WO2016108288A1 - Procédé de production de cellules progénitrices de muscle squelettique - Google Patents

Procédé de production de cellules progénitrices de muscle squelettique Download PDF

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WO2016108288A1
WO2016108288A1 PCT/JP2015/086585 JP2015086585W WO2016108288A1 WO 2016108288 A1 WO2016108288 A1 WO 2016108288A1 JP 2015086585 W JP2015086585 W JP 2015086585W WO 2016108288 A1 WO2016108288 A1 WO 2016108288A1
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cells
medium
days
inhibitor
cell
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英俊 櫻井
真 池谷
佐藤　貴彦
了 高山
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国立大学法人京都大学
旭化成株式会社
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    • 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
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • 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
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses

Definitions

  • the present invention relates to a novel method for producing skeletal muscle progenitor cells, a reagent kit for inducing differentiation used in the method, and a therapeutic agent for myogenic diseases including skeletal muscle progenitor cells produced by the method.
  • pluripotent cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) obtained by introducing undifferentiated cell-specific genes into somatic cells
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • Non-patent Document 1 Human ES cells treated with 5-Azacytidine (demethylating agent) were suspended in culture (1) Method of further adhesion culture after formation of embryoid bodies (Non-patent Document 2), (3) Method of suspension culture of pluripotent stem cells, followed by adhesion culture, then dissociation and adhesion culture again (Patent Document 1) has been developed.
  • An object of the present invention is to provide a novel method for producing skeletal muscle precursor cells from pluripotent stem cells.
  • the object of the present invention is also to provide skeletal muscle progenitor cells suitable for transplantation.
  • the present inventors have adopted a stepwise differentiation induction method using a specific factor to increase skeletal muscle progenitor cells from pluripotent stem cells. We succeeded in guiding with efficiency. In addition, the present inventors have found that skeletal muscle progenitor cells obtained by such a method show a very high engraftment rate in transplantation, and have completed the present invention.
  • a method for producing skeletal muscle progenitor cells from pluripotent stem cells comprising the following steps (1) and (2A) or (2B).
  • the cell obtained in the step (1) contains HGF and further contains IGF1
  • Cells obtained in the step (2B) (1) of culturing in a good culture solution (i) in a culture solution containing a TGF- ⁇ inhibitor, a GSK3 ⁇ inhibitor, IGF1, HGF and bFGF, (ii) in a culture medium containing a TGF- ⁇ inhibitor, a GSK3 ⁇ inhibitor and IGF1, and (iii)
  • the method according to [1] further comprising the step [2] of sequentially culturing in a culture solution containing a TGF- ⁇ inhibitor, IGF1 and HGF.
  • a step of culturing the cells obtained in the step (2A) or (2B) in a culture solution containing a TGF- ⁇ inhibitor, IGF1 and serum [3] The steps (1), (2B) and The method according to [1] or [2], wherein the TGF- ⁇ inhibitor in (3) is SB431542. [4] The method according to any one of [1] to [3], wherein the GSK3 ⁇ inhibitor in the step (1) is CHIR99021, and the concentration of the CHIR99021 in the medium is 5 ⁇ M or more. [5] The method according to any one of [1] to [4], wherein the GSK3 ⁇ inhibitor in the step (2B) is LiCl.
  • the pluripotent stem cells are human iPS cells or human ES cells.
  • a reagent kit for inducing differentiation from pluripotent stem cells to skeletal muscle progenitor cells comprising SB431542, CHIR99021, IGF1, HGF, and horse serum, and optionally further comprising LiCl and bFGF.
  • the pluripotent stem cells are human iPS cells or human ES cells.
  • a therapeutic agent for myogenic diseases comprising skeletal muscle progenitor cells produced by the method according to any one of [1] to [10].
  • a method for treating a myogenic disease comprising administering an effective amount of a skeletal muscle progenitor cell produced by the method according to any one of [1] to [10] to a subject.
  • the method of the present invention makes it possible to efficiently produce skeletal muscle progenitor cells from pluripotent stem cells. Moreover, since the obtained skeletal muscle progenitor cells show a high engraftment rate in a living body, they are extremely useful in transplantation therapy.
  • (A) shows the result of FACS analysis in which the cell group on day 7 of differentiation induction was selected with CD271 (NGFR) and Pax3-GFP.
  • (B) shows Pax3, T, Tbx6, Mesp2 for cells in each compartment ((1) NGFR-Pax3 +, (2) NGFR + Pax3 +, (3) NGFR + Pax3- and (4) NGFR-Pax3-) , PDGFRa, KDR, CD56, Sox10, NGFR, Pax7 and Myf5 gene expression levels measured by PCR are shown.
  • the value indicates a comparative value with respect to the expression level in the teratoma on the 28th day.
  • the result of having investigated the positive rate of Pax3 in the 14th day at the time of performing a subculture in the differentiation induction 7th day to a skeletal muscle progenitor cell by FACS is shown. 75% in the figure indicates the content of Pax3-positive cells.
  • the result of having measured the expression level of Tbx6, Wnt3a, Mesp2, PDGFRa, Pax3, Meox1 in each day of the differentiation induction process to a skeletal muscle progenitor cell by PCR is shown.
  • the result of the fluorescence image of the cell on the 35th day of differentiation induction into skeletal muscle progenitor cells is shown.
  • (A) is an immunostained image of MHC (red) and DAPI (blue)
  • (B) is a superimposed image of Myogenin (red, immunostained image of DAPI (blue) and bright field image
  • (C) is The fluorescence image of Pax3-GFP (green) is shown.
  • the result of having measured the expression level of Myf5 and MyoD in each day of the differentiation induction process to a skeletal muscle progenitor cell by PCR is shown.
  • the result of the fluorescence image of the cell of the 57th day (A) and 60th day (B) of the differentiation induction to a skeletal muscle progenitor cell is shown.
  • A shows a fluorescent image of Pax3-GFP (green)
  • B shows an immunostained image using antibodies of Embryonic MHC and Myogenin.
  • the Pax3-GFP positive cells (A) and negative cells (B) on the 84th day after induction of differentiation into skeletal muscle progenitor cells are shown by immunostaining images of the cells 7 days after the separation.
  • the upper row shows immunostained images of MHC (red) and DAPI (blue), and the lower row shows a superimposed image of Myogenin (red) and a bright field image.
  • the immunostaining image using the Pax7 (green) and Myf5 (red) antibody of the cell group on the 84th day after induction of differentiation into skeletal muscle progenitor cells is shown.
  • FIG. B shows Pax3-GFP positive cells, human nucleus (red) and eosin (pink) immunostained images (left) and laminin (green) and mice at the 4th week after transplantation into NSG mice.
  • the immunostained image (right figure) of a nucleus (blue) is shown.
  • Myf5-tdTomato positive cells shown in C3 +, red
  • negative cells shown in C3-, blue
  • Myf5-tdTomato positive cells top and negative cells (bottom) on the 44th day of induction of differentiation into skeletal muscle progenitor cells, 1st day (left), 7th (middle), and 14th (after) (Right figure) shows immunostained images of the cells.
  • the vertical axis in the figure represents the ratio of the Myf5 positive rate under each condition when the Myf5 positive rate under the condition where all the additives are added is 1.
  • the present invention provides a method for producing skeletal muscle progenitor cells from pluripotent stem cells using a specific factor in a specific step, a differentiation-inducing reagent kit containing the specific factor used in the method, and the The present invention relates to a therapeutic agent for myogenic diseases comprising skeletal muscle progenitor cells produced by the method.
  • skeletal muscle means mature muscle and includes muscle fibers, that is, myocytes that are multinucleated cells.
  • skeletal muscle progenitor cell means a cell that has not reached mature muscle cells but is in its previous stage and has the ability to selectively differentiate into muscle cells.
  • skeletal muscle progenitor cells do not mean that they have no ability to differentiate into other mesoderm cells such as osteoblasts and adipocytes, and in some cases, differentiation into cells other than muscle cells. Cells having ability can also be included in the skeletal muscle progenitor cells of the present invention.
  • Skeletal muscle progenitor cells are characterized by the expression of specific genes such as MyoD, Myf5, Pax7, Myogenin, myosin heavy chain, NCAM, Desmin, SkMAct, MF20, M-Cadherin, Fgfr4 and VCAME1 It can be identified by detecting expression.
  • the skeletal muscle progenitor cells in the present invention can be preferably Myf5-positive cells, more preferably Myf5 and Pax7-positive cells.
  • skeletal muscle progenitor cells in the present invention include skeletal muscle stem cells or satellite cells, and at least a part thereof can be engrafted as skeletal muscle stem cells or satellite cells after transplantation.
  • the differentiation-induced skeletal muscle progenitor cells may be provided as a cell population containing other cell types or may be a purified cell population.
  • Skeletal muscle progenitor cells need not have the same characteristics as naturally occurring skeletal muscle progenitor cells in humans, but they should have characteristics that are quite similar to those of the skeletal muscle progenitor cells. Those having properties that are capable of substitution are particularly desirable.
  • the skeletal muscle progenitor cell produced by the method of the present invention may be a cell population containing at least skeletal muscle progenitor cells, and may include a cell population containing other types of cells together with skeletal muscle progenitor cells.
  • the proportion of skeletal muscle progenitor cells is, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc., but is not limited thereto.
  • skeletal muscle progenitor cells are isolated from the cell population.
  • the pluripotent stem cell that can be used in the present invention is a stem cell that has pluripotency that can be differentiated into all cells existing in a living body and also has a proliferative ability.
  • ES embryonic stem
  • GS sperm stem
  • EG embryonic germ
  • iPS induced pluripotent stem
  • embryonicity derived from cloned embryos obtained by nuclear transfer Stem (ntES) cells, Muse cells and the like are included.
  • Preferred pluripotent stem cells are ES cells, iPS cells, and ntES cells, and more preferably human ES cells and human iPS cells, and more preferably human iPS cells, from the viewpoint of use in the treatment of myogenic diseases. It is.
  • Embryonic stem cells ES cells are stem cells established from the inner cell mass of early embryos (for example, blastocysts) of mammals such as humans and mice, and having pluripotency and proliferation ability by self-replication.
  • ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, the embryo after the morula, in the 8-cell stage of a fertilized egg, and have the ability to differentiate into any cell that constitutes an adult, so-called differentiation. And ability to proliferate by self-replication.
  • ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and then ES cell lines were established in primates such as humans and monkeys (JA Thomson et al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod 55: 254-259; JA JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).
  • ES cells can be established by taking an inner cell mass from a blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. In addition, maintenance of cells by subculture is performed using a culture solution to which substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (basic fibroblast growth factor (bFGF)) are added. It can be carried out.
  • LIF leukemia inhibitory factor
  • bFGF basic fibroblast growth factor
  • DMEM / F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng / ml bFGF is used as the culture medium for ES cell production.
  • Human ES cells can be maintained in a humid atmosphere of 37 ° C., 2% CO 2 /98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26: 215-224).
  • ES cells also need to be passaged every 3-4 days, where passage is eg 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 1 mM CaCl 2 and 20% KSR. Can be used.
  • ES cells can be generally selected by Real-Time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog as an index.
  • gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog
  • OCT-3 / 4, NANOG, and ECAD can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443). -452).
  • Human ES cell lines for example, WA01 (H1) and WA09 (H9) are obtained from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from the Institute of Regenerative Medicine, Kyoto University (Kyoto, Japan) Is possible.
  • sperm stem cells are testis-derived pluripotent stem cells that are the origin of spermatogenesis. Like ES cells, these cells can be induced to differentiate into various types of cells, and have characteristics such as the ability to create chimeric mice 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).
  • GDNF glial cell line-derived neurotrophic factor
  • Embryonic germ cells are cells that are established from embryonic primordial germ cells and have the same pluripotency as ES cells, such as LIF, bFGF, stem cell factor, etc. It can be established by culturing primordial germ cells in the presence of these substances (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550 -551).
  • iPS Artificial pluripotent stem cells
  • somatic cells in the form of DNA or protein, which is almost equivalent to ES cells
  • It is an artificial stem cell derived from a somatic cell having the characteristics of, for example, differentiation pluripotency and proliferation ability by 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; Nakagawa, M. et al., Nat. Biotechnol.
  • the reprogramming factor is a gene specifically expressed in ES cells, its gene product or non-cording RNA, a gene that plays an important role in maintaining undifferentiation of ES cells, its gene product or non-cording RNA, or It may be constituted by a low molecular compound.
  • genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, etc. are exemplified, and these reprogramming factors may be used alone or in combination.
  • the reprogramming factors include histone deacetylase (HDAC) inhibitors [for example, small molecule inhibitors such as valproate (VPA), trichostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA against HDAC (eg Nucleic acid expression inhibitors such as HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene) etc.], MEK inhibitors (eg PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen synthase-kinase 3 inhibitors (eg, Bio and CHIR99021), DNA methyltransferase inhibitors (eg, 5-azacytidine), histone methyltransferase inhibitors (eg, small molecule inhibitors such as BIX-01294, Suv39hl, Suv39h2, SetDBl and G9a nucleic acid expression inhibitors such as siRNA and
  • the reprogramming factor may be introduced into a somatic cell by a technique such as lipofection, fusion with a cell membrane-permeable peptide (for example, TAT and polyarginine derived from HIV), or microinjection.
  • a cell membrane-permeable peptide for example, TAT and polyarginine derived from HIV
  • DNA it can be introduced into somatic cells by techniques such as vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, and microinjection.
  • viral vectors examples include retroviral vectors and lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (WO 2010/008054) and the like.
  • artificial chromosome vectors examples include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC).
  • a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008).
  • the vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. so that a nuclear reprogramming substance can be expressed.
  • Selective marker sequences such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as green fluorescent protein (GFP), ⁇ -glucuronidase (GUS), FLAG, etc. Can be included.
  • the above vector has a LoxP sequence before and after the introduction of the gene into a somatic cell in order to excise the gene or promoter encoding the reprogramming factor and the gene encoding the reprogramming factor that binds to it. May be.
  • RNA it may be introduced into somatic cells by techniques such as lipofection and microinjection, and in order to suppress degradation, RNA incorporating 5-methylcytidine and pseudouridine® (TriLink® Biotechnologies) is used. Yes (Warren L, (2010) Cell Stem Cell. 7: 618-630).
  • culture media for iPS cell induction examples include DMEM, DMEM / F12, or DME culture media containing 10-15% FBS (these culture media include LIF, penicillin / streptomycin, puromycin, L-glutamine). , Non-essential amino acids, ⁇ -mercaptoethanol, etc.) or commercially available culture media (eg, culture media for mouse ES cell culture (TX-WES culture solution, Thrombo X), primate ES cells) Culture medium for culture (primate ES / iPS cell culture medium, Reprocell), serum-free medium (mTeSR, Stemcell Technology).
  • the somatic cell and the reprogramming factor are contacted on DMEM or DMEM / F12 containing 10% FBS for about 4 to 7 days. Then, re-spread the cells on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.), and use bFGF-containing primate ES cell culture medium about 10 days after contact of the somatic cells with the reprogramming factor. Culturing and generating iPS-like colonies about 30 to about 45 days or more after the contact.
  • feeder cells e.g., mitomycin C-treated STO cells, SNL cells, etc.
  • 10% FBS-containing DMEM culture medium including LIF, penicillin / streptomycin, etc.
  • feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
  • 5% CO 2 at 37 ° C. can contain puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.
  • somatic cells to be reprogrammed themselves are used (Takahashi K, et al. (2009), PLoS One.
  • extracellular matrix eg, Laminin- 5 (WO2009 / 123349) and Matrigel (BD)
  • a method of culturing using a medium not containing serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci USA 106.15720-15725).
  • iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845).
  • the culture medium is exchanged with a fresh culture medium once a day from the second day onward.
  • the number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 ⁇ 10 3 to about 5 ⁇ 10 6 cells per 100 cm 2 of culture dish.
  • IPS cells can be selected according to the shape of the formed colonies.
  • a drug resistance gene that is expressed in conjunction with a gene that is expressed when somatic cells are initialized for example, Oct3 / 4, Nanog
  • a culture solution containing the corresponding drug selection The established iPS cells can be selected by culturing with the culture medium.
  • the marker gene is a fluorescent protein gene
  • iPS cells are selected by observing with a fluorescence microscope, in the case of a luminescent enzyme gene, by adding a luminescent substrate, and in the case of a chromogenic enzyme gene, by adding a chromogenic substrate can do.
  • the term “somatic cell” refers to any animal cell (preferably, a mammalian cell including a human) except a germ line cell such as an egg, oocyte, ES cell, or totipotent cell.
  • Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells. , Passage cells, and established cell lines.
  • somatic cells include, for example, (1) neural stem cells, hematopoietic stem cells, mesenchymal stem cells, tissue stem cells such as dental pulp stem cells (somatic stem cells), (2) tissue progenitor cells, (3) lymphocytes, epithelium Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, enterocytes, spleen cells, pancreatic cells (exocrine pancreas cells, etc.), brain cells, lung cells, kidney cells Examples thereof include differentiated cells such as fat cells.
  • the mammal individual from which somatic cells are collected is not particularly limited, but is preferably a human.
  • somatic cells should be collected from the patient or another person with the same or substantially the same HLA type from the viewpoint that no rejection occurs.
  • the type of HLA is “substantially the same” means that when the cells obtained by inducing differentiation from iPS cells derived from the somatic cells are transplanted into a patient by using an immunosuppressant or the like, the transplanted cells are This means that the HLA types match to the extent that they can be engrafted.
  • the main HLA for example, 3 loci of HLA-A, HLA-B and HLA-DR, or 4 loci plus HLA-C) are the same (hereinafter the same).
  • E Cloned embryo-derived ES cells obtained by nuclear transfer nt ES cells are cloned embryo-derived ES cells produced by nuclear transfer technology and have almost the same characteristics as ES cells derived from fertilized eggs (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. (2007) , Nature, 450: 497-502).
  • an ES cell established from an inner cell mass of a clonal embryo-derived blastocyst obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell is an nt ES (nuclear transfer ES) cell.
  • nt ES nuclear transfer ES
  • nuclear transfer technology JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (above) is used (Wakayama). Seika et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 47-52).
  • Nuclear transfer can be initialized by injecting a somatic cell nucleus into a mammal's enucleated unfertilized egg and culturing for several hours.
  • Muse cells are pluripotent stem cells produced by the method described in WO2011 / 007900. Specifically, fibroblasts or bone marrow stromal cells are treated with trypsin for a long time, preferably 8 or 16 hours. It is a pluripotent cell obtained by suspension culture after treatment, and is positive for SSEA-3 and CD105.
  • ⁇ Method for producing skeletal muscle progenitor cells from pluripotent stem cells> in producing skeletal muscle progenitor cells from pluripotent cells, a method comprising the following steps (1) and (2A) or (2B) can be used.
  • Step of culturing a pluripotent stem cell in a culture solution containing a TGF- ⁇ inhibitor and a GSK3 ⁇ inhibitor The cell obtained in the step (1) contains HGF and further contains IGF1 Cells obtained in the step (2B) (1) of culturing in a good culture solution (i) in a culture solution containing a TGF- ⁇ inhibitor, a GSK3 ⁇ inhibitor, IGF1, HGF and bFGF, (ii) in a culture medium containing a TGF- ⁇ inhibitor, a GSK3 ⁇ inhibitor and IGF1, and (iii) Step of sequentially culturing in a culture medium containing a TGF- ⁇ inhibitor, IGF1 and HGF
  • the present invention also provides a cell obtained in the step (3), (2A) or (2B), a TGF- ⁇ inhibitor, IGF1 and A method including a step of culturing in a culture solution containing serum can be used.
  • the present invention includes a method comprising the step of isolating cells expressing Myf5 from the cell population obtained in the steps (4) and (3) in addition to the steps (1) to (3). Can be used.
  • the present invention can use a method including a step of re-culturing the cells obtained in the steps (5) and (4) in addition to the steps (1) to (4).
  • This step may include a step of culturing pluripotent stem cells prior to induction of differentiation from pluripotent stem cells to skeletal muscle progenitor cells.
  • pluripotent stem cells may be isolated by any method and cultured by suspension culture, or may be cultured by adhesion culture using a coated culture dish.
  • adhesion culture is preferably used.
  • a method for separating pluripotent stem cells for example, a method of separating mechanically, a separation solution having protease activity and collagenase activity (for example, Accutase (TM) and Accumax (TM)) or only collagenase activity is used.
  • TM Accutase
  • TM Accutase
  • TM Accumax
  • a separation method using a separation solution a dissociation method using Trypsin / EDTA, and the like.
  • a method of dissociating cells using Trypsin / EDTA is used.
  • the floating culture means that an embryoid body is formed by culturing cells in a non-adhering state on a culture dish, and is not particularly limited.
  • artificial culture is intended to improve adhesion to cells.
  • Culture dishes that have not been treated (eg, coated with an extracellular matrix) or artificially suppressed adhesion (eg, coated with polyhydroxyethyl methacrylic acid (poly-HEMA)) Can be done using.
  • the adhesion culture can be performed by culturing on a feeder cell or using a culture vessel coated with an extracellular matrix.
  • the coating treatment can be performed by placing a solution containing an extracellular matrix in a culture container and then removing the solution as appropriate.
  • the feeder cell means another cell that plays an auxiliary role used for adjusting the culture condition of the target cell.
  • the extracellular matrix is a supramolecular structure existing outside the cell and may be naturally derived or an artificial product (recombinant). Examples thereof include substances such as collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, laminin or fragments thereof.
  • These extracellular substrates may be used in combination, and may be prepared from cells such as BD-Matrigel (TM). Examples of the artifact include laminin fragments.
  • laminin is a protein having a heterotrimeric structure having one ⁇ chain, one ⁇ chain, and one ⁇ chain, and is not particularly limited.
  • the ⁇ chain includes ⁇ 1, ⁇ 2, ⁇ 3, It is ⁇ 4 or ⁇ 5, the ⁇ chain is ⁇ 1, ⁇ 2 or ⁇ 3, and the ⁇ chain is exemplified by ⁇ 1, ⁇ 2 or ⁇ 3.
  • the laminin fragment is not particularly limited as long as it is a laminin fragment having integrin-binding activity, and examples thereof include an E8 fragment which is a fragment obtained by digestion with elastase.
  • the step of culturing pluripotent stem cells is preferably adhesion culture, and the adhesion culture is a culture vessel coated with Matrigel (TM). Can be an adherent culture.
  • the culture medium to be used can be prepared using a medium used for culturing animal cells as a basal medium.
  • basal medium examples include IMDM medium, MediumMedi199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 medium , RPMI-base medium, mTeSR1 and mixed media thereof.
  • EMEM Eagle's Minimum Essential Medium
  • DMEM Dulbecco's modified Eagle's Medium
  • Ham's F12 medium Ham's F12 medium
  • RPMI 1640 medium Fischer's medium
  • StemPro34 medium RPMI-base medium
  • mTeSR1 is preferably used.
  • the medium may contain serum or may be serum-free.
  • the medium can be, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen It may contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol (2ME), thiol glycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, small molecule compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts.
  • KSR Knockout Serum Replacement
  • the culture period of the step of culturing pluripotent stem cells is exemplified by 1 day or more and 10 days or less, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 Day, 10 days, etc., preferably 4 days.
  • the medium contains a ROCK inhibitor.
  • the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK).
  • ROCK Rho kinase
  • Y-27632 can be preferably used in the present invention.
  • the concentration of Y-27632 in the medium is not particularly limited, but preferably 1 ⁇ M to 50 ⁇ M, for example, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 11 ⁇ M, 12 ⁇ M, 13 ⁇ M, 14 ⁇ M , 15 ⁇ M, 16 ⁇ M, 17 ⁇ M, 18 ⁇ M, 19 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, and 50 ⁇ M, but are not limited thereto. More preferably, it is 10 ⁇ M.
  • the period during which the ROCK inhibitor is added to the medium may be the culture period of the step of culturing pluripotent stem cells, and may be a period that suppresses cell death at the time of single dispersion, for example, at least 1 Day.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is about 2-5%, preferably 5%.
  • the medium can be changed during the culture period.
  • the medium used for the medium exchange may be a medium having the same components as the medium before the medium exchange or a medium having different components.
  • a medium having the same components is used.
  • the timing of the medium exchange is not particularly limited, but is performed, for example, every 1 day, every 2 days, every 3 days, every 4 days, or every 5 days after the start of cultivation with a fresh medium.
  • the medium exchange is preferably performed every 2 days.
  • Step (1) Step of culturing pluripotent stem cells in a culture solution containing a TGF- ⁇ inhibitor and a GSK3 ⁇ inhibitor
  • This step (1) is a step of culturing pluripotent stem cells with a TGF- ⁇ inhibitor and a GSK3 ⁇ inhibitor. It is the process of culturing in the culture solution containing.
  • the culture solution used in this step (1) can be prepared using a medium used for culturing animal cells as a basal medium.
  • IMDM medium for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen) , RPMI-base medium, and mixed media thereof.
  • EMEM Eagle's Minimum Essential Medium
  • DMEM Dulbecco's modified Eagle's Medium
  • Ham's F12 medium for example, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen) , RPMI-base medium, and mixed media thereof.
  • a mixed medium of IMDM medium and Ham's F12 medium is preferable.
  • the basal medium may contain serum or may be serum-free.
  • albumin, transferrin, Knockout Serum Replacement (KSR) serum substitute for FBS during ES cell culture
  • N2 supplement Invitrogen
  • B27 supplement Invitrogen
  • fatty acid insulin
  • selenium sub-selenium
  • N2 supplement Invitrogen
  • B27 supplement Invitrogen
  • fatty acid insulin
  • selenium sub-selenium
  • serum substitutes such as sodium acid
  • collagen precursor such as collagen precursor, trace elements
  • thiol glycerol lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non It may also contain one or more substances such as essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.
  • a preferred basal medium is a mixed medium of IMDM medium and Ham's F12 medium supplemented
  • the TGF ⁇ inhibitor is not particularly limited as long as it is a substance that inhibits signal transduction from binding of TGF ⁇ to the receptor to SMAD. For example, it inhibits binding of TGF ⁇ to the ALK family of receptors. And substances that inhibit the phosphorylation of SMAD by the ALK family.
  • TGF ⁇ inhibitors include, for example, Lefty-1 (NCBI Accession No., mouse: NM_010094, human: NM_020997 is exemplified), SB431542, SB202190 (above, RKLindemann et al., Mol.
  • the TGF ⁇ inhibitor used in this step (1) can be preferably SB431542.
  • the concentration of SB431542 in the medium is not particularly limited, but preferably 1 ⁇ M to 50 ⁇ M, for example, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 11 ⁇ M, 12 ⁇ M, 13 ⁇ M, 14 ⁇ M, 15 ⁇ M 16 ⁇ M, 17 ⁇ M, 18 ⁇ M, 19 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M ⁇ , 45 ⁇ M, and 50 ⁇ M, but are not limited thereto. More preferably, it is 2 ⁇ M to 10 ⁇ M, particularly preferably 5 ⁇ M.
  • a GSK-3 ⁇ inhibitor is defined as a substance that inhibits the kinase activity of GSK-3 ⁇ protein (for example, phosphorylation ability for ⁇ -catenin), and many of them are already known.
  • Lithium chloride (LiCl) first discovered as a -3 ⁇ inhibitor, BIO (also known as GSK-3 ⁇ inhibitor IX; 6-bromoindirubin 3'-oxime), SB216763 (3 -(2,4-Dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione), a GSK-3 ⁇ inhibitor VII which is a phenyl ⁇ bromomethyl ketone compound VII (4-dibromoacetophenone), L803-mts (also known as GSK-3 ⁇ peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2), which is a cell membrane permeation type phosphorylated peptide, and CHIR
  • the GSK-3 ⁇ inhibitor used in this step (1) may preferably be CHIR99021.
  • the concentration of CHIR99021 in the medium is not particularly limited, but preferably a concentration higher than 1 ⁇ M, which is the concentration used in GSK-3 ⁇ inhibition, is used, for example, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, Examples include, but are not limited to, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, and 50 ⁇ M. More preferably, it is 5 ⁇ M or more (for example, 5 ⁇ M to 50 ⁇ M, preferably 5 ⁇ M to 10 ⁇ M).
  • the culture period is exemplified by 10 days or more and 30 days or less, for example, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, etc., preferably 16 days to 21 days Between, more preferably 16 days.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is about 2-5%, preferably 5%.
  • passaging is an operation of dissociating cells in culture from a container and reseeding.
  • cells that are adhered to each other to form a population are dissociated (separated) into individual cells.
  • a method for dissociating cells for example, a method of dynamically dissociating, a dissociation solution having protease activity and collagenase activity (for example, Accutase (TM) and Accumax (TM)), or a dissociation solution having only collagenase activity is used.
  • TM Accutase
  • TM Accutase
  • TM Accumax
  • dissociation solution having only collagenase activity is used.
  • the dissociation method using Trypsin / EDTA for example, a method of dissociating cells using Trypsin / EDTA is used.
  • the passage is not particularly limited, but every 4 to 10 days from the start of this step, for example, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, preferably Is carried out every 7 days.
  • the culture solution immediately after the passage may contain a ROCK inhibitor for the purpose of preventing cell death of the replated cells.
  • the ROCK inhibitor can be used under the same conditions as described above.
  • the timing of the medium exchange is not particularly limited, but is performed, for example, every 1 day, every 2 days, every 3 days, every 4 days, or every 5 days after the start of cultivation with a fresh medium.
  • the medium exchange is preferably performed every 2 days.
  • the cells obtained in the step (1) may contain HGF and further contain IGF1. It is a process of culturing in a good culture solution.
  • the culture solution used in this step (2A) can be prepared using a medium used for culturing animal cells as a basal medium.
  • IMDM medium for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen) , RPMI-base medium, SF-O3 medium (Adeia Co., Ltd.) and mixed media thereof.
  • SF-O3 medium is preferably used.
  • the basal medium may contain serum or may be serum-free.
  • serum substitutes such as sodium acid
  • N2 supplement Invitrogen
  • B27 supplement Invitrogen
  • fatty acid insulin
  • selenium sub-selenium
  • serum substitutes such as sodium acid
  • collagen precursor such as collagen precursor
  • trace elements such as collagen precursor
  • thiol glycerol lipids
  • amino acids such as L-glutamine
  • Glutamax Invitrogen
  • non It may also contain one or more substances such as essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.
  • a preferred basal medium is SF-O3 medium supplemented with albumin and 2-mercaptoethanol.
  • the concentration of HGF in the medium used in this step (2A) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / ml , 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but not limited thereto. More preferably, it is 10
  • the concentration of IGF1 in the medium used in this step (2A) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / ml , 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml
  • the culture period is exemplified by 1 day or more and 40 days or less.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is about 2-5%, preferably 5%.
  • the cells obtained in the step (1) are (i) in a culture solution containing a TGF- ⁇ inhibitor, a GSK3 ⁇ inhibitor, IGF1, HGF and bFGF, (ii) in a culture medium containing a TGF- ⁇ inhibitor, a GSK3 ⁇ inhibitor and IGF1, and (iii) A step of sequentially culturing in a culture medium containing a TGF- ⁇ inhibitor, IGF1 and HGF.
  • This step is a step performed instead of the previous step (2A).
  • sub-steps (i) to (iii) will be described in order.
  • Sub-step (i) Step of culturing in a culture solution containing TGF- ⁇ inhibitor, GSK3 ⁇ inhibitor, IGF1, HGF and bFGF
  • the culture solution used in this sub-step (i) is a medium used for culturing animal cells Can be prepared as a basal medium.
  • the basal medium for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen) , RPMI-base medium, SF-O3 medium (Adeia Co., Ltd.) and mixed media thereof.
  • SF-O3 medium is preferably used.
  • the basal medium may contain serum or may be serum-free.
  • serum substitutes such as sodium acid
  • N2 supplement Invitrogen
  • B27 supplement Invitrogen
  • fatty acid insulin
  • selenium sub-selenium
  • serum substitutes such as sodium acid
  • collagen precursor such as collagen precursor
  • trace elements such as collagen precursor
  • thiol glycerol lipids
  • amino acids such as L-glutamine
  • Glutamax Invitrogen
  • non It may also contain one or more substances such as essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.
  • a preferred basal medium is SF-O3 medium supplemented with albumin and 2-mercaptoethanol.
  • the TGF ⁇ inhibitor used in this sub-step (i) can be the same as described above, and preferably SB431542.
  • the concentration of SB431542 in the medium used in this substep (i) is not particularly limited, but is preferably 1 ⁇ M to 50 ⁇ M, for example, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, It is not limited to 11 ⁇ M, 12 ⁇ M, 13 ⁇ M, 14 ⁇ M, 15 ⁇ M, 16 ⁇ M, 17 ⁇ M, 18 ⁇ M, 19 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M ⁇ , 45 ⁇ M, and 50 ⁇ M. More preferably, it is 10 ⁇ M.
  • the GSK-3 ⁇ inhibitor used in this sub-step (i) can be the same as described above, but can be preferably LiCl.
  • the concentration of LiCl in the medium used in this substep (i) is not particularly limited, but is preferably 1 mM to 50 mM, for example, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, Although it is 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, it is not limited to these. More preferably, it is 5 mM.
  • the concentration of IGF1 in the medium used in this substep (i) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, such as 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / Examples include, but are not limited to, ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml. More preferably, it
  • the concentration of HGF in the medium used in this substep (i) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, such as 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / Examples include, but are not limited to, ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml. More preferably, it is
  • the concentration of bFGF in the medium used in this substep (i) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, such as 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / Examples include, but are not limited to, ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml. More preferably,
  • the culture period is exemplified by 1 day or more and 10 days or less, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days. , 10 days, etc., preferably 4 days.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is about 2-5%, preferably 5%.
  • Sub-step (ii) a step of culturing in a culture medium containing a TGF- ⁇ inhibitor, a GSK3 ⁇ inhibitor and IGF1
  • the cells obtained in the sub-step (i) are treated with TGF- ⁇ .
  • the culture solution used in this substep (ii) can be prepared using a medium used for animal cell culture as a basal medium.
  • IMDM medium for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen) , RPMI-base medium, SF-O3 medium and mixed medium thereof.
  • EMEM Eagle's Minimum Essential Medium
  • DMEM Dulbecco's modified Eagle's Medium
  • Ham's F12 medium Ham's F12 medium
  • RPMI 1640 medium Fischer's medium
  • StemPro34 invitrogen
  • RPMI-base medium for example, SF-O3 medium is preferably used.
  • the basal medium may contain serum or may be serum-free.
  • serum substitutes such as sodium acid
  • N2 supplement Invitrogen
  • B27 supplement Invitrogen
  • fatty acid insulin
  • selenium sub-selenium
  • serum substitutes such as sodium acid
  • collagen precursor such as collagen precursor
  • trace elements such as collagen precursor
  • thiol glycerol lipids
  • amino acids such as L-glutamine
  • Glutamax Invitrogen
  • non It may also contain one or more substances such as essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.
  • a preferred basal medium is SF-O3 medium supplemented with albumin and 2-mercaptoethanol.
  • the TGF ⁇ inhibitor used in this sub-step (ii) can be the same as described above, and preferably SB431542.
  • the concentration of SB431542 in the medium used in this substep (ii) is not particularly limited, but is preferably 1 ⁇ M to 50 ⁇ M, for example, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, It is not limited to 11 ⁇ M, 12 ⁇ M, 13 ⁇ M, 14 ⁇ M, 15 ⁇ M, 16 ⁇ M, 17 ⁇ M, 18 ⁇ M, 19 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M ⁇ , 45 ⁇ M, and 50 ⁇ M. More preferably, it is 10 ⁇ M.
  • the GSK-3 ⁇ inhibitor used in this sub-step (ii) the same one as described above can be used, but preferably it can be LiCl.
  • the concentration of LiCl in the medium used in this substep (ii) is not particularly limited, but is preferably 1 mM to 50 mM, for example, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, Although it is 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, it is not limited to these. More preferably, it is 5 mM.
  • the concentration of IGF1 in the medium used in this substep (ii) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but not limited thereto. More preferably, it is 10
  • the culture period is exemplified by 1 day or more and 10 days or less, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days. , 10 days, etc., preferably 3 days.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is about 2-5%, preferably 5%.
  • Sub-step (iii) a step of culturing in a culture solution containing a TGF- ⁇ inhibitor, IGF1 and HGF
  • This sub-step (iii) is a step of treating the cells obtained in the sub-step (ii) with a TGF- ⁇ inhibitor. Culturing in a culture solution containing IGF1 and HGF.
  • the culture solution used in this substep (iii) can be prepared using a medium used for culturing animal cells as a basal medium.
  • IMDM medium for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen) , RPMI-base medium, SF-O3 medium and mixed medium thereof.
  • EMEM Eagle's Minimum Essential Medium
  • DMEM Dulbecco's modified Eagle's Medium
  • Ham's F12 medium for example, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen) , RPMI-base medium, SF-O3 medium and mixed medium thereof.
  • SF-O3 medium is preferably used.
  • the basal medium may contain serum or may be serum-free.
  • serum substitutes such as sodium acid
  • N2 supplement Invitrogen
  • B27 supplement Invitrogen
  • fatty acid insulin
  • selenium sub-selenium
  • serum substitutes such as sodium acid
  • collagen precursor such as collagen precursor
  • trace elements such as collagen precursor
  • thiol glycerol lipids
  • amino acids such as L-glutamine
  • Glutamax Invitrogen
  • non It may also contain one or more substances such as essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.
  • a preferred basal medium is SF-O3 medium supplemented with albumin and 2-mercaptoethanol.
  • the TGF ⁇ inhibitor used in this substep (iii) may be the same as described above, but may preferably be SB431542.
  • the concentration of SB431542 in the medium used in this substep (iii) is not particularly limited, but is preferably 1 ⁇ M to 50 ⁇ M, for example, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, It is not limited to 11 ⁇ M, 12 ⁇ M, 13 ⁇ M, 14 ⁇ M, 15 ⁇ M, 16 ⁇ M, 17 ⁇ M, 18 ⁇ M, 19 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M ⁇ , 45 ⁇ M, and 50 ⁇ M. More preferably, it is 10 ⁇ M.
  • the TGF ⁇ inhibitor used in can preferably be SB431542.
  • the concentration of IGF1 in the medium used in this substep (iii) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but not limited thereto. More preferably, it is
  • the concentration of HGF in the medium used in this substep (iii) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but not limited thereto. More preferably, it is 10
  • the culture period is exemplified by 7 days or more and 20 days or less, for example, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days. 16 days, 17 days, 18 days, 19 days, 20 days, etc., preferably 14 days.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is about 2-5%, preferably 5%.
  • the timing of the medium exchange is not particularly limited, but is performed, for example, every 1 day, every 2 days, every 3 days, every 4 days, or every 5 days after the start of cultivation with a fresh medium. Preferably, it is performed every 2 days.
  • the medium exchange can be performed, for example, once a week, twice a week, three times a week, or four times a week after starting culture with a fresh medium. Preferably, it is performed twice a week.
  • Step (3) Step of culturing in a culture solution containing a TGF- ⁇ inhibitor, IGF1 and serum
  • the cells obtained in the step (2A) or (2B) are treated with TGF- ⁇ inhibition. It is a step of culturing in a culture solution containing an agent, IGF1 and serum.
  • the culture solution used in this step (3) can be prepared using a medium used for culturing animal cells as a basal medium.
  • IMDM medium for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen) , RPMI-base medium and mixed media thereof are included.
  • a DMEM medium is preferably used.
  • the basal medium may contain serum or may be serum-free.
  • serum substitutes such as sodium acid
  • N2 supplement Invitrogen
  • B27 supplement Invitrogen
  • fatty acid insulin
  • selenium sub-selenium
  • serum substitutes such as sodium acid
  • collagen precursor such as collagen precursor
  • trace elements such as collagen precursor
  • thiol glycerol lipids
  • amino acids such as L-glutamine
  • Glutamax Invitrogen
  • non It may also contain one or more substances such as essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.
  • a preferred basal medium is a DMEM medium supplemented with serum, L-glutamine and 2-mercaptoethanol.
  • the serum used in this step (3) is horse serum, and the concentration in the basal medium is 1 to 10%, more preferably 2%.
  • the TGF ⁇ inhibitor used in this step (3) may be the same as described above, but may preferably be SB431542.
  • the concentration of SB431542 in the medium used in this step (3) is not particularly limited, but is preferably 500 nM to 50 ⁇ M, for example, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, Examples include, but are not limited to, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M, and 50 ⁇ M. More preferably, it is 5 ⁇ M.
  • the concentration of IGF1 in the medium used in this step (3) is not particularly limited, but is preferably 1 ng / ml to 50 ng / ml, such as 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35g / ml, 40ng / ml, 45ng / Examples include, but are not limited to, ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml. More preferably, it is 10
  • the culture period is exemplified by 9 days to 50 days, preferably 20 days to 40 days and preferably 30 days to 40 days.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is about 2-5%, preferably 5%.
  • the timing of the medium exchange is not particularly limited, but is performed, for example, every 1 day, every 2 days, every 3 days, every 4 days, or every 5 days after the start of cultivation with a fresh medium. Preferably, it is performed every 2 days.
  • the medium exchange can be performed, for example, once a week, twice a week, three times a week, or four times a week after starting culture with a fresh medium. Preferably, it is performed twice a week.
  • Step (4) Step of isolating cells expressing Myf5
  • cells expressing Myf5 are isolated from the cell population containing the skeletal muscle progenitor cells obtained in step (3). It is a process.
  • Myf5 expressing cells are isolated by detecting the expression of Myf5 in the cells and selectively expressing cells expressing Myf5 based on the expression.
  • Myf5 There is no particular limitation as long as it is a method that can detect cells that are not expressed and selectively remove cells that do not express Myf5 based on the detected cells. Therefore, you may isolate using the gene which substitutes Myf5.
  • a gene substituting Myf5 a gene existing on the cell surface is preferable, and for example, CD34 or M-cadherin is exemplified.
  • Isolation of cells expressing Myf5 can be performed using, for example, an antibody that specifically binds to Myf5 or a gene that substitutes for Myf5. In addition, it can also be carried out by detecting a marker gene operably linked to the Myf5 promoter.
  • a “marker gene” is a gene that encodes a protein that can be translated in a cell and function as an index. For example, the protein assists fluorescent protein, photoprotein, fluorescence, luminescence, or coloration. But not limited to those encoded by drug resistance genes.
  • fluorescent proteins include blue fluorescent proteins such as Sirius, BFP, and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; TurboGFP, AcGFP, TagGFP, Azami-Green (for example, hmAG1 ), Green fluorescent proteins such as ZsGreen, EmGFP, EGFP, GFP2, HyPer; yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana; KusabiraOrange (for example, hmKO2), mOrange Orange fluorescent proteins such as: TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry and other red fluorescent proteins; TurboFP602, mRFP1, JRed, KillerRed, mCh
  • the photoprotein can be exemplified by aequorin, but is not limited thereto.
  • proteins that assist fluorescence, luminescence, or coloration include, but are not limited to, enzymes that degrade fluorescence, luminescence, or color precursors such as luciferase, phosphatase, peroxidase, and ⁇ -lactamase.
  • the protein encoded by the drug resistance gene may be any protein that is resistant to the corresponding drug.
  • the drug resistance gene in the present invention includes, for example, an antibiotic resistance gene, but is not limited thereto.
  • antibiotic resistance genes include kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, blasticidin resistance gene, gentamicin resistance gene, kanamycin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, etc. .
  • a marker gene when a marker gene is detected, it can be carried out, for example, by introducing into the cell a vector in which the promoter region sequence of Myf5 gene is linked upstream of the marker gene.
  • cells expressing Myf5 can be identified with cells expressing the marker gene, so depending on whether the marker gene is positive, cells expressing Myf5 can be isolated. it can.
  • isolation of cells is not particularly limited, but can be performed by, for example, a flow cytometry method.
  • a flow cytometry method cell particles are flowed at a high speed in a very thin flow, irradiated with laser light, and light such as fluorescence (when cells are pre-fluorescently labeled) and scattered light are emitted. If a cell sorter is provided for measurement, a target cell can be isolated.
  • Step (5) Step of Reculturing Isolated Cells
  • This step (5) is a step of reculturing the skeletal muscle progenitor cells isolated in the step (4) in a culture solution.
  • the culture solution used in this step (5) can be prepared using a medium used for culturing animal cells as a basal medium.
  • the same medium as in the above step (3) is used, but is not limited thereto.
  • the culture period may be any period as long as the engraftment rate at the transplant destination is increased when the skeletal muscle progenitor cells are transplanted, and is exemplified by 6 hours to 60 hours.
  • the time may be 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, etc., preferably 24 hours.
  • a reagent kit for inducing differentiation of skeletal muscle progenitor cells from pluripotent stem cells includes a skeletal muscle progenitor cell inducer (for example, a lyophilized product, a frozen solution dissolved in an appropriate buffer, etc.), cells, reagents and A culture medium may be included.
  • the kit may further include a document or instructions describing the differentiation induction procedure.
  • the skeletal muscle progenitor cells derived from pluripotent stem cells produced by the method of the present invention can be used for various purposes. For example, autologous or allogeneic transplantation into a patient with skeletal muscle progenitor cells differentiated from iPS cells induced using somatic cells collected from a subject or from another person with the same or substantially the same type of HLA Stem cell therapy by transplantation becomes possible.
  • the target disease in the present invention is exemplified by myogenic diseases (myopathy).
  • Myogenic diseases include, for example, muscular dystrophy (eg, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, congenital muscular dystrophy, limb girdle muscular dystrophy, myotonic muscular dystrophy, etc.), congenital myopathy, distal myopathy, mitochondrial myopathy And hereditary myopathy such as polymyositis, dermatomyositis, myasthenia gravis, and other non-hereditary myopathy muscular dystrophy, glycogenosis, and periodic limb paralysis.
  • a more preferred subject is muscular dystrophy.
  • the skeletal muscle progenitor cells differentiated from the subject-derived iPS cells are more likely to reflect the state of the muscle cells in the subject's body. It can also be suitably used for a toxicity in-vitro evaluation system. Furthermore, it can be preferably used as a tool for pathological research of undiscovered myopathy.
  • the present invention provides a therapeutic agent for myogenic diseases comprising skeletal muscle progenitor cells produced from pluripotent stem cells by the above-described method.
  • a therapeutic agent for myogenic diseases comprising skeletal muscle progenitor cells produced from pluripotent stem cells by the above-described method.
  • CTx Cardiotoxin
  • engraftment of the transplanted skeletal muscle progenitor cells was observed. . Therefore, the skeletal muscle progenitor cells provided in the present invention are useful for treating myogenic diseases.
  • Skeletal muscle progenitor cells for the treatment of hereditary muscular diseases such as muscular dystrophy include skeletal muscle progenitors derived from pluripotent stem cells derived from other patients with the same or substantially the same HLA type as the patient Cells are preferably used.
  • human regenerative medicine it is not easy to obtain human ES cells of the same or substantially the same type of HLA, so human iPS cells are used as pluripotent stem cells for inducing skeletal muscle progenitor cells. It is preferable to use it.
  • skeletal muscle progenitor cells differentiated from iPS cells derived from the patient's own somatic cells can be used as skeletal muscle progenitor cells for the treatment of hereditary muscle disease.
  • iPS cells derived from somatic cells of a DMD patient are deficient in the dystrophin gene
  • a normal dystrophin gene is introduced into the iPS cells.
  • the dystrophin cDNA is 14 kb in length, and the optimal adeno-associated virus (AAV) vector for gene transfer into muscle cells has only about 4.5 kb capacity, so the current gene therapy strategy is a shortened functional dystrophin gene.
  • AAV adeno-associated virus
  • the mutation site of the causative gene can be repaired using the endogenous DNA repair mechanism or homologous recombination of iPS cells. That is, a chimeric RNA / DNA oligonucleotide (chimeraplast) having a sequence with a normal mutation site is introduced and bound to a target sequence to form a mismatch, and an endogenous DNA repair mechanism is activated to induce gene repair.
  • gene repair can be performed by introducing a single-stranded DNA having 400-800 bases homologous to the mutation site to cause homologous recombination.
  • the normal skeletal muscle progenitor cell derived from the patient can be produced by inducing differentiation of the iPS cell obtained by repairing the disease-causing gene thus obtained into the skeletal muscle progenitor cell through the above-described steps.
  • an immune response to normal gene products may occur even in the patient's own skeletal muscle progenitor cells Therefore, in any case, when transplanting skeletal muscle progenitor cells, it is preferable to use an immunosuppressant in combination.
  • a dystrophin function may be substituted by introducing a utrophin gene, which is a dystrophin homolog expressed in patient skeletal muscle.
  • the skeletal muscle progenitor cells produced by the method of the present invention can be formulated in the form of cells, or can be formulated as it is without selection.
  • desirable skeletal muscle progenitor cells for transplantation are cells produced through the production steps (1) to (5) of the present invention.
  • the survival rate of the transplanted cells at the transplant destination can be increased.
  • the culture period in the step (5) is preferably 24 hours, but is not limited thereto, and may be any period as long as the cell engraftment rate at the transplant destination is increased.
  • the skeletal muscle progenitor cells of the present invention are mixed with a pharmaceutically acceptable carrier according to conventional means, for example, injections, suspensions, drops, etc. It is manufactured as a parenteral formulation.
  • a pharmaceutically acceptable carrier for example, injections, suspensions, drops, etc.
  • examples of pharmaceutically acceptable carriers that can be included in the parenteral preparation include isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants.
  • An aqueous liquid for injection can be mentioned.
  • the agent of the present invention includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, lidocaine hydrochloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum albumin, polyethylene glycol) Etc.), preservatives (eg, sodium benzoate, benzalkonium chloride, etc.), antioxidants (eg, ascorbic acid, sodium edetate, etc.) and the like.
  • a buffer eg, phosphate buffer, sodium acetate buffer
  • a soothing agent eg, lidocaine hydrochloride, procaine hydrochloride, etc.
  • a stabilizer eg, human serum albumin, polyethylene glycol
  • preservatives eg, sodium benzoate, benzalkonium chloride, etc.
  • antioxidants eg, ascorbic acid, sodium edetate, etc.
  • skeletal muscle progenitor cells may be suspended in the aqueous solution so as to be about 1.0 ⁇ 10 5 to about 1.0 ⁇ 10 7 cells / mL. Since the thus obtained preparation is stable and has low toxicity, it can be safely administered to mammals such as humans.
  • the administration method is not particularly limited, it is preferably injection or drip administration, and examples include intravenous administration, intraarterial administration, intramuscular administration (affected local administration) and the like.
  • the dose of the agent of the present invention varies depending on the administration subject, treatment target site, symptom, administration method, etc., but usually in DMD patients (weight 60 kg), for example, in the case of intravenous injection, Conveniently, about 1.0 ⁇ 10 5 to about 1 ⁇ 10 7 cells as skeletal muscle progenitor cells are administered about 4 to about 8 times at intervals of about 1 to about 2 weeks.
  • the present invention provides a method for screening candidate agents that are useful for treating or preventing myogenic diseases.
  • the screening method for a therapeutic or prophylactic agent for myogenic diseases can include the following steps: (1) contacting a candidate substance with a skeletal muscle progenitor cell differentiated from an iPS cell derived from a myogenic disease patient; (2) A step of selecting as a therapeutic or prophylactic agent for myogenic diseases when the pathological state of the skeletal muscle progenitor cells is alleviated as compared with the case where they are not contacted with a candidate substance.
  • the pathological state of skeletal muscle progenitor cells can be observed as a dystrophin protein deficiency or mutation in the skeletal muscle progenitor cells, or a positive inflammatory marker.
  • a dystrophin protein deficiency or mutation in the skeletal muscle progenitor cells or a positive inflammatory marker.
  • an inflammation marker the activity of prostaglandin D2 or NFkB is exemplified.
  • the alleviation of the pathological condition can be confirmed by, for example, the expression of short dystrophin protein by dystrophin protein or exon skipping or the decrease of inflammatory marker.
  • candidate substances are, for example, cell extracts, cell culture supernatants, microbial fermentation products, marine organism-derived extracts, plant extracts, purified proteins or crude proteins, peptides, non-peptide compounds, synthetic low molecular compounds And natural compounds.
  • candidate substances are also (1) biological library, (2) synthetic library method using deconvolution, (3) “one-bead one-compound” library And (4) any of a number of approaches in combinatorial library methods known in the art, including synthetic library methods using affinity chromatography sorting.
  • Biological library methods using affinity chromatography sorting are limited to peptide libraries, but the other four approaches can be applied to peptide, non-peptide oligomer, or small molecule compound libraries of compounds (Lam (1997 ) Anticancer Drug Des. 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci.
  • Pluripotent stem cell human iPS cells were cultivated by a conventional method (Takahashi K, et al. Cell. 131: 861-72, 2007) provided by Professor Yamanaka of Kyoto University.
  • the iPS cell line (Pax3-GFP iPSCs) expressing EGFP was produced by linking EGFP sequence to the 3 'side of the start codon of Pax3 locus by homologous recombination according to conventional methods using 201B7.
  • human iPS cells (Pax3-GFP iPSCs or Myf5-tdTomato C3 iPSCs) were subjected to low-density culture on a culture vessel coated with MatriGel. Briefly, one 6 cm dish of human iPS cells was treated in the same manner as normal passage. After dissociating from the MSNL feeder with CTK solution and washing twice with PBS, 1 mL 0.25% Trypsin / 1 mM EDTA was added and incubated at 37 ° C. for 5 minutes.
  • MatriGel is diluted 50-fold (when left for 2 hours) or 100-fold (when left overnight) in human iPS cell maintenance medium (without bFGF), added to the culture vessel, allowed to stand, and MatriGel coated Went.
  • ⁇ Process (1) (Day 0 to 20)> CDMi basal medium supplemented with 10 ⁇ M SB431542 and 10 ⁇ M CHIR99021 (1% Albumin from bovine serum (SIGMA), 1% Penicillin-Streptomycin Mixed Solution (Nacalai Tesque), 1% CD Lipid Concentrate (Invitrogen), 1% Insulin- IMDM (1X) Iscove's Medified Dullbecco's Medium (+) L-Glutamine (+) 25 mM HEPES (Invitorogen) and F-12 (1X) Nutrient Mixture (Ham) with Transferrin-Selenium (Invitrogen) and 450 ⁇ M 1-Thioglycerol (SIGMA) ) (+) L-Glutamine (Invitrogen) mixed with 1: 1) was exchanged at 2 ml / well. Thereafter, the medium was changed on the second day (Day 2) and the fifth day (Day 5).
  • the subculture was performed by the following method. After the medium was aspirated and washed twice with PBS, 500 ⁇ L 0.25% Trypsin / 1 mM EDTA was added and incubated at 37 ° C. for 5 minutes. Subsequently, 2.5 ml of CDMi basal medium was added and pipetted to dissociate the cells into single cells. After centrifugation at 900 rpm and 4 ° C. for 5 minutes, the supernatant was removed and resuspended in CDMi basal medium. Thereafter, the number of cells was counted.
  • CDMi basal medium supplemented with 10 ⁇ M SB431542, 5 ⁇ M CHIR99021, and 10 ⁇ M Y-27632 to a culture container coated with MatriGel, and add the above cells at 1 x 10 6 per 6 cm dish or 4 x 10 5 per well of a 6- well plate. Sowing. On the 8th day (Day8), 10th day (Day10) and Day12, the medium was changed with a CDMi basal medium supplemented with 10 ⁇ M SB431542 and 5 ⁇ M CHIR99021.
  • the medium was exchanged with a CDMi basal medium supplemented with 10 ⁇ M SB431542 and 5 ⁇ M CHIR99021.
  • the cell culture medium obtained in step (1) was 0.2% BSA, 200 ⁇ M 2-ME (2-Mercaptoethanol), 5 mM LiCl (Nacarai), 10 ⁇ M SB431542, 10 ng / ml IGF-1 (Peprotech), 10 ng / ml HGF. (Peprotech) and SF-O3 (Sanko Junyaku) supplemented with 10 ng / ml bFGF (oriental yeast) were added, and the culture was continued.
  • Step (2B) (ii) Day 25-27)> The cell culture medium obtained in step (2B) (i) was replaced with SF-O3 supplemented with 0.2% BSA, 200 ⁇ M 2-ME, 5 mM LiCl, 10 ⁇ M SB431542 and 10 ng / ml IGF-1, and cultured for 3 days. did.
  • Step (2B) (iii) (Day 28-41)> The medium of the cells obtained in step (2B) (ii) was replaced with SF-O3 supplemented with 0.2% BSA, 200 ⁇ M 2-ME, 10 ⁇ M SB431542, 10 ng / ml IGF-1 and 10 ng / ml HGF, Cultured for a week. At this time, the medium was changed twice a week in the same fresh medium.
  • HS Horse Serum
  • FACS analysis and comparison of marker gene expression levels were performed. Briefly, for FACS analysis, Day7 cells in the above step (1) were collected, and CD271 negative Pax-GFP positive cells were focused on using CD271 (NGFR) and Pax3-GFP as indices.
  • the marker gene expression level was measured by measuring the expression levels of each gene of Pax3, T, Tbx6, Mesp2, PDGFRa, KDR, CD56, Sox10, NGFR, Pax7 and Myf5 by RT-PCR.
  • FACS analysis was performed to examine the effect of subculture on Day 7 of differentiation induction . Briefly, as shown in the above step (1), the cells were subcultured on Day 7 and then cultured until Day 14 was collected, and the cells were selected by FACS using Pax3-GFP as an index. It was.
  • step (1) cells cultured until Day 14 in a medium in which CHIR99021 and SB431542 are mixed with several addition amounts are collected, and the cells are collected by FACS using Pax3-GFP as an index. This was done by sorting.
  • CH and SB on the graph indicate CHIR99021 and SB431542, respectively, and the numbers on the right side indicate their concentrations ( ⁇ M).
  • RT-PCR analysis was performed. Briefly, the expression status of Tbx6, Wnt3a, Mesp2, PDGFRa, Pax3, and Meox1 genes was examined by RT-PCR.
  • Pax3-GFP negative cells on differentiation induction day 84 contained cells that strongly stained MHC and Myogenin (FIG. 7). From this, it was suggested that cells differentiated in myotubes in vitro in Pax3-GFP negative were suggested on the 84th day of induction of differentiation.
  • Transplant experiment (1) The effect of differentiation-induced Day51 on transplantation of skeletal muscle progenitor cells to mice was examined. Briefly, the day51 cell group in the above step (3) was transplanted intramuscularly into the anterior tibial muscle of NSG mice one day after CTx treatment. Four weeks after transplantation, the periphery of the transplant site was excised, sections were prepared according to a conventional method, antibody staining of human nuclei, Spectrin, eMYH and laminin was performed, and engraftment and muscle regeneration were evaluated.
  • Transplant experiment (2) The effect of differentiation-inducing day 84 in transplanting skeletal muscle progenitor cells to mice was examined. Briefly, Pax3-GFP positive cells were selected by FACS for the day84 cell group in the above step (3). Subsequently, 2.6 ⁇ 10 5 Pax3-GFP positive cells were transplanted intramuscularly into the anterior tibial muscle of NSG mice one day after CTx treatment. Four weeks after transplantation, antibody staining of human nuclei, eosin and laminin was performed to evaluate the engraftment and muscle regeneration of the transplanted cells.
  • An iPS cell line (Myf5-tdTomato C3) that expresses tdTomato by linking pluripotent stem cells 201B7 to the 5 'side of the start codon of the Myf5 locus by homologous recombination according to conventional methods and linking to the expression of Myf5 iPSCs) were prepared.
  • iPS cells obtained above were prepared as in Example 1 prior to differentiation induction, and differentiation induction of skeletal muscle progenitor cells was performed as follows.
  • the gene expression state Myf5-tdTomato C3 iPSCs was used to induce differentiation into muscle progenitor cells by the method described in Example 1.
  • the cell group (Myf5-tdTomato positive cells and negative cells) at this time was analyzed by RT-PCR in order to examine the expression state of the marker gene over time from Day 34 to Day 85. Briefly, Myf5-tdTomato positive or negative cells were selected by FACS for Day84 cell group in the above step (3 ′), and Myf5, MyoD, Pax3, and Pax7 genes were expressed by RT-PCR, respectively. I checked the condition.
  • Myf5-tdTomato positive cells and negative cells The cell group (Myf5-tdTomato positive cells and negative cells) on the 44th day (Day 44) was subjected to antibody staining in order to examine myotube differentiation ability. Briefly, MHC and Myogenin antibody staining of each cell group cultured under the conditions of step (3 ') for 1 day, 7 days or 14 days after sorting by FACS using Myf5-tdTomato as an index on Day44 Went.
  • Myf5-tdTomato positive cells on the 44th day of differentiation induction contained many cells that changed to cells strongly stained with MHC and Myogenin (FIG. 12). This suggests that Myf5-tdTomato positive cells contain cells that can differentiate into myocytes.
  • Transplant experiment (1 ') In order to examine the effect of transplantation on skeletal muscle progenitor cells (day 71 Myf5-positive cells) induced by the differentiation induction method of this example, transplantation experiments were performed. Briefly, Myf5-tdTomato positive cells were selected by FACS for the day71 cell group in the above step (3 ′). 7.5 ⁇ 10 5 Myf5-tdTomato positive cells immediately after selection or 6.1 ⁇ 10 5 Myf5-tdTomato negative cells immediately after selection were transplanted intramuscularly into the anterior tibial muscle of NSG mice one day after CTx treatment . Four weeks after transplantation, human spectrin antibody staining was performed to evaluate muscle regeneration.
  • Transplant experiment (2 ') In order to examine the effect of transplantation on skeletal muscle progenitor cells (Myf5 positive cells on day 78) induced by the differentiation induction method of this example, transplantation experiments were performed. Briefly, Myf5-positive cells were selected by FACS for the day78 cell group in the above step (3 ′). Subsequently, 5 ⁇ 10 5 Myf5-positive cells were re-cultured for 24 hours, and then transplanted intramuscularly into the anterior tibial muscle of NSG mice one day after CTx treatment. Four weeks after transplantation, human spectrin antibody staining was performed to evaluate muscle regeneration.
  • An iPS cell line (Myf5-tdTomato C3) that expresses tdTomato by linking pluripotent stem cells 201B7 to the 5 'side of the start codon of the Myf5 locus by homologous recombination according to conventional methods and linking to the expression of Myf5 iPSCs and Myf5-tdTomato E16 iPSCs).
  • iPS cells obtained above were prepared as in Example 1 prior to differentiation induction, and differentiation induction of skeletal muscle progenitor cells was performed as follows.
  • ⁇ Process (1 ”) (Day 0 to 17)> Culture was performed for 18 days under the same conditions as in step (1) of Example 1. In addition, the passage was performed on the 7th day (Day7) and the 14th day (Day14) by the same method as described above.
  • ⁇ Process (2A) (Day 18-39)>
  • the cell culture medium obtained in step (1 ′′) was added to SF-O3 (Sanko Junyaku) supplemented with 0.2% BSA and 200 ⁇ M 2-ME (2-Mercaptoethanol), 10 ⁇ M SB431542 and / or 10 ng / ml IGF-
  • the culture medium was replaced with a medium supplemented with 1 (Peprotech) and / or 10 ng / ml HGF (Peprotech) and / or 10 ng / ml bFGF (oriental yeast), or none added, and cultured for 21 days.
  • Myf5-tdTomato C3 iPSCs and Myf5-tdTomato E16 iPSCs were used to induce differentiation into muscle progenitor cells by the methods of steps (1 ", (2A) and (3") above. . With respect to the cell groups (Myf5-tdTomato positive cells and negative cells) at this time, the ratio of the Myf5-tdTomato positive cell group was evaluated.
  • Transplant experiment (1 ) In order to examine the effect of transplantation on skeletal muscle progenitor cells (Myf5-positive cells on day 35) induced by the differentiation induction method of this example, transplantation experiments were performed. Briefly, Myf5-tdTomato positive cells were selected by FACS for the day35 cell group in the above step (2A). Immediately transplanted 3.7 ⁇ 10 5 Myf5-tdTomato positive cells immediately after selection or 3.7 ⁇ 10 6 Myf5-tdTomato negative cells immediately after selection into the anterior tibial muscle of NSG mice 1 day after CTx treatment . Four weeks after transplantation, human Spectrin antibody staining was performed to evaluate muscle regeneration.
  • the method of the present invention can provide an efficient method for inducing differentiation from pluripotent stem cells to skeletal muscle progenitors.
  • the skeletal muscle precursor obtained by the method of the present invention exhibits a high engraftment rate in a living body and is therefore extremely useful in transplantation treatment.

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Abstract

La présente invention concerne un procédé de production de cellules progénitrices de muscle squelettique à partir de cellules souches pluripotentes, ledit procédé comprenant les étapes suivantes (1) et (2A) ou (2B) : (1) une étape de culture des cellules souches pluripotentes dans un milieu de culture liquide contenant un inhibiteur du TGF-β et un inhibiteur de GSK3β ; (2A) une étape de culture des cellules obtenues dans l'étape (1) dans un milieu de culture liquide contenant HGF éventuellement conjointement à IGF1 ; ou (2B) une étape de culture des cellules obtenues dans l'étape (1) successivement (i) dans un milieu de culture liquide contenant un inhibiteur du TGF-β, un inhibiteur de GSK3β, IGF1, HGF et bFGF, (ii) dans un milieu de culture liquide contenant un inhibiteur du TGF-β, un inhibiteur de GSK3β et IGF1, et (iii) dans un milieu de culture liquide contenant un inhibiteur du TGF-β, IGF1 et HGF. En outre, ce procédé peut comprendre (3) une étape de culture des cellules obtenues dans l'étape (2A) ou (2B) dans un milieu de culture liquide contenant un inhibiteur du TGF-β, IGF1 et du sérum.
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Cited By (9)

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WO2016168890A1 (fr) * 2015-04-22 2016-10-27 Genea Ip Holdings Pty Ltd Génération de cellules de lignées musculaires à partir de cellules souches
WO2018088501A1 (fr) 2016-11-11 2018-05-17 国立大学法人大阪大学 Procédé pour induire la différenciation de cellules souches pluripotentes en cellules somatiques
EP3246398A4 (fr) * 2015-01-14 2018-06-06 Kyoto University Procédé de sélection de cellule progénitrice des muscles squelettiques
US10258628B2 (en) 2016-10-26 2019-04-16 Genea Biocells USA (Holdings), Inc. Generation of muscle lineage cells and therapeutic uses thereof
WO2019177118A1 (fr) * 2018-03-15 2019-09-19 国立大学法人京都大学 Méthode pas-à-pas de production de diverses cellules à partir de cellules souches pluripotentes
JP2020068775A (ja) * 2018-10-30 2020-05-07 国立大学法人京都大学 細胞の製造方法
EP3565887A4 (fr) * 2017-01-06 2020-12-02 The Regents of The University of California Méthodes permettant la production de cellules progénitrices de muscle squelettique
WO2021015086A1 (fr) * 2019-07-19 2021-01-28 国立大学法人京都大学 Procédé de fabrication de cellules de myotube mâtures à partir de cellules souches du muscle squelettique
JPWO2022014604A1 (fr) * 2020-07-13 2022-01-20

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JP7548494B2 (ja) 2018-10-30 2024-09-10 国立大学法人京都大学 細胞の製造方法
WO2021015086A1 (fr) * 2019-07-19 2021-01-28 国立大学法人京都大学 Procédé de fabrication de cellules de myotube mâtures à partir de cellules souches du muscle squelettique
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JP7429294B2 (ja) 2020-07-13 2024-02-07 国立大学法人京都大学 骨格筋前駆細胞及びその精製方法、筋原性疾患を治療するための組成物、並びに骨格筋前駆細胞を含む細胞群の製造方法

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