMEM or DMEM/F12 medium containing 10% FBS at 37° C. in the presence of 5% CO2 and are cultured for about 4 to 7 days. Subsequently, the cells are reseeded on feeder cells (e.g., mitomycin C-treated STO cells or SNL cells). About 10 days after contact between the somatic cells and the nuclear reprogramming substances, cells are cultured in a bFGF-containing medium for primate ES cell culture. After about 30 to about 45 days or more of the contact, iPS cell-like colonies can be formed. Cells may also be cultured under conditions in which the oxygen concentration is as low as 5%-10% in order to increase the efficiency for inducing iPS cells.
Alternatively, cells may be cultured using DMEM containing 10% FBS (which may further appropriately contain LIF, penicillin/streptomycin, L-glutamine, nonessential amino acids, β-mercaptoethanol, and the like) on feeder cells (e.g., mitomycin C-treated STO cells or SNL cells). After about 25 to about 30 days or more of such culture, ES cell-like colonies can be formed.
During the above culture, medium exchange with a fresh medium is performed once a day from day 2 after the start of culture. In addition, the number of somatic cells to be used for nuclear reprogramming is not limited, but ranges from about 5×103 to about 5×106 cells per culture dish (100 cm2).
When a gene including a drug resistance gene is used as a marker gene, cells expressing the marker gene can be selected by culturing the cells in a medium (selective medium) containing the relevant drug. Also, cells expressing the marker gene can be detected when the marker gene is a fluorescent protein gene, through observation with a fluorescence microscope, by adding a luminescent substrate in the case of a luminescent enzyme gene, or adding a chromogenic substrate in the case of a chromogenic enzyme gene.
The term “somatic cells” as used herein may refer to any cells other than germ cells from mammals (e.g., humans, mice, monkeys, pigs, and rats). Examples of such somatic cells include keratinizing epithelial cells (e.g., keratinizing epidermal cells), mucosal epithelial cells (e.g., epithelial cells of the surface layer of tongue), exocrine epithelial cells (e.g., mammary glandular cells), hormone-secreting cells (e.g., adrenal medullary cells), cells for metabolism and storage (e.g., hepatocytes), boundary-forming luminal epithelial cells (e.g., type I alveolar cells), luminal epithelial cells of internal tubules (e.g., vascular endothelial cells), ciliated cells having carrying capacity (e.g., airway epithelial cells), cells for secretion of extracellular matrix (e.g., fibroblasts), contractile cells (e.g., smooth muscle cells), cells of blood and immune system (e.g., T lymphocytes), cells involved in sensation (e.g., rod cells), autonomic nervous system neurons (e.g., cholinergic neurons), sense organ and peripheral neuron supporting cells (e.g., satellite cells), nerve cells and glial cells of the central nervous system (e.g., astroglial cells), chromocytes (e.g., retinal pigment epithelial cells), and progenitor cells thereof (tissue progenitor cells). Without particular limitation concerning the degree of cell differentiation, the age of an animal from which cells are collected, or the like, both undifferentiated progenitor cells (also including somatic stem cells) and terminally-differentiated mature cells can be similarly used as origins for somatic cells in the present invention. Examples of undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
In the present invention, mammals from which somatic cells are collected are not particularly limited and are preferably humans.
(E) Clone embryo-derived ES cells obtained by nuclear transplantation
ntES (nuclear transfer ES) cells are clone embryo-derived ES cells prepared by nuclear transplantation techniques, having properties almost the same as those of fertilized egg-derived ES cells (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). Specifically, ntES cells are the ES cells which are established from the inner cell mass of a blastocyst from a clone embryo that is obtained via substitution of the nucleus of an unfertilized egg with the nucleus of a somatic cell. For preparation of ntES cells, nuclear transplantation techniques (J. B. Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and the above ES cell preparation techniques are used in combination (Kiyoka Wakayama et al., (2008), Experimental Medicine, Vol. 26, No. 5 (Extra Number), pp. 47-52). Upon nuclear transplantation, the nucleus of a somatic cell is injected into a mammalian enucleated unfertilized egg and then the resultant is cultured for several hours, so that reprogramming can be carried out.
(F) Fusion Stem Cells
Fusion stem cells are prepared by fusing somatic cells to ova or ES cells, so that they have pluripotency similar to that of the ES cells to be fused. Moreover, fusion stem cells also have a gene peculiar to somatic cells (Tada M et al. Curr Biol. 11: 1553-8, 2001; Cowan CA et al. Science. 2005 Aug 26; 309 (5739): 1369-73).
<Method for Inducing Differentiation into Skeletal Muscle or Skeletal Muscle Progenitor Cells>
According to the present invention, the method comprising the following steps can be used for artificially inducing the differentiation of pluripotent cells such as ES cells or iPS cells into skeletal muscle or skeletal muscle progenitor cells;

    (1) culturing human pluripotent stem cells by suspension culture,
    (2) culturing a cell population after suspension culture by adhesion culture,
    (3) dissociating cells after adhesion culture; and
    (4) culturing dissociated cells by adhesion culture.


The term “skeletal muscle” as used herein refers to mature muscle including muscle fiber; that is, muscle cells that are multinuclear cells. Also, the term “skeletal muscle progenitor cells” refers to cells that can selectively differentiate into muscle cells, but this does not mean that such cells completely lack differentiation potency into other mesodermal cells such as osteoblasts or fat cells. In the present invention, skeletal muscle progenitor cells include skeletal muscle stem cells (and, possibly, satellite cells).
In the present invention, artificial skeletal muscle or skeletal muscle progenitor cells obtained via induced differentiation may be provided as a cell population containing other cell species or as a purified cell population. Artificial skeletal muscle or skeletal muscle progenitor cells can be identified by detecting the expression of marker genes for skeletal muscle progenitor cells and skeletal muscle, such as MyoD, MyfS, Pax7, myogenin, myosin heavy chain, NCAM, desmin, SkMAct, MF20, M-cadherin, Fgfr4, and VCAME1 . In addition to this method, artificial skeletal muscle or skeletal muscle progenitor cells can also be identified using the formation of myotube cells having contractile capacity (e.g., expression of an MRF4 gene) or the like as an index. Here the term “artificial” is used for skeletal muscle or skeletal muscle progenitor cells resulting from in vitro induction of differentiation of human pluripotent cells. Artificial skeletal muscle or skeletal muscle progenitor cells may have properties not identical to, but significantly analogous to those of skeletal muscle or skeletal muscle progenitor cells naturally generated in humans. Artificial skeletal muscle or skeletal muscle progenitor cells that are biocompatible when transplanted in vivo and are capable of compensating and functioning as an alternative for a site of damage are particularly desirable.
(A) Step of Culturing Human Pluripotent Stem Cells by Suspension Culture
In this step, human pluripotent cells obtained as described above are dissociated each other cell or obtained in colony form by an arbitrary method and then suspension culture is carried out. In the preferable invention, colony form of pluripotent stem cells are directly used for suspension culture. In a method for dissociation, cells may be mechanically dissociated or dissociated using a dissociation solution having protease activity and collagenase activity (e.g., Accutase (TM) and Accumax (TM)). Preferably, such a method involves mechanically removing colonies formed under conditions in which cells are cultured while maintaining pluripotency and then subjecting the removed cells directly to suspension culture. Here, colonies to be preferably used herein are cultured for at least 3 days and preferably 5 days before removal.
Here the term “suspension culture” refers to the culture of cells not adhering to a culture dish. Suspension culture can be carried out using a culture dish that has not been artificially treated (e.g., via coating treatment using an extracellular matrix, or the like) in order to improve its properties of adhering to cells or treated (e.g., via coating treatment using polyhydroxyethyl methacrylate (poly-HEMA)) to artificially suppress adhesion. However, the examples are not limited to them.
A medium for suspension culture can be prepared using a medium to be used for culturing animal cells, as a basal medium. Examples of a basal medium include IMDM, a Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), aMEM, Doulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, an RPMI 1640 medium, Fischer's medium, and mixtures thereof. Preferably, such a mixture is prepared by mixing DMEM and F12 at 1:1 . A medium may contain serum or may be serum free. If necessary, for example, a medium may contain one or more serum substitutes, such as albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS upon culture of ES cells), an N2 supplement (Invitrogen), a B27 supplement (Invitrogen), fatty acid, insulin, a collagen progenitor, a trace element, 2-mercaptoethanol, 3′-thiolglycerol, as well as one or more substances such as lipids, amino acid, nonessential amino acid, a vitamin, a growth factor, a cytokine, an antibiotic, an antioxidant, pyruvic acid, a buffering agent, and inorganic salts.
An example of such a medium is DMEM/Ham's F12 (mixture) medium containing 20% knockout serum replacement (KSR), 2 mM L-glutamine, and nonessential amino acids.
The temperature for culture ranges from about 30° C. to 40° C. and is preferably about 37° C., but the examples are not limited thereto. Culture is carried out under an atmosphere containing CO2. CO2 concentration preferably ranges from about 2% to 5%. The time for culture ranges from 5 to 9 days, for example, and is more preferably 7 days. Medium exchange is preferably carried out every 2 to 5 days.
(B) Step of Culturing Cell Population by Adhesion Culture
In this step, the cell population after suspension culture, which is obtained in the previous step, is directly cultured in an arbitrary medium using a coated culture dish. Examples of a coating agent include collagen, gelatin, laminin, heparan sulfate proteoglycan, and entactin, or combinations thereof. A preferable example of a coating agent is gelatin.
A medium to be used in this step can be prepared using a medium to be used for culturing animal cells as a basal medium. Examples of a basal medium include IMDM, a Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), aMEM, Doulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, an RPMI 1640 medium, Fischer's medium, and mixtures thereof. Preferably, such a basal medium is DMEM. A medium desirably contains no serum. If necessary, a medium may contain one or more serum substitutes such as albumin, insulin, transferrin, sodium selenite, ITS-X (Gibco) (containing insulin, transferrin, and sodium selenite), Knockout Serum Replacement (KSR) (serum substitute for FBS upon culture of ES cells), an N2 supplement (Invitrogen), a B27 supplement (Invitrogen), fatty acid, a collagen progenitor, a trace element, 2-mercaptoethanol, and 3′-thiolglycerol, as well as one or more substances such as lipids, amino acid, Glutamax (Gibco), nonessential amino acid, a vitamin, a growth factor, a cytokine, an antibiotic, an antioxidant, pyruvic acid, a buffering agent, and inorganic salts.
An example of a preferable medium contains insulin, transferrin, and sodium selenite, in addition to glutamine and 2-mercaptoethanol.
The temperature for culture ranges from about 30° C. to 40° C. and is preferably about 37° C., but the examples are not limited thereto. Culture is carried out under an atmosphere containing CO2. CO2 concentration preferably ranges from about 2% to 5%. The time for culture ranges from 7 to 21 days, for example, and is more preferably 14 days. Medium exchange is desirably carried out every 2 to 5 days.
(C) Step of Dissociating Cells and Culturing Cells by Adhesion Culture Again
In this step, the cell population after adhesion culture (obtained in the previous step) is dissociated and then cultured in an arbitrary medium using a culture dish coated at a low initial cell density. Examples of a coating agent include collagen, gelatin, laminin, heparan sulfate proteoglycan or entactin, and combinations thereof. In this step, a preferable coating agent is type I collagen.
As a method for dissociating a cell population, mechanical dissociation or a method using a dissociation solution having Trypsin/EDTA or protease activity and collagenase activity (e.g., Accutase (Trademark) and Accumax (Trademark)) may be used.
A medium to be used in this step can be prepared using a medium to be used for culturing animal cells as a basal medium. Examples of a basal medium include IMDM, a Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), aMEM, Doulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, an RPMI 1640 medium, Fischer's medium, and mixtures thereof. Preferably, such a medium is DMEM. A medium may contain serum. Examples of serum include, but are not particularly limited to, human serum, monkey serum, fetal calf serum, bovine serum, swine serum, horse serum, donkey serum, chicken serum, quail serum, sheep serum, goat serum, dog serum, cat serum, rabbit serum, rat serum, guinea pig serum, and mouse serum. If necessary, a medium may contain one or more serum substitutes such as albumin, insulin, transferrin, sodium selenite, ITS-X (Gibco), Knockout Serum Replacement (KSR) (serum substitute for FBS upon culture of ES cells), an N2 supplement (Invitrogen), a B27 supplement (Invitrogen), fatty acid, a collagen progenitor, a trace element, 2-mercaptoethanol, and 3′-thiolglycerol, as well as one or more substances such as lipids, amino acid, Glutamax (Gibco), nonessential amino acid, a vitamin, a growth factor, a cytokine, an antibiotic, an antioxidant, pyruvic acid, a buffering agent, and inorganic salts.
An example of a preferable medium contains fetal calf serum and horse serum, in addition to nonessential amino acid, glutamine, and 2-mercaptoethanol.
Also, a low cell density ranges from about 1,000 to 3,000 cells/cm2, for example.
The temperature for culture ranges from about 30° C. to 40° C. and is preferably about 37° C., but the examples are not limited thereto. Culture is carried out under an atmosphere containing CO2. CO2 concentration preferably ranges from about 2% to 5%. The time for culture ranges from 21 to 35 days, for example, and is more preferably 28 days. Medium exchange is desirably carried out every 2 to 5 days.
In this step, adhesion culture may further be carried out for 14 to 21 days, after medium exchange with a serum free medium. Such a medium to be used herein preferably contains insulin, transferrin, and sodium selenite, in addition to glutamine and 2-mercaptoethanol.
<Kit for Induction of Differentiation into Skeletal Muscle or Skeletal Muscle Progenitor Cells>
The present invention provides a kit for inducing the differentiation of pluripotent stem cells into skeletal muscle or skeletal muscle progenitor cells. This kit may contain the above medium to be used for induction of differentiation, a dissociation solution (e.g., Trypsin/EDTA or buffer having protease activity and collagenase activity), a coating agent for coating a culture dish (e.g., collagen, gelatin, laminin, heparan sulfate proteoglycan or entactin, or combinations thereof), and the like. This kit may further contain written procedures or instructions for induction of differentiation.
<Skeletal Muscle or Skeletal Muscle Progenitor Cells>
The present invention provides artificial skeletal muscle or skeletal muscle progenitor cells prepared by the method for induction of differentiation as described above.
Artificial skeletal muscle or skeletal muscle progenitor cells can be identified using markers for skeletal muscle progenitor cells and skeletal muscle, such as MyoD, Myf5, Pax7, myogenin, myosin heavy chain, NCAM, Desmin, SkMAct, MF20, M-Cadherin, Fgfr4, and VCAME1.
Skeletal muscle or skeletal muscle progenitor cells may be directly transplanted into a living body after preparation. Preferably, skeletal muscle progenitor cells may be transplanted into a living body. At this time, skeletal muscle cells or skeletal muscle progenitor cells can be purified and separated by a conventional technique such as a flow cytometry method using a fluorescence-labeled specific antibody as described below.
<Application to Screening for Agents for Treating Myogenic Diseases>
The skeletal muscle or the skeletal muscle progenitor cells of the present invention can also be used for screening for compounds (e.g., pharmaceutical compounds, solvents, small molecules, peptides, or polynucleotides) for treating myogenic diseases (e.g., muscular dystrophy and myopathies). For example, a candidate pharmaceutical compound alone or a combination of a candidate pharmaceutical compound and another medical agent is added to the above-induced skeletal muscle or skeletal muscle progenitor cells, so that evaluation can be made based on a change in number of the cells. Here, skeletal muscle or skeletal muscle progenitor cells are preferably cells that present a phenotype similar to that of a myogenic disease to be treated. Particularly preferable skeletal muscle or skeletal muscle progenitor cells are cells obtained by inducing the differentiation of iPS cells prepared from somatic cells from a patient affected with a given disease or ntES cells into which the nuclei of somatic cells from a patient with such a disease have been transplanted.
<Use for Regenerative Medicine>
The artificial skeletal muscle or skeletal muscle progenitor cells of the present invention can be effectively used in the field of regenerative medicine in order to normalize damaged skeletal muscle tissue. Therefore, the cells can be used for treating myogenic diseases.
Examples of myogenic diseases include muscular dystrophy (Duchenne, Becker, congenital, and limb-girdle muscular dystrophy), congenital myopathies, mitochondrial diseases, glycogenosis, myasthenia gravis, and myasthenic syndrome.
Also, when the cells are used as therapeutic medicaments or composition, cell purity is desirably increased. An example of such a method for increasing cell purity is a method for sorting a target cell such as a flow cytometry method. A flow cytometry method involves causing cell particles to flow through the very thin stream of a solution at a high rate, irradiating the same with a laser beam, and then measuring light such as fluorescence emitted from the particles (when the cells are fluorescence-labeled in advance) or scattered light. A cell sorter is provided so as to make it possible to sort and separate a target cell. Fluorescence labeling of cells can be carried out using an antibody (fluorescence-labeled) specific to the skeletal muscle or skeletal muscle progenitor cells, such as an SM/C-2.6 antibody (Fukada S, et al, Exp Cell Res. 296: 245-55, 2004), an anti-M-cadherin antibody, an anti-FGFR4 antibody, an anti-NCAM antibody, and an anti-VCAM1 antibody.
Such cells to be used for regenerative medicine may be patient-derived ES cells, patient-derived iPS cells, or ntES cells prepared using patient-derived nuclei or pluripotent stem cells from others having the same or substantially the same type of HLA. When patient-derived pluripotent stem cells are used for treating a myogenic disease such as muscular dystrophy, a normal disease-related gene such as normal human dystrophin-coding DNA (including a gene, genomic DNA, cDNA, and the like) (e.g., GenBank Accession No. BC150141(SEQ ID NO: 21)) is directly or indirectly introduced into the thus established ES cells, ntES cells, or iPS cells, and then the differentiation of the cells into target skeletal muscle or skeletal muscle progenitor cells can be induced using the method of the present invention. The full-length of a dystrophin gene is 14 kb and the introduction of the gene with the use of a vector such as an artificial chromosome (preferably, the human chromosome (Republication 2008/013067)) is exemplified. In addition to such a method, a method that involves introducing a shortened functional dystrophin gene using an adeno-associated virus vector, a method that involves introducing a mini-dystrophin gene using a retrovirus vector, a Sendai virus vector, or a lentivirus vector, and the like are exemplified.
Techniques described herein for illustrative purposes can be carried out by known methods. ES cells or ntES cells can be directly transformed or transfected with a vector containing DNA encoding normal human dystrophin. Meanwhile, iPS cells can be directly transformed or transfected using such a vector, or iPS cells may be induced after transformation or transfection of somatic cells with such a vector in advance for induction of iPS cells. Transformation or transfection and other general techniques (e.g., gene recombination techniques and PCR amplification techniques) are described in academic documents, patent documents, academic books (Sambrook et al., Molecular Cloning A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology), and the like, and thus the disclosures therein can be referred to.
EXAMPLES
The present invention will be further described in detail by examples as follows, but the scope of the present invention is not limited by these examples. ES cells
Human ES cells (KhES-1) received from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan) were cultured by a conventional method (Suemori H, et al. Biochem Biophys Res Commun. 345: 926-32, 2006). Human iPS cells (253G4) received from prof. Yamanaka were cultured by a conventional method (Nakagawa M, et al. Nat Biotechnol. 26: 101-6, 2008).
Induction of differentiation (with feeder cells) into skeletal muscle or skeletal muscle progenitor cells
Human ES cells were cultured for 5 days using STO cells as feeder cells, removed in the form of colonies using a dissociation solution for human ES cells [containing Trypsin, 10 mg/ml Collagenase IV, KSR (Invitrogen), 1 M CaCl2/PBS, and PBS], and then subjected to 7 days of suspension culture (embryoid body formation) in a human embryonic stem cell maintenance medium [DMEM/F12 (Sigma)+20% KSR+1% NEAA+2 mM L-glutamine] in a Petri dish. Adhesion culture was then carried out for 14 days in a serum free medium (DMEM+1×ITS-X (Gibco)+1×Glutamax (Gibco)+100 μM 2-mercaptoethanol) on a 0.1% gelatin-coated cell culture dish. Subsequently, embryoid bodies were dissociated using Trypsin/EDTA and then seeded onto a type I collagen-coated dish (BD Bioscience) at a low density (about 1,000 to 3,000 cells/cm2), followed by 3 to 4 weeks of culture using a serum medium [DMEM+10% FCS (fetal calf serum; Sigma)+5% HS (horse serum; Sigma)+1% NEAA+(1×Glutamax)+100 μM 2-mercaptoethanol]. Induction of differentiation might be discontinued for cell transplantation; however, medium exchange with a serum free medium (DMEM+1×ITS-X+(1×Glutamax)+100 μM 2-mercaptoethanol) was carried out and then cells were further cultured for 2 to 3 weeks. The outline of the above processes is shown in FIG. 1. Medium exchange was carried out every 5 days under all conditions.
The cells resulting from the induction of differentiation were immunostained using the antibody for a myosin heavy chain II (Sigma, myosin Skeletal M7523) that is a mature skeletal muscle marker, and the antibody for a skeletal muscle transcription factor myogenin (Dako, M3559 (F5D)) thought to be involved in fusion of skeletal muscle cells. Thus, expression of them could be confirmed (FIG. 2 and FIG. 3). Furthermore, spontaneous contractile activity was also observed. As described above, it was demonstrated that preparation of mature skeletal muscle in vitro from human pluripotent cells was successful using the method. At the same time, it was suggested that skeletal muscle progenitor cells had been prepared.
5.0×105/20 μL Solution of cells, which were differentiated from human ES cells for 49 days with above method and dissociated with trypsin/EDTA, were directly transplanted into tibialis anterior muscle of skeletal muscle injury model NOG mouse which was treated with Cardiotoxin at 24 hours prior to transplanting and 1.2 Gy total body irradiation. On 4 weeks after transplantation, the engraftment was identified by immunostaining with human specific laminin (merosin) antibody (Novocastra Laboratories) and laminin A/C antibody (Dako) (FIG. 4). Same result could be obtained from the cells differentiated from iPS cells (253G4).
Industrial Applicability
The present invention makes it possible to reduce the frequency of complicated isolation work involving pluripotent stem cells such as ES cells or iPS cells and to prepare skeletal muscle or skeletal muscle progenitor cells. The thus prepared skeletal muscle or skeletal muscle progenitor cells can be used in the field of regenerative medicine for treating myogenic diseases.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

## Claims

1. A method for producing skeletal muscle or skeletal progenitor cells from human pluripotent stem cells comprising:
a) culturing human pluripotent stem cells in suspension culture such that an embryoid body is formed;
b) culturing the embryoid body of step (a) on gelatin-coated culture dish in a serum-free medium, wherein the embryoid body adheres to the gelatin-coated culture dish;
c) dissociating the embryoid body of step (b) such that dissociated cells are obtained; and
d) culturing the dissociated cells of step (c) on a type I collagen-coated culture dish, wherein the dissociated cells of step (c) adhere to the type I collagen-coated culture dish, and in a differentiation medium comprising serum, thereby producing skeletal muscle or skeletal muscle progenitor cells.

2. The method according to claim 1, wherein the human pluripotent stem cells of step (a) are in the form of pluripotent stem cell colonies produced on an adhesion substrate and the colonies are directly placed into the suspension culture.

3. The method according to claim 1, wherein the serum free medium comprises insulin, transferrin, and sodium selenite.

4. The method according to claim 1, wherein the culturing of the dissociated cells comprises culturing the dissociated cells at an initial density of 1,000-3,000 cells/cm2.

5. The method according to claim 1, wherein in step (d) the differentiation medium comprises fetal calf serum and horse serum.

6. The method according to claim 1, wherein the culturing of step (a) occurs for 7 days.

7. The method according to claim 1, wherein the culturing of (b) occurs for 14 days.

8. The method according to claim 1, wherein the culturing step of (d) occurs for at least 21 days.

9. The method according to claim 5, wherein in step (d) further comprises exchanging the differentiation medium with a serum-free medium comprising insulin, transferrin and selenite and further culturing the dissociated cells.

10. The method according to claim 1, wherein the human pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.

11. The method according to claim 2, wherein the serum-free medium of step (b) comprises insulin, transferrin and selenite.

12. The method according to claim 2, wherein the culturing of the dissociated cells comprises culturing the dissociated cells at an initial density of 1,000-3,000 cells/cm 2.

13. The method according to claim 9, wherein the dissociated cells in step (d) are cultured in the differentiation medium for 28 days and then further cultured in the serum free differentiation medium for 21 days.