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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
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  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
  • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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  • C12N2500/00—Specific components of cell culture medium
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/40—Regulators of development
  • C12N2501/41—Hedgehog proteins; Cyclopamine (inhibitor)
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/415—Wnt; Frizzeled
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

• the present invention relates to a method of inducing differentiation from a pluripotent stem cell, particularly from an induced pluripotent stem cell, to a skeletal muscle
• progenitor cell a progenitor cell
• reagent kit for use in the method a reagent kit for use in the method
• skeletal muscle progenitor cell obtained by the method, and a treatment of myopathy using the skeletal muscle progenitor cell.
• muscular diseases involve a wide variety of pathologic conditions, the symptoms manifested are for the most part muscular atrophy and associated weakness of muscles.
• Muscular atrophy can occur in two types: myogenic diseases
• muscle in which muscles are disordered, and neurogenic diseases, in which nerves that control muscle motors are
• Muscular dystrophy generically refers to hereditary muscular diseases characterized by gradual progression of muscular atrophy and weakness of muscles in repeated cycles of muscle fiber necrosis and regeneration, which are classified under a wide variety of disease types, involving different causal genes for respective disease types and different modes of mutations.
• stem cell transplantation As a possible radical therapy for muscular dystrophy, stem cell transplantation has been proposed. Satellite cells between muscle fibers and basement membrane are muscular stem cells that had been known before. Later, it was found that stem cells capable of differentiating into muscles are present in marrow cells, which are relatively easy to collect, and which can be proliferated to some extent in vitro, so muscular stem cell transplantation therapy attracted attention. Because muscular dystrophy is a hereditary disease, however, the patient's own bone marrow cannot be used in the therapy, and even marrow cells are unable to proliferate infinitely.
• Embryonic stem (ES) cells are capable of differentiating into almost all types of tissues, and can be proliferated nearly infinitely while maintaining the undifferentiated state.
• Darabi et al. recently succeeded in restoring some normal muscular function in a mouse model of muscular dystrophy by transferring the transcription factor Pax3, which promotes differentiation into myocytes, to mouse ES cells to induce muscle formation, sorting out skeletal muscle progenitor cells, and transplanting the sorted cells to the mouse model of muscular dystrophy [Darabi, R. et al., Nat. Med., 14: 134-143 (2008) ] . Because of the unavoidable involvement of genetic manipulation for differentiation into skeletal muscle
• iPS cells induced pluripotent stem cells
• An object of the present invention is to provide a method of inducing differentiation from pluripotent stem cells, including iPS cells, to skeletal muscle progenitor cells, without gene manipulation, by optimizing the culture conditions, and a differentiation induction reagent kit that comprises a medium ingredient to be added to the medium in the method. It is another object of the present invention to provide a
• dystrophy using skeletal muscle progenitor cells derived from pluripotent stem cells as obtained by the method.
• mouse ES cells can be induced to differentiate into skeletal muscle progenitor cells under serum-free conditions by culturing mouse ES cells in a medium containing bone morphogenetic protein 4 (BMP4), and then further culturing the cells in a medium
• BMP4 bone morphogenetic protein 4
• the present inventors conducted differentiation induction experiments with various growth factors in
• the present inventors found that the differentiation induction efficiency could be increased by further adding BMP and/or insulin-like growth factor-1 (IGF-I) to Medium A, and/or further adding sonic hedgehog (Shh) and/or IGF-I to Medium B.
• IGF-I insulin-like growth factor-1
• sh sonic hedgehog
• the present invention provides the
• [1] A method of producing a skeletal muscle progenitor cell with the use of an iPS cell.
• a method of producing a PDGFR ⁇ -positive mesodermal cell from a pluripotent stem cell wherein the pluripotent stem cell is cultured under serum-free conditions and in the presence of Activin A.
• a method of producing a skeletal muscle progenitor cell from a PDGFR ⁇ -positive mesodermal cell wherein the mesodermal cell is cultured under serum-free conditions and in the
• Wntl, Wnt3a and Wnt7a are Wntl, Wnt3a and Wnt7a.
• Wnt signal inducer comprises at least one selected from among LiCl, Wntl, Wnt3a and Wnt7a.
• kits comprises Activin A, BMP4 and IGF-I.
• a reagent kit for inducing differentiation from a PDGFR ⁇ - positive mesodermal cell to a skeletal muscle progenitor cell wherein the kit comprises LiCl, Shh and IGF-I.
• a skeletal muscle regeneration promoting agent comprising as an active ingredient a skeletal muscle progenitor cell contained in the cell population according to any one of [24] to [26] above.
• a satellite cell formation promoting agent comprising as an active ingredient a skeletal muscle progenitor cell
• regeneration and/or satellite cell formation comprising administering an effective amount of the cell population according to any one of [24] to [26] above to the subject.
• a pluripotent stem cell can be induced to differentiate into a skeletal muscle progenitor cell without gene manipulation by adding
• the present invention also makes it possible to induce
• pluripotent stem cells can be differentiated into skeletal muscle progenitor cells under serum-free conditions; therefore, lot-to-lot variation is small, skeletal muscle progenitor cells can be efficiently obtained using any cell clone, and applications to medical practice are possible.
• skeletal muscle progenitor cells obtained by the present invention have a muscle inflammation suppressing effect and muscle tissue repair effect, they are expected to find applications for skeletal muscle regenerative medicine in muscular dystrophy and other muscular diseases.
• FIG. IA shows conditions for inducing differentiation from an iPS cell to a primitive streak mesodermal cell and a method of evaluation.
• FIG. IB shows charts of a FACS analysis in which the effects of Activin A, IGF-I and HGF on the induction of differentiation from an iPS cell to a primitive streak mesodermal cell were evaluated by the percentage of PDGFR ⁇ -positive cells (left) , and a graph obtained by evaluating the effects by viable cell count (right) .
• FIG. 1C shows charts of a FACS analysis in which the influence of seeded cell density on the induction of differentiation from an iPS cell to a primitive streak mesodermal cell was evaluated by the percentage of PDGFR ⁇ -positive cells.
• FIG. 2A is a chart of a FACS analysis in which the influence of Activin A concentration on the induction of differentiation from an iPS cell to a primitive streak
• FIG. 2B is a chart of a FACS analysis in which the influence of BMP4 concentration on the induction of differentiation from an iPS cell to a primitive streak
• FIG. 2C is a graphic representation of the influence of Activin A and BMP4 concentrations on the induction of differentiation from an iPS cell to a primitive streak mesodermal cell, as evaluated by viable cell count.
• FIG. 3A shows conditions for inducing differentiation from an iPS cell to a primitive streak mesodermal cell and conditions for inducing differentiation from a primitive streak mesodermal cell to a skeletal muscle progenitor cell, and a method of evaluation.
• FIG. 3B shows charts of a FACS analysis in which the effect of Sonic Hedgehog (Shh) on the induction of differentiation from a primitive streak mesodermal cell to a skeletal muscle progenitor cell was evaluated by the percentage of SM/C-2.6-positive cells (left), and a photograph of an RT- PCR in which the effect was evaluated by the expression of Myf5 (right) .
• FIG. 3C shows charts of a FACS analysis in which the effect of IGF-I on the induction of differentiation from a primitive streak mesodermal cell to a skeletal muscle
• progenitor cell was evaluated by the percentage of SM/C-2.6- positive cells (left) , and a photograph of an RT-PCR in which the effect was evaluated by the expression of Myf5 (right) .
• FIG. 4A is a graphic representation showing results of a FACS analysis of a mesodermal cell population derived from iPS cells obtained by the differentiation induction method of the present invention with PDGFR ⁇ as a marker (left), and results of separation of the cell population into a PDGFR ⁇ -positive fraction and a negative fraction (right) .
• FIG. 4B is a
• FIG. 4C is a graphic representation of a FACS analysis showing results of a comparison of the ratio of SM/C-2.6-positive cells and
• FIG. 4D is a photographic image
• FIG. 4E compares the numbers of DsRed-positive cells and Pax7- positive cells obtained after intramuscular injection (i.m.) and intravenous injection (i.v.) of PDGFR ⁇ -positive cells.
• FIG. 5A is a photographic representation of results of an analysis of various tissues performed 4 weeks after
• FIG. 5B is a photographic representation of results of fluorescent
• FIG. 5C is a photographic representation of results of fluorescent
• FIG. 5D is a photographic representation of results of fluorescent immunostaining of DsRed and Pax7 in similar tissues.
• FIG. 6A is a photographic representation showing the results of an analysis of various tissues performed 4 weeks after intramuscular injection of skeletal muscle progenitor cells derived from iPS cells (PDGFR ⁇ -positive cells) into the left tibialis anterior muscle (T.A.) of DMD-null mice.
• FIG. 6B is a photographic representation of results of fluorescent immunostaining of dystrophin in similar tissues.
• FIG. 6C is a photographic representation of results of fluorescent
• Fig. 7 (a - d) are photographic representations showing the results of Myogenin immunostaining of mature skeletal muscle resulting from differentiation induction, in a test tube, of the PDGFR ⁇ -positive fraction and negative fraction obtained by the differentiation induction method of the present
• Fig. 7 (e) is a graph showing the ratio of the
• Fig. 8A is a graph showing the induction conditions and evaluation method when the differentiation induction method of the present invention is performed at different timing of LiCl addition.
• Fig. 8B is a graphic representation showing the results of FACS analysis of changes of the ratio of the
• the present invention provides a method of producing a skeletal muscle progenitor cell from a pluripotent stem cell, comprising the step 1) of differentiating a pluripotent stem cell into a PDGFR ⁇ -positive mesodermal cell, and the step 2) of differentiating the mesodermal cell into a skeletal muscle progenitor cell.
• a first aspect of the present invention relates to a method of producing a PDGFR ⁇ -positive mesodermal cell from a pluripotent stem cell, comprising culturing a pluripotent stem cell under serum-free conditions, and in the presence of Activin A.
• the material may be any undifferentiated cell possessing a "self- renewal” that enables it to proliferate while retaining the undifferentiated state, and "pluripotency” that enables it to differentiate into all the three primary germ layers of the embryo. Examples include iPS cells, ES cells, embryonic germ (EG) cells, embryonic cancer (EC) cells and the like, with preference given to iPS cells or ES cells.
• the method of the present invention is applicable to any mammalian species for which any pluripotent stem cell line has been established or can be established.
• mice examples include humans, mice, rats, monkeys, dogs, pigs, bovines, cats, goat, sheep, rabbits, guinea pigs, hamsters and the like, with preference given to humans, mice, rats, monkeys, dogs and the like, more preferably humans or mice.
• Pluripotent stem cells can be acquired by methods known per se.
• available methods of preparing ES cells include, but are not limited to, methods in which a mammalian inner cell mass in the blastocyst stage is cultured [see, for example, Manipulating the Mouse Embryo: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)] and methods in which an early embryo prepared by somatic cell nuclear transfer is cultured [Wilmut et al., Nature, 385, 810 (1997); Cibelli et al., Science, 280, 1256 (1998); Iritani et al., Protein, Nucleic Acid and Enzyme, 44, 892 (1999); Baguisi et al., Nature Biotechnology, 17, 456 (1999); Wakayama et al., Nature, 394, 369 (1998); Wakayama et al . , Nature Genetics, 22, 127 (1999); Wakayama et al., Proc. Natl. Acad. Sci
• An iPS cell can be prepared by transferring a nuclear reprogramming substance to a somatic cell.
• Any cells other than germ cells of mammalian origin e.g., mice, humans
• iPS cells can be used as starting material for the production of iPS cells. Examples include keratinizing
• epithelial cells e.g., keratinized epidermal cells
• mucosal epithelial cells e.g., epithelial cells of the superficial layer of tongue
• exocrine gland epithelial cells e.g., mammary gland cells
• hormone-secreting cells e.g.,
• adrenomedullary cells cells for metabolism or storage (e.g., liver cells) , intimal epithelial cells constituting interfaces (e.g., type I alveolar cells), intimal epithelial cells of the obturator canal (e.g., vascular endothelial cells), cells having cilia with transporting capability (e.g., airway epithelial cells) , cells for extracellular matrix secretion (e.g., fibroblasts), constrictive cells (e.g., smooth muscle cells), cells of the blood and the immune system (e.g., T lymphocytes), sense-related cells (e.g., bacillary cells), autonomic nervous system neurons (e.g., cholinergic neurons), sustentacular cells of sensory organs and peripheral neurons (e.g., satellite cells), nerve cells and glia cells of the central nervous system (e.g., astroglia cells), pigment cells (e.g., retinal pigment epithelial cells
• undifferentiated progenitor cells including somatic stem cells
• differentiated mature cells can be used alike as sources of somatic cells in the present invention.
• tissue stem cells sematic stem cells
• nerve stem cells such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
• the choice of mammal individual as a source of somatic cells is not particularly limited; however, when the iPS cells obtained are to be used for the treatment of nonhereditary myopathy in humans, it is preferable, from the viewpoint of prevention of graft rejection, that somatic cells are patient's own cells or collected from another person having the same or substantially the same HLA type as that of the patient. Meanwhile, if the iPS cell is to be used for the treatment of a hereditary muscular disease such as muscular dystrophy, it is preferable that the somatic cell be collected from a person, other than the patient, who has the normal gene, and whose HLA type is the same or substantially the same as the patient's.
• substantially the same HLA type means that the HLA type of donor matches with that of patient to the extent that the transplanted cells, which have been obtained by inducing differentiation of iPS cells derived from the donor' s somatic cells, can be engrafted when they are transplanted to the patient with use of immunosuppressor and the like.
• HLA type includes an HLA type wherein major HLAs (the three major loci of HLA-A, HLA-B and HLA-DR) are identical
• a nuclear reprogramming substance refers to any substance (s) capable of inducing an iPS cell from a somatic cell, which may be composed of any substance such as a proteinous factor or a nucleic acid that encodes the same (including forms incorporated in a vector), or a low-molecular compound.
• the nuclear reprogramming substance is a proteinous factor or a nucleic acid that encodes the same, the following combinations, for example, are
• 2007/069666 for information on replacement of Sox2 with Soxl8 and replacement of Klf4 with Klfl or Klf5 in the combination (2) above, see Nature Biotechnology, 26, 101-106 (2008)); for the combination "Oct3/4, KIf4, c-Myc, Sox2", see also Cell, 126, 663-676 (2006), Cell, 131, 861-872 (2007) and the like; for the combination "Oct3/4, Klf2 (or Klf5) , c-Myc, Sox2", see also Nat. Cell Biol., 11, 197-203 (2009); for the combination w Oct3/4, KIf4, c-Myc, Sox2, hTERT, SV40 LT”, see also Nature, 451, 141- 146 (2008).]
• Oct3/4 may be replaced with another member of the Oct family, for example, OctlA, Oct ⁇ or the like.
• Sox2 (or Soxl, Sox3, Soxl5, Soxl7, Soxl8) may be replaced with another member of the Sox family, for example, Sox7 or the like.
• Lin28 may be replaced with another member of the Lin family, for example, Lin28b or the like.
• nuclear reprogramming substances in the present invention.
• the somatic cell to undergo nuclear reprogramming is endogenously expressing one or more of the constituents of any one of (1) to (24) above at a level sufficient to cause nuclear reprogramming, a combination of only the remaining constituents excluding the one or more constituents can also be included in the scope of "nuclear reprogramming substances" in the present invention.
• a combination of at least one, preferably two or more, more preferably three or more, selected from among Oct3/4, Sox2, KIf4, c-Myc, Nanog, Lin28 and SV40LT, is a preferable nuclear reprogramming substance.
• the iPS cells obtained are to be used for therapeutic purposes, a combination of the three factors Oct3/4, Sox2 and KIf4 [combination (9) above] are preferably used.
• the four factors Oct3/4, Sox2, KIf4 and c-Myc, or the five factors Oct3/4, KIf4, c-Myc, Sox2 and Lin28, or the six factors consisting of the five factors and Nanog [combination (12) above] are preferably used.
• the four factors Oct3/4, Sox2, KIf4 and c-Myc, or the five factors Oct3/4, KIf4, c-Myc, Sox2 and Lin28, or the six factors consisting of the five factors and Nanog [combination (12) above] and further, the seven factors consisting of the six factors and SV40 Large T
• a proteinous factor for use as a nuclear reprogramming substance can be prepared by inserting the cDNA obtained into an appropriate expression vector, introducing the vector into a host cell, and recovering the recombinant proteinous factor from the cultured cell or its conditioned medium.
• the nuclear reprogramming substance used is a nucleic acid that encodes a proteinous factor
• the cDNA obtained is inserted into a viral vector, plasmid vector, episomal vector etc. to construct an expression vector, and the vector is subjected to the step of nuclear reprogramming.
• somatic cell can be achieved using a method known per se for protein transfer into a cell, provided that the substance is a proteinous factor.
• An advantage of the method of the present invention for producing a skeletal muscle progenitor cell resides in the possibility of inducing differentiation into a skeletal muscle progenitor cell without gene manipulation.
• the starting material iPS cell be also prepared without gene manipulation.
• Such methods include, for example, the method using a protein transfer reagent, the method using a protein transfer domain (PTD)- or cell penetrating peptide (CPP)- fusion protein, the microinjection method and the like.
• Protein transfer reagents are commercially available, including those based on a cationic lipid, such as BioPOTER Protein Delivery Reagent (Gene Therapy Systems), Pro-JectTM Protein Transfection Reagent
• PIERCE ProVectin
• IGENEX ProVectin
• lipid such as Profect-1 (Targeting Systems)
• membrane- permeable peptide such as Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif), GenomONE (ISHIHARA SANGYO KAISHA, LTD. ) utilizing HVJ envelope (inactivated hemagglutinating virus of Japan) and the like.
• HVJ envelope inactivated hemagglutinating virus of Japan
• Nuclear reprogramming substance (s) is (are) diluted in an appropriate solvent (e.g., a buffer solution such as PBS or HEPES) , a transfer reagent is added, the mixture is incubated at room temperature for about 5 to 15 minutes to form a complex, this complex is added to cells after exchanging the medium with a serum-free medium, and the cells are incubated at 37°C for one to several hours. Thereafter, the medium is removed and replaced with a serum-containing medium.
• an appropriate solvent e.g., a buffer solution such as PBS or HEPES
• Developed PTDs include those using transcellular domains of proteins such as drosophila-derived AntP, HIV-derived TAT (Frankel, A. et al, CeJl 55, 1189-93 (1988) or Green, M. &
• Buforin II (Park, C. B. et al. Proc. Natl Acad. Sci. USA 97, 8245-50 (2000)), Transportan (Pooga, M. et al. FASEB J. 12, 67- 77 (1998)), MAP (model amphipathic peptide) (Oehlke, J. et al. Biochim. Biophys. Acta. 1414, 127-39 (1998)), K-FGF (Lin, Y. Z. et al. J. Biol. Chem. 270, 14255-14258 (1995)), Ku70 (Sawada, M. et al. Nature Cell Biol. 5, 352-7 (2003)), Prion (Lundberg, P. et al.
• CPPs derived from the PTDs include polyarginines such as HR (Cell Stem Cell, 4,381-384 (2009)) and 9R (Cell Stem Cell, 4, 472-476 (2009)).
• a fused protein expression vector incorporating cDNA of a nuclear reprogramming substance and PTD or CPP sequence is prepared, and recombination expression is performed using the vector.
• the fused protein is recovered and used for transfer. Transfer can be performed in the same manner as above except that a protin transfer reagent is not added.
• Microinjection a method of placing a protein solution in a glass needle having a tip diameter of about 1 ⁇ m
• injecting the solution into a cell ensures the transfer of the protein into the cell.
• nuclear reprogramming substance may also be used preferably in the form of a nucleic acid that encodes a proteinous factor, rather than the factor as it is.
• the nucleic acid may be a DNA or an RNA, or a DNA/RNA chimera, and may be double-stranded or single-stranded.
• the nucleic acid is a double-stranded DNA, particularly a cDNA.
• a cDNA of a nuclear reprogramming substance is inserted into an appropriate expression vector comprising a promoter capable of functioning in a host somatic cell.
• expression vectors include, for example, viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus and Sendai virus, plasmids for the expression in animal cells (e.g., pAl-11, pXTl, pRc/CMV, pRc/RSV, pcDNAI/Neo) and the like.
• viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus and Sendai virus
• plasmids for the expression in animal cells e.g., pAl-11, pXTl, pRc/CMV, pRc/RSV, pcDNAI/Neo
• a vector for this purpose can be chosen as appropriate according to the intended use of the iPS cell to be obtained.
• Useful vectors include adenovirus vector, plasmid vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, Sendai virus vector, episomal vector and the like.
• promoters used in expression vectors include the EFl ⁇ promoter, the CAG promoter, the SRa promoter, the SV40 promoter, the LTR promoter, the CMV (cytomegalovirus) promoter, the RSV (Rous sarcoma virus) promoter, the MoMuLV
• HSV-TK herpes simplex virus thymidine kinase promoter and the like, with preference given to the EFl ⁇ promoter, the CAG promoter, the MoMuLV LTR, the CMV promoter, the SRa promoter and the like.
• the expression vector may contain as desired, in addition to a promoter, an enhancer, a polyadenylation signal, a
• selectable marker gene a SV40 replication origin and the like.
• selectable marker genes include the dihydrofolate reductase gene, the neomycin resistant gene, the puromycin resistant gene and the like.
• An expression vector harboring a nucleic acid as a nuclear reprogramming substance can be introduced into a cell by a technique known per se according to the choice of the vector.
• a viral vector for example, a plasmid containing the nucleic acid is introduced into an appropriate packaging cell (e.g., Plat-E cells) or a complementary cell line (e.g., 293-cells) , the viral vector produced in the culture supernatant is recovered, and the vector is infected to the cell by a method suitable for the viral vector.
• an appropriate packaging cell e.g., Plat-E cells
• a complementary cell line e.g., 293-cells
• specific means using a retroviral vector are disclosed in WO2007/69666, Cell, 126, 663-676 (2006) and Cell, 131, 861-872 (2007).
• a nucleic acid encoding a nuclear reprogramming substance is preferably expressed transiently, without being integrated into the chromosome of the cells.
• adenoviral vector is capable of being stably present outside the chromosome, and can be mentioned as another preferred vector. Because Sendai viral vector is capable of being stably present outside the chromosome, and can be
• Sendai viral vector one described in J. Biol. Chem. , 282, 27383-27391 (2007) and JP-3602058 B can be used.
• a method can be used preferably wherein a nucleic acid encoding a nuclear reprogramming substance is cut out using the Cre-loxP system, when becoming unnecessary. That is, with loxP sequences arranged on both ends of the nucleic acid in advance, iPS cells are induced, thereafter the Cre reconnbinase is allowed to act on the cells using a plasmid vector or adenoviral vector, and the region sandwiched by the loxP sequences can be cut out.
• the enhancer-promoter sequence of the LTR U3 region possibly upregulates a host gene in the vicinity thereof by insertion mutation, it is more preferable to avoid the
• a plasmid vector can be transferred into a cell using the lipofection method, liposome method, electroporation method, calcium phosphate co- precipitation method, DEAE dextran method, microinjection method, gene gun method and the like.
• lipofection method liposome method
• electroporation method calcium phosphate co- precipitation method
• DEAE dextran method DEAE dextran method
• microinjection method gene gun method and the like.
• Specific means using a plasmid as a vector are described in, for example, Science, 322, 949-953 (2008) and the like.
• the transfection can be performed once or more plasmid vector, an adenovirus vector and the like.
• the transfection can be performed once or more optionally chosen times (e.g., once to 10 times, once to 5 times or the like), preferably the transfection can be repeatedly performed twice or more (e.g., 3 times or 4 times).
• transgene can get integrated into chromosome; therefore, it is eventually necessary to confirm the absence of insertion of the gene into chromosome by Southern blotting or PCR. For this reason, like the aforementioned Cre-loxP system, it can be advantageous to use a means wherein the transgene is integrated into chromosome, thereafter the gene is removed.
• a method can be used wherein the transgene is integrated into chromosome using a transposon, thereafter a transposase is allowed to act on the cell using a plasmid vector or adenoviral vector so as to completely
• transposons piggyBac, a transposon derived from a lepidopterous insect, and the like can be mentioned. Specific means using the piggyBac transposon is disclosed in Kaji, K. et al., Nature, 458: Ill-lib (2009), Woltjen et al., Nature, 458: 766-770 (2009).
• Another preferable non-integration type vector is an episomal vector, which is autonomously replicable outside chromosome. Specific means using an episomal vector is
• the nuclear reprogramming substance is a low- molecular compound
• introduction thereof into a somatic cell can be achieved by dissolving the substance at an appropriate concentration in an aqueous or non-aqueous solvent, adding the solution to a medium suitable for cultivation of somatic cells isolated from human or mouse [e.g., minimal essential medium (MEM) comprising about 5 to 20% fetal bovine serum, Dulbecco' s modified Eagle medium (DMEM), RPMI1640 medium, 199 medium, F12 medium, and the like] so that the nuclear reprogramming
• MEM minimal essential medium
• DMEM Dulbecco' s modified Eagle medium
• RPMI1640 medium e.g., fetal bovine serum
• 199 medium fetal bovine serum
• F12 medium RPMI1640
• the nuclear reprogramming substance concentration will fall in a range that is sufficient to cause nuclear reprogramming in somatic cells and does not cause cytotoxicity, and culturing the cells for a given period.
• the nuclear reprogramming substance concentration varies depending on the kind of nuclear reprogramming substance used, and is chosen as appropriate over the range of about 0.1 nM to about 100 nM. Duration of contact is not particularly limited, as far as it is sufficient to cause nuclear reprogramming of the cells; usually, the nuclear reprogramming substance may be allowed to be co-present in the medium until a positive colony- emerges .
• establishment efficiency improvers are expected to further raise the efficiency of establishment of iPS cells.
• iPS cell establishment efficiency improvers include, but are not limited to, histone deacetylase (HDAC) inhibitors [e.g., valproic acid (VPA) (Nat. Biotechnol., 26(1): 795-797 (2008)], low-molecular inhibitors such as trichostatin A, sodium butyrate, MC 1293, and M344, nucleic acid-based expression inhibitors such as siRNAs and shRNAs against HDAC (e.g., HDACl siRNA Smartpool ® (Millipore) , HuSH 29mer shRNA Constructs against HDACl (OriGene) and the like) , and the like] , DNA methyltransferase inhibitors (e.g., 5' -azacytidine) [Nat. Biotechnol. , 26(1): 795-797 (2008)], G9a histone deacetylase (HDAC) inhibitors [e.g., valproic acid (VPA) (Nat. Biotech
• methyltransferase inhibitors e.g., low-molecular inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), nucleic acid-based expression inhibitors such as siRNAs and shRNAs against G9a (e.g., G9a siRNA (human) (Santa Cruz Biotechnology) and the like) and the like], 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)), UTFl [Cell Stem Cell, 3, 475-479 (2008)], Wnt
• low-molecular inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)
• nucleic acid-based expression inhibitors such as siRNAs and shRNAs against G9a (e.g., G9a siRNA (human) (S
• nucleic acid-based expression inhibitors may be in the form of expression vectors harboring a DNA that encodes an siRNA or shRNA.
• siRNA siRNA
• shRNA a DNA that encodes an siRNA or shRNA.
• SV40 large T and the like can also be included in the scope of iPS cell
• somatic cell nuclear reprogramming process is understood as an overall event resulting from contact of nuclear reprogramming
• contact of an iPS cell establishment efficiency improver with a somatic cell can be achieved as described above for each of three cases: (a) the improver is a proteinous factor, (b) the improver is a nucleic acid that encodes the proteinous factor, and (c) the improver is a low-molecular compound.
• An iPS cell establishment efficiency improver may be brought into contact with a somatic cell simultaneously with a nuclear reprogramming substance, or either one may be contacted in advance, as far as the efficiency of establishment of iPS cells from the somatic cell is significantly improved, compared with the absence of the improver.
• the nuclear reprogramming substance is a nucleic acid that encodes a proteinous factor and the iPS cell
• the iPS cell establishment efficiency improver is a chemical inhibitor, the iPS cell establishment efficiency improver can be added to the medium after the cell is cultured for a given length of time after the gene transfer treatment, because the nuclear
• reprogramming substance involves a given length of time lag from the gene transfer treatment to the mass-expression of the proteinous factor, whereas the iPS cell establishment
• efficiency improver is capable of rapidly acting on the cell.
• a nuclear reprogramming substance and an iPS cell establishment efficiency improver are both used in the form of a viral or plasmid vector, for example, both may be simultaneously introduced into the cell.
• Somatic cells separated from a mammal can be pre-cultured using a medium known per se suitable for the cultivation thereof, depending on the kind of the cells.
• a medium known per se suitable for the cultivation thereof include, but are not limited to, a minimal essential medium (MEM) containing about 5 to 20% fetal calf serum,
• Dulbecco' s modified Eagle medium DMEM
• RPMI1640 medium 199 medium
• F12 medium F12 medium
• a transfection reagent such as a cationic liposome in contacting the cell with nuclear reprogramming substance (s) and iPS cell establishment efficiency improver (s)
• the medium be previously replaced with a serum- free medium to prevent a reduction in the transfer efficiency.
• LIF Leukemia Inhibitory Factor
• fibroblast growth factor bFGF
• SCF stem cell factor
• the cells are cultured in the co-presence of mouse embryo-derived fibroblasts (MEFs) treated with radiation or an antibiotic to terminate the cell division thereof, as feeder cells.
• MEFs mouse embryo-derived fibroblasts
• STO cells and the like are commonly used as MEFs, but for inducing iPS cells, SNL cells [McMahon, A. P. & Bradley, A. Cell 62, 1073-1085 (1990)] and the like are commonly used.
• Co-culture with feeder cells may be started before contact of the nuclear reprogramming substance, at the time of the contact, or after the contact
• a candidate colony of iPS cells can be selected by a method with drug resistance and reporter activity as indicators, and also by a method based on visual examination of morphology.
• resistance and/or reporter activity is selected using a
• a drug resistance gene and/or a reporter gene is targeted to the locus of a gene highly expressed specifically in pluripotent cells (e.g., Fbxl5, Nanog, Oct3/4 and the like, preferably Nanog or Oct3/4) .
• pluripotent cells e.g., Fbxl5, Nanog, Oct3/4 and the like, preferably Nanog or Oct3/4 .
• Examples of such recombinant somatic cells include MEFs from a mouse having the ⁇ geo (which encodes a fusion protein of ⁇ -galactosidase and neomycin phosphotransferase) gene knocked-in to the Fbxl5 locus [Takahashi & Yamanaka, Cell, 126, 663-676 (2006)], MEFs from a transgenic mouse having the green fluorescent protein (GFP) gene and the puromycin resistance gene integrated in the Nanog locus [Okita et al., Nature, 448, 313-317 (2007)] and the like. Meanwhile, examples of the method of selecting candidate
• colonies based on visual examination of morphology include the method described by Takahashi et al. in Cell, 131, 861-872
• the number of clones established decreases but the resulting colonies are mostly of iPS cells of high quality comparable to ES cells, so that iPS cells can efficiently be established even without using reporter cells.
• the identity of the cells of a selected colony as iPS cells can be confirmed by positive responses to a Nanog (or Oct3/4) reporter (puromycin resistance, GFP positivity and the like) as well as by the formation of a visible ES cell-like colony, as described above.
• a Nanog or Oct3/4 reporter
• puromycin resistance or GFP positivity and the like
• Mouse pluripotent stem cells can exit in two functionally distinct states, LIF-dependent ES cells and bFGF-dependent epiblast stem cells (EpiSCs) .
• EpiSCs bFGF-dependent epiblast stem cells
• human ES and iPS cells in a mouse ES cell-like pluripotent state have been established by ectopic induction of Oct3/4, Sox2, KIf4, c- Myc and Nanog in the presence of LIF (see Cell Stem Cells, 6: 535-546, 2010), or ectopic induction of Oct3/4, Klf4 and Klf2 combined with LIF and inhibitors of GSK3 ⁇ and ERK1/2 pathway (see Proc. Natl. Acad. Sci. USA, online publication
• naive human ES and iPS cells may be prefarable starting materials for the present invention due to their pluripotent more immature compared to that of conventional human ES and iPS cells.
• Basal media for differentiation induction include, but are not limited to, serum-free minimal essential medium (MEM) , Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, 199 medium, F12 medium, mixtures thereof, and media prepared by supplementing any one of the aforementioned media with
• the differentiation induction medium of the present invention for inducing differentiation from a pluripotent stem cell to a PDGFR ⁇ -positive mesodermal cells contains Activin A as an essential additive in the basal medium.
• Activin A increases cell survival dose-dependently in the induction of differentiation from a pluripotent stem cell to a PDGFR ⁇ -positive mesodermal cell.
• the Activin A concentration is, for example, about 1 ng/ml or more, preferably about 3 ng/ml or more, more preferably about 5 ng/ml or more, and is, for example, about 20 ng/ml or less, preferably about 15 ng/ml or less, more preferably 10 ng/ml or less.
• the medium A preferably further contains BMP and/or IGF- 1.
• BMP remarkably increases the induction efficiency for
• BMP examples include BMP2, BMP4, BMP7 and the like.
• One kind of BMP may be used alone, and 2 kinds or more may be used in combination.
• 1 kind of BMP4 is used alone, or in combination with another or more kinds of BMP.
• the BMP concentration, as calculated for all kinds of BMP together is, for example, about 5 ng/ml or more, preferably about 7.5 ng/ml or more, more preferably about 10 ng/ml or more, and is, for example, about 30 ng/ml or less, preferably about 20 ng/ml or less, more preferably about 15 ng/ml or less.
• the IGF-I concentration is, for example, about 1 ng/ml or more, preferably about 5 ng/ml or more, more
• ng/ml preferably about 10 ng/ml or more, and is, for example, about 30 ng/ml or less, preferably about 20 ng/ml or less, more preferably about 15 ng/ml or less.
• the medium A contains Activin A, BMP4 and IGF-I in addition to the basal medium.
• concentrations of these ingredients can be chosen over the range of about 3 to 15 ng/ml, preferably about 5 to 10 ng/ml, for Activin A, about 7.5 to 20 ng/ml, preferably about 10 to 15 ng/ml, for BMP4, and about 5 to 20 ng/ml, preferably about 10 to 15 ng/ml, for IGF-I.
• pluripotent stem cells are seeded to a culture vessel known per se (e.g., gelatin- or collagen- coated 10 cm cell culture dish) to obtain a cell density of, for example, about 3 to 10 x 10 4 cells/mL, preferably about 4 to 8 x 10 4 cells/mL (about 3 to 10 x 10 5 cells/10 cm dish, preferably about 4 to 8 x 10 5 cells/10 cm dish) , and cultured in an incubator under atmospheric conditions of 5% CC> 2 /95% air at about 30 to 40 0 C, preferably about 37°C, for about 2 to 7 days, preferably about 3 to 4 days.
• a culture vessel known per se e.g., gelatin- or collagen- coated 10 cm cell culture dish
• differentiation into PDGFR ⁇ -positive mesodermal cells can be confirmed by, for example, analyzing the phenotype of a cell surface antigen using an antibody against PDGFR ⁇ and a cell sorter. As required, the expression of another cell surface antigen or transcription factor can also be examined. Examples of the other surface antigen include Flkl and VEGFR2. Examples of the transcription factor include brachyury(T) and Mixll.
• the pluripotent stem cells first differentiate into primitive streak mesodermal cells, which are the most immature type of PDGFR ⁇ /Flkl double-positive cells, and then into PDGFR ⁇ - positive/Flkl-negative paraxial mesodermal cells, which are destined to differentiate into muscle cells. Meanwhile, lateral plate mesodermal cells, which are destined to
• the differentiate into hemocytes and myocardial cells exhibit the PDGFR ⁇ -negative/Flkl-positive phenotype. Because the PDGFR ⁇ - positive cell fraction obtained by this step of differentiation induction expresses Flkl and brachyury (T) , the majority of the cells contained in the fraction are thought to be in the stage of differentiation into primitive streak mesodermal cells.
• the present invention also provides a reagent kit for induction of differentiation from a pluripotent stem cell to a PDGFR ⁇ -positive mesodermal cell, the kit comprising Activin A, BMP4 and IGF-I.
• Activin A, BMP4 and IGF-I are contained in the medium A.
• the present invention also provides a reagent kit for induction of differentiation from a pluripotent stem cell to a PDGFR ⁇ -positive mesodermal cell, the kit comprising Activin A, BMP4 and IGF-I.
• These ingredients may be supplied in a state dissolved in water or an appropriate buffer solution, and may also be supplied as a lyophilized powder which may be used after being freshly dissolved in an appropriate solvent.
• These ingredients may be supplied as individual reagents in respective kits, and, as far as they do not adversely affect each other, they can be supplied as a single mixed reagent of 2 kinds or more.
• mesodermal cells under serum-free conditions and in the presence of a Wnt signal inducer, it is possible to induce differentiation into skeletal muscle progenitor cells.
• a second aspect of the present invention relates to a method of producing a skeletal muscle progenitor cell from a PDGFR ⁇ -positive mesodermal cell.
• the PDGFR ⁇ -positive mesodermal cells to be treated in this step of differentiation induction are not limited to those obtained in the step 1) described in detail in (2) above, and may be prepared by any method.
• PDGFR ⁇ -positive mesodermal cells obtained by culturing ES cells in a BMP4-containing medium can be used.
• the PDGFR ⁇ -positive mesodermal cells to be treated in the step 2) are PDGFR ⁇ -positive mesodermal cells of pluripotent stem cell derivation, preferably of iPS cell or ES cell derivation, prepared in the step 1) .
• a serum-free medium of the same composition as the foregoing step 1) is likewise preferably used.
• the differentiation induction medium of the present invention for induction of differentiation from PDGFR ⁇ - positive mesodermal cells to skeletal muscle progenitor cells
• (medium B) contains at least 1 kind of Wnt signal inducer as an essential additive in the basal medium.
• Wnt signal inducer examples include LiCl, Wntl, Wnt3a, Wnt7a and the like.
• the Wnt signals mediated by Wntl, Wnt3a, Wnt7a and the like positively control the expression of Myf5 and MyoD, which are transcription factors involved in muscle genesis, and LiCl is known as a classical activator of Wnt signals.
• LiCl is used as a Wnt signal inducer.
• the concentration of Wnt signal inducer is, for example, about 1 mM or more, preferably about 3 mM or more, more preferably about 5 mM or more.
• the concentration of Wnt signal inducer is, for example, about 20 mM or less, preferably about 15 mM or less, more preferably 10 mM or less.
• the medium B preferably further contains Shh and/or IGF-I.
• Shh and IGF-I remarkably increase skeletal muscle progenitor cell induction efficiency when present in ranges of effective concentrations.
• the concentration of Shh is, for example, about 5 ng/ml or more, preferably about 10 ng/ml or more, more preferably about 15 ng/ml or more.
• the concentration of Shh is, for example, about 50 ng/ml or less, preferably about 30 ng/ml or less, more preferably about 25 ng/ml or less.
• the concentration of IGF-I is, for example, about 1 ng/ml or more, preferably about 5 ng/ml or more. Also, the concentration of IGF-I is, for example, about 40 ng/ml or less, preferably about 20 ng/ml or less.
• the medium B contains LiCl, Shh and IGF-I in addition to the basal medium.
• concentrations of these ingredients can be chosen as appropriate over the range of about 3 to 15 mM, preferably about 5 to 10 mM, for LiCl, about 10 to 30 ng/ml, preferably about 15 to 25 ng/ml, for Shh, and about 1 to 40 ng/ml, preferably about 5 to 20 ng/ml, for IGF-I.
• PDGFR ⁇ -positive mesodermal cells are seeded to a culture vessel known per se (e.g., gelatin- or collagen-coated 10 cm cell culture dish and the like) to obtain a cell density of, for example, about 3 to 10 x 10 4 cells/mL, preferably about 4 to 8 x 10 4 cells/mL (about 3 to 10 x 10 5 cells/10 cm dish, more preferably about 4 to 8 x 10 5 cells/10 cm dish) , and cultured in an incubator in an atmosphere of 5% CO 2 /95% air at about 30 to 40 0 C, preferably about 37°C, for about 1 to 7 days, preferably about 2 to 4 days.
• a culture vessel known per se e.g., gelatin- or collagen-coated 10 cm cell culture dish and the like
• Wnt signal inducer such as LiCl may also be added to a culture medium during induction of differentiation from pluripotent stem cells to PDGFR ⁇ -positive mesodermal cells.
• Wnt signal inducer is added to a culture medium day 0 to day 3, more preferably day 1 or day 2 from the beginning of the induction of differentiation of pluripotent stem cells.
• progenitor cells can be confirmed by, for example, analyzing the expression of the transcription factors Myf5 and MyoD by RT-PCR and the like. As required, furthermore, the expression of other transcription factors and cell surface antigens can also be examined. Examples of other transcription factors include Pax3 and Pax7. Examples of cell surface antigens include SM/C-2.6 and PDGFR ⁇ .
• a more highly purified skeletal muscle progenitor cell population can be obtained by selecting and separating a PDGFR ⁇ -positive cell fraction from a cell culture obtained by this differentiation induction step. Because the PDGFR ⁇ -negative cell fraction expresses Pax3 and Pax7, as well as Soxl, a marker of neurons in the developmentalstage, it is thought to have differentiated into nervous cells; the non-fractionated cell population
• obtained by this induction step is potentially preferably useful as a source of graft cells, for example, when nerve regeneration is required in addition to skeletal muscle regeneration.
• the present invention also provides a reagent kit for induction of differentiation from a PDGFR ⁇ - positive mesodermal cell to a skeletal muscle progenitor cell, comprising LiCl, Shh and IGF-I.
• a reagent kit for induction of differentiation from a PDGFR ⁇ - positive mesodermal cell to a skeletal muscle progenitor cell comprising LiCl, Shh and IGF-I.
• These ingredients may be supplied in a state dissolved in water or an appropriate buffer solution, and may also be supplied as a dried (lyophilized) powder which may be used after being freshly dissolved in an appropriate solvent.
• These ingredients may be supplied as individual reagents in respective kits, and, as far as they do not adversely affect each other, they can be supplied as a single mixed reagent of 2 kinds or more.
• the present invention also provides a cell population containing skeletal muscle progenitor cells derived from pluripotent stem cells, produced by the foregoing step 2) .
• the cell population may be a purified population of skeletal muscle progenitor cells, and 1 kind or more of cells other than skeletal muscle progenitor cells may be co-present.
• a skeletal muscle progenitor cell is defined as a cell that is both Myf5-positive and MyoD-positive. As stated above, cells that are Myf5-positive and MyoD-positive are contained only in the PDGFR ⁇ -positive cell fraction, and the great majority of PDGFR ⁇ -positive cells are also positive for SM/C-2.6;
• purified skeletal muscle progenitor cells can be obtained by sorting out the cell culture obtained in the foregoing step 2) , using an anti-PDGFR ⁇ antibody and/or anti- SM/C-2.6 antibody.
• the cell population containing skeletal muscle progenitor cells of the present invention is a cell population of iPS cell or ES cell derivation produced through the foregoing steps 1) and 2) .
• the iPS cell has been produced by, for example, transferring a reprogramming gene to a somatic cell by means of a retrovirus vector or lentivirus vector, the reprogramming gene is integrated in the genome of the cell; therefore, the skeletal muscle progenitor cells derived from the iPS cell also have the reprogramming gene integrated in the genome thereof.
• the skeletal muscle progenitor cells derived from iPS cells have been established for the first time by the present invention, the skeletal muscle progenitor cells having an extraneous reprogramming gene integrated in the genome thereof are of course novel cells.
• a reprogramming gene to be integrated in the genome of skeletal muscle progenitor cells is a nucleic acid that encodes one of the nuclear reprogramming substances described above with respect to preparing iPS cells, preferably 3 genes consisting of Oct3/4, Sox2, and KIf4, or 4 genes consisting of the foregoing three and c-Myc.
• pluripotent stem cells thus established can be used for varied purposes.
• the cell enables a stem cell therapy by autologous or allogeneic transplantation in which skeletal muscle progenitor cells differentiated from an iPS cell induced using a somatic cell collected from a muscular disease patient or another person having the same or substantially the same type of HLA are transplanted to the patient to regenerate skeletal muscles.
• skeletal muscle progenitor cells differentiated from an iPS cell of a muscular disease patient are believed to reflect the status of muscle cells in the actual patient's body more than do the corresponding existing cell line, they can also be suitably used in in vitro evaluation systems for the pharmacological efficacy and toxicity of therapeutic drugs for muscular diseases. They can further be preferably used as a tool for pathological research into muscular diseases of unknown causes.
• the skeletal muscle progenitor cell promotes skeletal muscle
• muscular diseases that can be treated with the skeletal muscle progenitor cell of the present invention include, but are not limited to, muscular dystrophy [e.g., Duchenne's muscular dystrophy (DMD), Becker type muscular dystrophy, congenital muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy and the like] ,
• DMD Duchenne's muscular dystrophy
• Becker type muscular dystrophy congenital muscular dystrophy
• limb-girdle muscular dystrophy limb-girdle muscular dystrophy
• myotonic muscular dystrophy myotonic muscular dystrophy and the like
• hereditary myopathies such as congenital myopathy, distal myopathy and mitochondrial diseases
• non-hereditary myopathies such as multiple myositis, dermatomyositis and myasthenia gravis
• neurogenic muscular diseases such as spinal amyotrophy, bulbar amyotrophy and amyotrophic lateral sclerosis, and the like.
• the skeletal muscle progenitor cell of the present invention can be used to promote skeletal muscle
• myodystrophies such as DMD.
• a skeletal muscle progenitor cell for treating muscular dystrophy and other hereditary muscular diseases a skeletal muscle progenitor cell differentiated from a
• pluripotent stem cell induced from a person, other than the patient, having the same or substantially the same type of HLA as the patient's is preferably used.
• human regenerative medicine it is difficult to obtain human ES cells having the same or substantially the same type of HLA; therefore, it is preferable to use a human iPS cell as a pluripotent stem cell for inducing a skeletal muscle progenitor cell.
• a skeletal muscle progenitor cell differentiated from an iPS cell derived from a somatic cell of the patient as a skeletal muscle progenitor cell for the treatment of a hereditary muscular disease.
• a skeletal muscle progenitor cell differentiated from an iPS cell derived from a somatic cell of the patient, as a skeletal muscle progenitor cell for the treatment of a hereditary muscular disease.
• the normal dystrophin gene is transferred to the iPS cell.
• the dystrophin cDNA is 14 kb in total length, and the adeno-associated virus (AAV) vector, which is best suited for transfection to muscle cells, can only accommodate a length of up to about 4.5 kb. For this reason, current strategic attempts of gene therapy include transfer of a shortened functional dystrophin gene
• micro-dystrophin gene (3.7 kb) using the AAV vector, transfer of a 6.4 kb mini-dystrophin gene using a retrovirus/lentivirus vector enabling insertion of a larger DNA, or transfer of the full-length dystrophin gene in a bare state or using a Gutted adenovirus vector.
• a retrovirus/lentivirus vector enabling insertion of a larger DNA
• transfer of the full-length dystrophin gene in a bare state or using a Gutted adenovirus vector In case of an iPS cell, the highest
• the mutated site in the causal gene can be repaired on the basis of the endogenous DNA repair mechanism of the iPS cell or homologous recombination.
• a chimeric RNA/DNA oligonucleotide (chimeraplast) having the normalized sequence at the mutated site is transferred and allowed to bind to the target sequence and form a mismatch, whereby the endogenous mechanism for DNA repair is activated to induce gene repair.
• gene repair can also be achieved by transferring a 400-800-base single-stranded DNA that is homologous to the mutated site to cause homologous recombination.
• the thus-obtained iPS cell with the repaired causal gene is induced to differentiate into a skeletal muscle progenitor cell via the foregoing steps 1) and 2) , whereby a normal skeletal muscle progenitor cell derived from the patient can be produced.
• the eutrophin gene a dystrophin homologue also expressed in the patient's skeletal muscles, may be transferred as a substitute for the dystrophin function.
• the skeletal muscle progenitor cell differentiated from an iPS cell induced from a somatic cell of the patient is possibly a normal cell; therefore, the skeletal muscle progenitor cell can
• the cell population containing skeletal muscle progenitor cells obtained by the foregoing step 2) can be prepared as a preparation of purified cells obtained by sorting skeletal muscle progenitor cells, and can also be prepared as a
• PDGFR ⁇ -negative fraction is considered to be a cell population of the nervous system; therefore, this fraction can be
• the skeletal muscle progenitor cells (including a cell population containing skeletal muscle progenitor cells; the same applies below) of the present invention are produced as a parenteral preparation, preferably as an injection, suspension, or drip infusion, in a mixture with a pharmaceutically
• the pharmaceutically acceptable carrier by a conventional means.
• the pharmaceutically acceptable carrier that can be contained in the parenteral preparation include aqueous liquids for
• the agent of the present invention may be formulated with, for example, a buffering agent (e.g., phosphate buffer solution, sodium acetate buffer solution), a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride and the like) , a stabilizer (e.g., human serum albumin, polyethylene glycol and the like), a preservative, an anti-oxidant and the like.
• a buffering agent e.g., phosphate buffer solution, sodium acetate buffer solution
• a soothing agent e.g., benzalkonium chloride, procaine hydrochloride and the like
• a stabilizer e.g., human serum albumin, polyethylene glycol and the like
• preservative e.g., an anti-oxidant and the like.
• skeletal muscle progenitor cells are suspended in one of the aforementioned aqueous liquids to obtain a cell density of about 1.0 ⁇ l0 6 to about 1.0 ⁇ l0 7
• the preparation thus obtained is stable and less toxic, it can be safely administered to mammals such as humans.
• the method of administration is not particularly limited, the preparation is preferably administered by
• Useful routes of administration include intravenous administration, intra-arterial
• intramuscular administration topical administration to affected site
• the agent of the present invention is capable of selective engrafting at a site of muscle damage, and exhibiting inflammation suppressive action and skeletal muscle
• the agent of the present invention is preferably administered using a systemic route for administration such as intravenous administration or intra-arterial administration, particularly when symptoms are manifested in many sites.
• the dose of the agent of the present invention varies depending on the subject of administration, target site, symptoms, method of administration and the like.
• intravenous administration for example, it is usually convenient to administer the agent in an amount of about 1.0*10 5 to about l*10 7 cells, based on the amount of skeletal muscle progenitor cells per dose, about 4 to about 8 times at about 1- to 2-week intervals.
• iPS-Nanog-20D-17 obtained by infecting mouse MEF with the 4 genes consisting of Oct3/4, KIf4, Sox2 and c-Myc by means of retrovirus [Okita, K. et al., Nature 448, 313-317 (2007)]
• iPS-DsRed obtained by infecting mouse TTF with the 3 genes consisting of Oct3/4, KIf4 and Sox2 by means of retrovirus
• a basal medium was prepared by adding 2 mM L-glutamine (Nacalai Tesque) , lxNon-essential amino acid (Invitrogen) , 100 ⁇ M 2-mercaptoethanol (Invitrogen) , 50 mU/L penicillin and 50 ⁇ g/L streptomycin to DMEM (Nacalai Tesque) , and this was supplemented with fetal bovine serum (Invitrogen) at 15% to obtain an iPS cell maintenance medium.
• iPS cells Differentiation of iPS cells was induced using a serum- free basal medium for differentiation induction prepared by adding 0.2% bovine serum albumin (Sigma), 100 ⁇ M 2- mercaptoethanol (Invitrogen) , 50 mU/L penicillin and 50 ⁇ g/L streptomycin to S-Clone SF-O3 (Sanko Junyaku Co., Ltd.).
• IGF-I (Peprotech) 10 ng/ml
• IGF-I (Peprotech) 5 ng/ml
• differentiation induction basal medium 10 ml of the culture medium for the first 3 days was added to a 10 cm cell culture dish coated with collagen type IV (Nitta Gelatin), and 500,000 undifferentiated iPS cells were seeded thereinto.
• the cells were cultured in an incubator adjusted to 37°C, 5% CO 2 , and 100% humidity for 3 days, after which the medium was replaced with the culture medium for the last 3 days.
• the cells were thus differentiation-induced for a total of 6 days, from which skeletal muscle progenitor cells were separated by the method of cell separation described below, and these were used in the subsequent experiments .
• the mouse iPS cells induced by the above-described differentiation induction method were divided into a population of skeletal muscle progenitor cells and a population of other cells using FACS Aria (Becton Dickinson) .
• the cells were stained using antibodies as described previously [Sakurai, H. et al., Stem Cells 24, 575-586 (2006)].
• Three different rat monoclonal antibodies were used: APA5 (anti-PDGFR ⁇ ) , ECCD2 (anti-ECD) and SM/C-2.6.
• the former two were supplied by Dr. Nishikawa [Sakurai, H. et al., Stem Cells 24, 575-586 (2006)], and the remaining one by Dr. Yamamoto [Fukada, S.
• the APA5 and SM/C-2.6 were conjugated with biotin (PIERCE) and fluorescently labeled using streptavidin-APC as a secondary antibody.
• the ECCD2 was directly conjugating with Alexa488 (Molecular Probes) by a conventional method and fluorescently labeled.
• fluorescently labeled cells were suspended in Hanks' balanced salt solution (Invitrogen) supplemented with 1% bovine serum albumin to obtain a cell density of 5,000,000 cells per mL, fluorescence was examined and analyzed using FACS Aria, and the PDGFR ⁇ -positive fraction was separated and recovered.
• Hanks' balanced salt solution Invitrogen
• bovine serum albumin 1% bovine serum albumin
• cDNA was synthesized from the RNA using the SuperScriptII reverse transcription kit (Invitrogen) .
• the PCR primers shown below were used.
• mice used in the experiment of induction of muscle regeneration with cardiotoxin were of the C57BL/6 line (Japan SLC) .
• DMD-null mice obtained from Dr. Hanaoka at the
• Cardiotoxin was administered 3 days before
• tissue section sample obtained was sliced into sections 10 ⁇ m thick using a cryostat (Leica) , and allowed to adhere to APS-coated glass slides (Matsunami Glass) and dried.
• Each tissue section prepared was fixed in 4% paraformaldehyde (Nacalai Tesque) /PBS at room temperature for 20 minutes, and washed with PBS for 5 minutes 3 times, after which blocking was performed using a PBS supplemented with 1% goat serum (Sigma), 0.1% bovine serum albumin, and 0.2% Triton X-100
• R&D monoclonal: R&D was conjugated directly with each secondary antibody using the Zenon-Alexa488 IgGl labeling kit (Molecular Probes) , and the conjugate was diluted to a concentration of 1:100 in a PBS supplemented with 0.2% Triton X-100, and reacted at room temperature for 1 hour. Subsequently, to stain the cell nuclei, 5 ⁇ g/ml DAPI (Sigma) was diluted in PBST 5000 folds, and the dilution was reacted at room temperature for 5 minutes and washed with PBS 3 times, after which cover glass was placed on the glass slide, and the sample was sealed. The stained tissue section was examined for data acquisition using the SP5 confocal microscopic system (Leica) .
• the differentiation induction basic medium was supplemented with 0.2% bovine serum albumin (Sigma Ltd.), 100 ⁇ M 2-mercaptoethanol (Invitrogen) , 50 mU/L Penicillin/50 ⁇ g/L Streptomycin was used as a differentiation induction basic medium, which was further added with the following growth factors. To remove dead cells, the medium was exchanged with a medium having the same composition 24 hr from the start of the differentiation induction.
• HGF (R&D) 10 ng/ml
• IGF-I (Peprotech) 2 ng/ml
• IGF-I (Peprotech) 2 ng/ml
• the cells differentiated into mature skeletal muscle by the above-mentioned differentiation induction were evaluated by immunostaining.
• the medium was discarded leaving the cells attached to the dish, and the cells were fixed in 4% paraformaldehyde (Nacalai Tesque) /PBS at 4°C for 10 minutes, and washed with PBS for 5 minutes 3 times, after which blocking was performed using a PBS supplemented with 1% goat serum (Sigma) , 0.1% bovine serum albumin, and 0.2% Triton X-100 (Nacalai)
• the nucleus was stained with a Giemsa staining solution (Merck) at room temperature for 10 min, washed 3 times with PBS, observed with All-in-One microscope BioZero (KEYENCE) and photographed.
• the positive cells were measured by analysis based on visual observation of the whole well.
• Example 1 Induction of differentiation from iPS cells to primitive streak mesodermal cells
• PDGFR ⁇ -positive cells being 50.2%, when 500,000 cells were seeded to a 10 cm dish. At lower numbers of cells seeded, few cells survived; at a higher number of cells (1,000,000 cells), the percentage of PDGFR ⁇ -positive cells was 26.5%,
• FIG. 1C representing an about half induction efficiency
• Example 2 Influences of BMP4 and Activin A concentrations in induction of differentiation from iPS cells to primitive streak mesodermal cells
• Example 3 Induction of differentiation from primitive streak mesodermal cells derived from iPS cells to skeletal muscle progenitor cells
• the results described in the previous section demonstrated that three factors consisting of Activin A, BMP4, and IGF-I were essential in the differentiation from iPS cells to primitive streak mesodermal cells (during the first 3 days) , the factor that promotes the differentiation from primitive streak mesodermal cells to skeletal muscle progenitor cells (during the last 3 days) remained unidentified.
• the present inventors investigated the potential of the Wnt signal inducer LiCl, used alone or in combination with other growth factors, for inducing the differentiation of iPS cells into skeletal muscle progenitor cells.
• Selected growth factor candidates i.e., Sonic Hedgehog (Shh) and IGF-I
• Sonic Hedgehog (Shh) and IGF-I were added to the differentiation medium for the last 3 days, and their influences on differentiation into skeletal muscle progenitor cells were examined (FIG. 3A) .
• the iPS cell used was iPS-DsRed.
• the results are shown in FIG. 3B.
• SM/C-2.6 is a marker capable of specifically staining satellite cells, which are skeletal muscle stem cells [Fukada, S. et al., Exp Cell Res., 296, 245-255 (2004)], and has been reported to enable the separation of skeletal muscle progenitor cells from a mouse ES cell induction system [Chang, H. et al., Generation of transplantable, functional satellite-like cells from mouse embryonic stem cells. Faseb J., 23: 1907-1919 (2009)].
• iPS-DsRed was induced to differentiate as
• FIG. 3A the cells were cultured in the presence of LiCl, alone or with the addition of Shh 20 ng/ml; the cells were induced to differentiate for a total of 6 days, stained with SM/C-2.6, and evaluated.
• the percentage of SM/C-2.6-positive cells was 28.5% in the Shh-free group, and 38.8% in the Shh 20 ng/ml addition group, showing an increase of about 10% (FIG. 3B) .
• RT-PCR analysis of the expression of Myf5 revealed that the expression of Myf5 was not observed at all in the Shh-free group, whereas Myf5 was expressed in the Shh addition groups, with the highest expression observed at a concentration of 20 ng/ml (FIG. 3B) .
• Example 4 Effect of skeletal muscle progenitor cells derived from iPS cells on damaged skeletal muscles
• PDGFR ⁇ was expressed in about 47% of the cells (FIG. 4A) .
• This cell population was divided by FACS Aria into a PDGFR ⁇ -positive fraction and a negative fraction.
• FIG. 4A 1. for the PDGFRa + fraction, 2. for the PDGFR ⁇ " fraction
• RNA was extracted from these two fractions, and the gene expression was analyzed by RT-PCR (FIG. 4B, 1 for PDGFRa + fraction, 2 for PDGFR ⁇ " fraction) .
• PDGFR ⁇ -positive fraction This demonstrated that the cell population having characters of skeletal muscle progenitor cells are for the most part contained in the PDGFR ⁇ -positive fraction. Additionally, Pax3 and Pax7, which are markers of the dermomyotome, the developmental origin of skeletal muscle progenitor cells, were expressed in both the PDGFR ⁇ -positive and -negative fractions; rather, Pax7 was strongly expressed in the PDGFR ⁇ -negative fraction.
• Pax3 and Pax7 are known to be expressed also in the early development of the nervous system, and also since the expression of Soxl, a marker of neurons in the developmental stage, was strongly expressed in the PDGFR ⁇ -negative fraction, it was thought to be likely that the Pax3 and Pax7 expressed in the PDGFR ⁇ -negative
• RNA fraction represent a cell population of the nervous system.
• the expression of ⁇ -actin served for control to indicate a constant amount of RNA.
• PDGFR ⁇ -negative fraction was a cell population free from skeletal muscle progenitor cells.
• iPS-DsRed cells characters as skeletal muscle progenitor cells in vivo as well.
• the iPS cells used were iPS-DsRed cells. Because iPS-DsRed cells are derived from the TTF of a mouse that is constantly expressing DsRed, their presence serves as an index of the fact of iPS derivation.
• PDGFR ⁇ -positive cells induced from an iPS-DsRed cell were transplanted by intramuscular injection to skeletal muscles of mice with muscle regeneration caused by cardiotoxin treatment. 4 weeks after the transplantation, the expression of DsRed was analyzed to evaluate the behavior of the cells in vivo (FIG. 4D) .
• FIG. 4E shows mean numbers of positive cells visible at five sites counted at the time of tissue examination at a magnifying rate of x400. Whether the cells were transplanted by intramuscular injection (i.m.) or intravenous injection (i.v.), the number of DsRed-positive cells apparent per visual field remained almost constant (2.4 versus 2.6, FIG. 4E, 2 upper rows) .
• Example 5 Effect of skeletal muscle progenitor cells derived from iPS cells on mouse model of muscular dystrophy
• mesodermal cells derived from iPS cells differentiate into satellite cells when transplanted to
• dystrophin was expressed in a mesh-like pattern around the peripheries of muscle fibers (FIG. 5B, rightmost) .
• T.A receiving skeletal muscle progenitor cells derived from iPS cells transplanted thereto, dystrophin was expressed in a mesh- like pattern around the peripheries of muscle fibers, as seen in the wild type (FIG. 5B, 2nd leftmost) .
• mice receiving skeletal muscle progenitor cells derived from iPS cells transplanted thereto were evaluated, a little expression of dystrophin in a mesh-like state was observed in all the forelimbs, quadriceps femoris muscles, and diaphragm, although a background corresponding to the inflamed tissue in the interstitium was observed.
• skeletal muscle progenitor cells derived from iPS cells were engrafted as cells that produce muscle fiber dystrophin when transplanted to dystrophin-deficient muscular dystrophy model mice, resulting in the expression of dystrophin in the muscle fibers, whereby muscle fiber collapse is suppressed, the inflammation in the skeletal muscles is cured, and completion of the tissue repair and over-regeneration state is promoted.
• mice and engrafted were transplanted to the muscular dystrophy model mice and engrafted.
• progenitor cells derived from iPS cells differentiated into satellite cells even in transplantation to the muscular
• the motor function of DMD-null mice transplanted with skeletal muscle progenitor cells was also evaluated.
• a hanging test was performed to determine how long each mouse can endure grasping a cage mesh by its limbs upside down. The test was repeated 3 times at 5-minute resting intervals, and mean
• the hanging time was calculated for a total of 3 repeats.
• the DMD- null mice not receiving the skeletal muscle progenitor cells at all exhibited a hanging time of about 2.3 seconds, whereas the mice receiving the skeletal muscle progenitor cells
• Example 6 Effect of skeletal muscle progenitor cells derived from iPS cells on mouse model of muscular dystrophy (2)
• Example 5 PDGFRa positive cells derived from iPS cells were transplanted to both T.A. of DMD-null mouse. In this Example, the same experiment as in Example 5 was performed except that the cells were transplanted to only one T.A.
• dystrophin fluorescent immunostaining of dystrophin was performed (FIG. 6B) .
• FIG. 6B As expected in control DMD-null mice, no expression of dystrophin (visualized in green) was observed (e, f) .
• dystrophin is expressed in a mesh-like pattern around the peripheries of muscle fibers (a, b) . In the T.A.
• progenitor cells derived from iPS cells were engrafted as cells that produce muscle fiber dystrophin when transplanted to dystrophin-deficient muscular dystrophy model mice, resulting in the expression of dystrophin in the muscle fibers, whereby muscle fiber collapse is suppressed, the inflammation in the skeletal muscles is cured, and completion of the tissue repair and over-regeneration state is promoted.
• DsRed-positive cells were observed mainly in the interstitium (FIG. 6C, c, red arrow) , although DsRed- positive cells were also observed in the peripheral muscle fiber (FIG. 6C, b, e, white arrow), and SM/C-2.6 which is a marker of satellite cells was also expressed in the same site. Therefore, it was demonstrated that the skeletal muscle
• progenitor cells derived from iPS cells differentiated into satellite cells in the body (Fig. 6C, a, c, d, f, white arrow) .
• Example 7 Induction of in vitro differentiation of skeletal muscle progenitor cell derived from iPS cells
• the Myogenin-positive part was identical with the nucleus in the PDGFR ⁇ -positive fraction, which certainly establishes that they were signals in the nucleus (c) . It was also found that the Myogenin-negative nucleus was also present and not all cells had differentiated into the skeletal muscle. On the other hand, in the PDGFR ⁇ - negative fraction, most nuclei were Myogenin-negative (d) . The proportion of the Myogenin-positive nuclei to the total number of nuclei on the culture dish is shown in the Table (e) .
• induction efficiency could be reproduced by continuous addition of LiCl from day 1 or day 2 from the start of the differentiation induction (Fig. 8B) .
• This method could also be reproduced with other iPS cell clone (iPS-Ng-20D-17) .

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