KR101496610B1 - A Composition for Inducing Myogenic-lineage Differentiation of Stem Cells - Google Patents

Abstract

The present invention relates to a composition for inducing muscle cell differentiation from stem cells and a pharmaceutical composition for preventing or treating diseases related to muscle loss, which efficiently induces muscle cells from stem cells by targeting histone deacetylase 6 (HDAC6) as a new differentiation control; and can be widely used for treating and preventing diseases related to various muscle loss like muscular dystrophy, myositis and fibrosis by regenerating a loss of muscle tissues.

Description

[0001] The present invention relates to a composition for inducing differentiation of stem cells into muscle cells,

The present invention relates to a composition for inducing differentiation of stem cells into muscle cells and a composition for preventing or treating muscle damage diseases using the same.

Histone deacetylase (HDAC) deacetylates histones to condense chromatin and inhibit gene transcription ( 1-2 ). HDAC can also regulate acetylation of non-histone proteins ( 1 ). There are 18 HDACs classified into four groups in mammals: Class I (HDAC1, 2, 3 and 8), Class Ⅱ (Class Ⅱa : HDAC4, 5, 7 and 9; Class Ⅱb: HDAC6 and 10), Class Ⅲ (Sirt1-7) , Class Ⅳ (HDAC11) (1) .

Appropriate regulation of HDAC is so important for the normal development and disease stage 3 cancer, which acts and their inhibition of HDAC and self-study to treat a variety of diseases, including inflammatory diseases, and degenerative diseases (3-4) . Interestingly, HDAC inhibition can promote the specific differentiation of stem cells into nerves, bone, and hepatocytes ( 5-7 ).

The present inventors investigated the mRNA levels of HDAC 1-HDAC11, which changes as the embryonic stem cell (ESC) differentiates, and sought to elucidate the mechanism by which the microenvironment of the tissue regulates ESC differentiation.

The present inventors have found that HDAC6 decreases during differentiation. The HDAC6 rust-down ESC markedly increased Pax7, a marker of muscle precursor cells, and promoted differentiation into muscle lineages. Although many interesting features of HDAC6 have been reported ( 8 ), their function in stem cell differentiation is almost unknown. The present inventors have found that muscle regeneration is increased when HDAC6 rust-down ESCs are transplanted into muscle injured mice. This finding shows that HDAC6 can be a regulator of stem cell differentiation into the muscle lineage and can be a target for treating muscle damage.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors sought to develop a method for inducing differentiation of stem cells into muscle-derived cells targeted by identifying new factors involved in the regulation of differentiation of stem cells. As a result, it was found that inhibition of the expression or activity of HDAC6 in stem cells efficiently differentiated into muscle groups, and that regeneration of injured muscle was increased in the transplantation of stem cells in which the expression or activity of HDAC6 was inhibited , Thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for inducing differentiation of stem cells into muscle cells.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating muscle damage diseases.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for inducing differentiation of stem cells into muscle cells comprising an inhibitor of HDAC6 (histone deacetylase 6) as an active ingredient.

The present inventors sought to develop a method for inducing differentiation of stem cells into muscle-derived cells targeted by identifying new factors involved in the regulation of differentiation of stem cells. As a result, it was found that when the expression or activity of HDAC6 in the stem cells is inhibited, it is efficiently differentiated into the muscular system, and the regeneration of injured muscle is increased in the transplantation of stem cells in which the expression or activity of HDAC6 is inhibited .

As used herein, the term " inhibitor of HDAC6 " encompasses both a series of natural or synthetic substances that inhibit or eliminate the expression or activity of HDAC6 at the gene or protein level.

According to a specific embodiment of the present invention, the inhibitor of HDAC6 of the present invention comprises a blocker of a protein consisting of the amino acid sequence of SEQ ID No. 2, an antibody specifically binding to the protein, and an aptamer specifically binding to the protein Lt; / RTI >

As used herein, the term " protein " refers to a molecule formed by peptide bonds and amino acid residues joined together. According to the present invention, the amino acid sequence of the second sequence of the sequence listing is the amino acid sequence of the HDAC6 (Histone deacetylase 6) protein.

As used herein, the term " blocker " refers to any substance that reduces or eliminates the activity of the target protein HDAC6, including both synthetic compounds and natural products.

The term " antibody " as used herein refers to a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to HDAC6 and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. According to the present invention, the decrease in HDAC6 leads to a decrease in the activity of differentiation markers and an increase in the number of mesenchymal and muscle marker genes, thereby promoting the differentiation into muscle cells. Thus, the antibody specifically binding to HDAC6 inhibits the intrinsic activity of HDAC6 Thereby promoting the differentiation of the stem cells into the muscular system.

Polyclonal antibodies can be produced by methods well known in the art for injecting HDAC6 antigen into animals and obtaining blood from animals to obtain sera containing antibodies. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

Monoclonal antibodies may be obtained from the hybridoma method (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519), or the phage antibody library (Clackson et al, Nature, 352: 624 Biol., 222: 58, 1-597, 1991) techniques. The antibody prepared by the above method can be isolated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like. The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

As used herein, the term " aptamer " refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity. The general contents of the Aptamer are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 142727 (1998). For example, the process of selecting an aptamer binding to a specific molecule can be performed by (i) preparing a nucleic acid library having various forms using DNA synthesis and an in vitro transcription method (in the case of RNA), and (ii) To selectively bind only the target nucleic acid to the target molecule, and (iv) finally isolating the target nucleic acid construct from the target molecule By elucidating the nucleic acid construct and then amplifying the nucleic acid, the aptamer exhibiting excellent binding ability and specificity can be found by repeating the above steps 5 to 15 times more.

According to a specific embodiment of the present invention, the inhibitor of HDAC6 of the present invention is a nucleic acid molecule which inhibits the expression of a nucleotide encoding the amino acid sequence of the second sequence of the Sequence Listing. More specifically, the nucleotide sequence encoding the amino acid sequence of the second sequence of Sequence Listing is comprised of the nucleotide sequence of Sequence Listing first sequence.

As used herein, the term " nucleotide " is a deoxyribonucleotide or ribonucleotide present in single or double stranded form and includes analogs of natural nucleotides unless otherwise specifically indicated (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)). According to the present invention, the nucleotide sequence of the first sequence of the sequence listing is the nucleotide sequence of the HDAC6 gene.

It is clear to a person skilled in the art that the nucleotide sequence in which expression is suppressed in the present invention is not limited to the nucleotide sequence described in the attached sequence listing.

Variations in nucleotides do not cause changes in the protein. Such nucleic acids include functionally equivalent codons or codons that encode the same amino acid (e.g., by codon degeneration, six codons for arginine or serine), or codons that encode biologically equivalent amino acids ≪ / RTI >

Considering the mutation having the above-mentioned biological equivalent activity, the nucleic acid molecule used in the present invention is interpreted to include a sequence showing substantial identity with the sequence described in the sequence listing. The above-mentioned substantial identity is determined by aligning the sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. Homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology.

Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv. Appl. Math. 2: 482 (1981) ; Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from National Center for Biological Information (NBCI) It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

As used herein, the term " expression inhibition " refers to a modification of a nucleotide sequence that results in the degradation of a target gene, and preferably thereby, the target gene expression is significantly reduced, or is undetectable or is present at a meaningless level .

According to a more specific embodiment of the present invention, the nucleic acid molecule for expression suppression used in the present invention is selected from the group consisting of shRNA, siRNA, miRNA, ribozyme, PNA (peptide nucleic acids) and antisense oligonucleotides . More specifically, the nucleic acid molecule is an shRNA.

As used herein, the term " shRNA (small hairpin RNA) " means a nucleotide consisting of 50 to 70 single strands, and forms a stem-loop structure in vivo . In other words, shRNA is an RNA sequence that creates a tight hairpin structure to inhibit gene expression through RNA interference. A long RNA of 19-29 nucleotides complementary to both the loop region of 5-10 nucleotides forms a double stranded stem with base pairs. The shRNA is transduced into the cell via a vector containing the U6 promoter so that it is always expressed, and is usually transferred to daughter cells to allow the gene expression inhibition to be inherited. The shRNA hairpin structure is cleaved by an intracellular mechanism to become siRNA and bind to RNA-induced silencing complex (RISC). These RISC bind to mRNA and cleave it. The shRNA is transcribed by RNA polymerase III.

As used herein, the term " siRNA " means a short double-stranded RNA capable of inducing RNAi (RNA interference) phenomenon through cleavage of a specific mRNA. A sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knockdown method or as a method of gene therapy.

The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases. The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of the target gene by the RNAi effect. The sticky end structure can have both a 3-terminal protruding structure and a 5-terminal protruding structure. The number of protruding bases is not limited. For example, the base water may be from 1 to 8 bases, preferably from 2 to 6 bases. In addition, the siRNA can be added to a protruding portion at one end in a range that can maintain the effect of suppressing the expression of a target gene, for example, a low molecular RNA (for example, a natural RNA molecule such as a tRNA, Molecules). The siRNA terminal structure does not need to have a cleavage structure on both sides, and a stem loop structure in which one terminal region of double-stranded RNA is connected by linker RNA may be used. The length of the linker is not particularly limited as long as it does not interfere with the pairing of the stem portions.

As used herein, the term " miRNA (microRNA) " regulates gene expression and refers to a nucleic acid molecule having a total length of 20-50 nucleotides, preferably 20-45 nucleotides, more preferably 20-40 nucleotides, Refers to a single stranded RNA molecule consisting of 30 nucleotides, most preferably 21-23 nucleotides. miRNAs are oligonucleotides that are not expressed in cells and have a short stem-loop structure. A miRNA has total or partial homology with one or more mRNA (messenger RNA) and inhibits target gene expression through a complementary binding to the mRNA.

As used herein, the term " ribozyme " refers to an RNA molecule that has the same function as an enzyme that recognizes a nucleotide sequence of a specific RNA and cleaves the nucleotide sequence of the specific RNA. The ribozyme is a region that specifically binds with a complementary base sequence of the target messenger RNA strand and cleaves the target RNA.

As used herein, the term " PNA (peptide nucleic acid) " means a molecule having both nucleic acid and protein properties, and capable of complementarily binding with DNA or RNA. PNA was first reported in 1999 as a pseudo-DNA in which a nucleobase is linked by a peptide bond (Nielsen PE, Egholm M, Berg RH, Buchardt O, "Sequence-selective recognition of DNA by strand displacement with a thymine- substituted polyamide ", Science 1991, Vol. 254: pp1497-1500). PNAs are synthesized by artificial chemical methods, not found in nature. PNA forms a double strand by causing a hybridization reaction with a natural nucleic acid of a complementary base sequence. When the length is the same, the PNA / DNA double strand is more stable than the DNA / DNA double strand, and the PNA / RNA double strand is more stable than the DNA / RNA double strand. The peptide backbone, which is repeatedly linked by N- (2-aminoethyl) glycine to amide bond, is most commonly used as the peptide backbone. In this case, unlike the basic structure of a negatively charged natural nucleic acid, Is neutral. The four nucleic acid bases present in the PNA have a spatial size and a distance between the nucleotide bases is almost the same as that of the native nucleic acid. PNA is chemically more stable than natural nucleic acid, and biologically stable because it is not degraded by nuclease or protease.

The term " antisense oligonucleotide " as used herein refers to DNA or RNA or a derivative thereof containing a nucleotide sequence complementary to the sequence of a specific mRNA, and binds to a complementary sequence in the mRNA to inhibit the translation of the mRNA into a protein The antisense nucleotide sequence of the present invention refers to a DNA or RNA sequence complementary to the mRNA of the target gene and capable of binding to the mRNA of the target gene, and is capable of translating the mRNA of the target gene, translocating it into the cytoplasm, It may inhibit essential activity for maturation or any other overall biological function.

The antisense oligonucleotides may be modified at one or more base, sugar or backbone sites to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol. , 5 (3): 343-55, 1995 ). The oligonucleotide backbone can be modified with phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic sugar, and the like. In addition, the antisense nucleic acid may comprise one or more substituted sugar moieties. Antisense oligonucleotides may comprise modified bases. Modified bases include hypoxanthane, 6-methyladenine, 5-methylpyrimidine (especially 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, -Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine .

According to a specific embodiment of the present invention, the nucleic acid molecule of the present invention is contained in a gene delivery system.

As used herein, the term " gene transporter " means an agent for introducing and expressing a desired target gene into a target cell. The ideal gene transporter is harmless to the human body, is easy to mass-produce, and must be able to efficiently transfer the gene.

As used herein, the term " gene transfer " means that a gene is carried into a cell and has the same meaning as transduction of a gene into a cell. At the tissue level, the term gene transfer has the same meaning as the spread of a gene. Accordingly, the gene carrier of the present invention can be described as a gene penetration system and a gene diffusion system.

To prepare the gene delivery vehicle of the present invention, the nucleotide sequence of the present invention is preferably present in a suitable expression construct. In such expression constructs, the nucleotide sequence of the invention is preferably operatively linked to a promoter. As used herein, the term " operably linked " means a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, The regulatory sequence will regulate transcription and / or translation of the other nucleic acid sequences. In the present invention, the promoter bound to the nucleotide sequence of the present invention is preferably capable of regulating transcription of the rilacsin gene by operating in an animal cell, more preferably a mammalian cell, and includes a promoter derived from a mammalian virus And a promoter derived from the genome of a mammalian cell, such as CMV (mammalian cytomegalovirus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha Promoter of a human IL-4 gene, a promoter of a human lymphotoxin gene, and a promoter of a human GM-CSF gene, or a promoter of a human GM-CSF gene, or a promoter of a promoter, a metallothionine promoter, a beta-actin promoter, U6 promoter, but are not limited thereto.

The nucleotide sequence of the present invention can be applied to all gene delivery systems used for conventional gene therapy, preferably plasmids, adenoviruses (Lockett LJ, et al., Clin. Cancer Res. 3: 2075-2080 (1997) ), Adeno-associated viruses (AAV, Lashford LS., Et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), retroviruses (Gunzburg WH, et al., Retroviral vectors .. Gene Therapy Technologies, Applications and Regulations Ed A. Meager, 1999), lentivirus (Wang G. et al, J. Clin Invest 104 (11...): R55-62 (1999)), herpes simplex virus (Chamber R., et al, Proc Natl Act Sci USA 92:.... 1411-1415 (1995)), Bash Catania virus (Puhlmann M. et al, Human Gene Therapy 10:. 649-657 (1999)) , Liposomes (Methosin in Molecular Biology, Vol. 199, SC Basu and M. Basu (Eds.), Human Press 2002) or niosomes. Most specifically, the gene carrier of the present invention is prepared by applying the nucleotide molecule of the present invention to a plasmid.

In the present invention, when the gene carrier is prepared based on a viral vector, the contacting step is carried out according to a virus infection method known in the art. Infection of host cells with viral vectors is described in the above cited documents.

If the gene delivery system in the present invention is a kit inner (naked) or recombinant DNA molecule is a plasmid, microinjection (Capecchi, MR, Cell, 22 :. 479 (1980); and Harland and Weintraub, J. Cell Biol 101: 1094- 1099 (1985)), calcium phosphate precipitation method (Graham, FL et al., Virology , 52: 456 (1973), and Chen and Okayama, Mol. Cell. Biol. 7: 2745-2752 6: 716-718 (1986)), liposome-mediated transfection (Eugene et al., EMBO J. , 1: 841 (1982), and Tur-Kaspa et al., Mol. Cell Biol. method (Wong, TK et al, Gene , 10:87 (1980); Nicolau etene, Biochim Biophys Acta, 721:.... 185-190 (1982); and Nicolau.et al, methods Enzymol, 149. .: (1987)), DEAE-dextran treatment (Gopal, MoI. Cell Biol. , 5: 1188-1190 (1985)), and gene bend buddhite (Yang et al., Proc. Natl. ., 87: 9568-9572 (1990)) and by the method can be a gene transfection into a cell.

According to a more specific embodiment of the present invention, the stem cells in which the differentiation into muscle cells are induced by the composition of the present invention may be embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) to be.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating muscle damage diseases comprising an inhibitor of HDAC6 (histone deacetylase 6) as an active ingredient.

Since the inhibitor of HDAC6 used in the present invention has already been described above, the description thereof is omitted in order to avoid excessive duplication.

As used herein, the term " muscle damage disorder " refers to any disease in which muscle tissue or muscle cells are damaged resulting in loss of muscle tissue. According to the present invention, since the composition of the present invention promotes the differentiation of stem cells into muscle cells, muscle tissue can be effectively restored in a degenerative muscle disease caused by damage of muscle tissue. Therefore, the term " composition for preventing or treating muscle damage diseases " is used in the same sense as " composition for muscle regeneration ".

Concretely, the muscle damage diseases prevented or treated with the composition of the present invention are muscular dystrophy, muscular atrophy, myositis, polymyositis, peripheral vascular disease, Fibrosis < / RTI >

As used herein, the term " treatment " includes (a) inhibiting the development of a disease, disorder or condition; (b) relief of the disease, disorder or condition; Or (c) eliminating the disease, disease or condition. The composition of the present invention plays a role of suppressing, eliminating or alleviating the development of muscle damage diseases or symptoms thereof by promoting regeneration of injured muscles by promoting differentiation of stem cells into muscle cells. Therefore, the composition of the present invention may be used as a therapeutic composition itself by being used for promoting the differentiation of endogenous autologous stem cells, or may be administered together with a therapeutic implantable stem cell, As a cell therapy adjuvant. Herein, the term " treatment " or " therapeutic agent " includes the meaning of " therapeutic aid "

As used herein, the term " prophylactic " means inhibiting the development of a disease or disease in a subject who has never been diagnosed as having a disease or disease, but is likely to suffer from such disease or disease.

The term " administering " or " administering " as used herein refers to the administration of an effective amount of a composition of the present invention directly to a subject so that the same amount is formed in the body of the subject.

As used herein, the term " subject " includes without limitation humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Specifically, the object of the present invention is human.

The composition of the present invention comprises (a) a pharmaceutically effective amount of the HDAC6 inhibitor of the present invention described above; And (b) a pharmaceutically acceptable carrier.

As used herein, the term " pharmaceutically effective amount " means an amount sufficient to achieve the inhibition of HDAC6 and thereby the efficacy or activity of muscle regeneration and muscle repair.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably parenterally administered, for example, by topical application to skin.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-1000 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets, capsules or gels (e.g., hydrogels), and may additionally contain dispersing or stabilizing agents .

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for inducing differentiation of stem cells into muscle cells and a pharmaceutical composition for preventing or treating muscle injury diseases.

(b) The present invention highly efficiently induces specific differentiation of stem cells into muscle cells by using HDAC6 (Histone deacetylase 6), which has not yet been known, as a new differentiation regulating target.

(c) The present invention can be usefully used for preventing or treating various muscular damage diseases such as muscular stiffness, muscle inflammation and fibrosis by regenerating the lost muscle tissue.

Figure 1 shows that HDAC6 decreases as EB is formed in the ES state and that the decrease in HDAC6 promotes the expression of the mesenchymal and muscle marker genes. In E14 embryonic stem cells, HDAC6 decreased in embryoid body (EB) compared to ES (Fig. 1a). The pre-differentiation marker gene (Oct4) was reduced after embryoid formation (N = 3, respectively). In C57 embryonic stem cells, the protein and mRNA of HDAC6 decreased in embryoid body (EB) compared to embryonic stem cells (ES, n = 3) (FIG. Transformation of shHDAC6 significantly reduced the protein and mRNA of HDAC6 as compared to the non-target shRNA control (shMock) (n = 3) (Fig. 1d). Specifically, the transformation of shHDAC6 specifically inhibited HDAC6 in the HDAC family gene (Fig. 1C). FIG. 1E is a graph showing the results of real-time PCR after incubating the cells on a gelatin-coated plate containing DMEM / 10% FBS for 5 days (n = 3). As shHDAC6 cells differentiated, the muscle marker genes (Pax3, Pax7 and MyoD) were significantly increased in the mesenchymal line, while the vascular marker genes (PECAM, SMA and VE-cadherin) (Fig. 1F).
FIG. 2 is a graph showing that the differentiation from HDAC6 rust-down cells into muscle is increased. The mRNA (Figure 2a) and protein (Figure 2b) of Pax7, the precursor cell markers of muscle, increased significantly in all HDAC6 rust-down C57 cells. Cells were plated on gelatin-coated plates containing DMEM / 10% FBS for one day, then cultured for 20 days and replaced with skeletal muscle induced media (Fig. 2C). Pax7 and muscle regulators (Myf5, MyoD and Myogenin) increased during induction of muscle differentiation in shMock-control cells, but increased in shHDAC6- cells (N = 3, Fig. 2d). FIG. 2E is a graph showing the results of immunofluorescence staining for Pax7 (red) / nucleus (blue) after culturing for 10 days or 20 days in SkIM. The parental cell line C2C12 was used as a positive control for Pax7 staining (x200).
Figure 3 shows that the chromatin structure of the Pax7 promoter in shHDAC6 cells has a more open structure and improves regeneration of damaged muscle. Pax7 mRNA and protein were significantly reduced in HDAC6 rust-down C57 cells (Fig. 3a). FIG. 3B is a schematic diagram illustrating the regulation of histone acetylation and the binding of the Pax7 promoter to RNA polymerase II (Pol II). The acetylation of H3 and H4 is known to be an indicator of activated chromatin structure ( 20 ). (N = 4, * p < 0.01) by real-time PCR and quantification of the binding of Pax7 promoter and RNA Pol II (Fig. 3d) with increasing acetylation of histone H3 and H4
Figure 3e is a schematic diagram of a rotarod experiment using a cardiotoxin (CTX) -infected mouse. One day before cell transplantation, the tibialis anterior muscle of both legs was damaged by CTX. (N = 13), mice transplanted with shX (mouse) (n = 13), mice injected with CTX alone (n = 14) (n = 12). (P < 0.001, ** p < 0.01 shMock-cells vs. shHDAC6-cells), and shHDAC6 stem cells were injected with weekly (Figure 3f) delayed detachment from rotating rods after cell transplantation It was found that the time to maintain without deterioration on the rotor rod was similar to that of the sham group, which does not cause muscle damage. At 2 weeks after cell transplantation, the rotarod delay time of all mice (n = 52) was measured (Fig. 3g), and shHDAC6 stem cell injected group showed significantly increased muscle regeneration effect than shMock stem cell injected group .
FIG. 3h shows immunostaining of the cross section of TA muscle 30 days after shHDAC6 cell transplantation against laminin (green) / nucleus (blue) (magnification = x400). DiI (+) / laminin (+) muscle fiber indicates that the injected stem cells have successfully differentiated into muscle cells before the stem cells were injected into the muscle injured mice. In particular, the center-nucleus with the nucleus at the center is a muscle fiber marker that causes muscle regeneration. As shown in FIG. 3h, most of the DiI (+) muscle fibers are center-nucleus, which is significantly higher in the shHDAC6 cell-injected group than in the shMock cell-injected group (FIG.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Cell culture and in vitro differentiation

(MESC) and C57BL / 6-based mESC (C57-mESC, accession number SCRC-1002; ATCC) were inoculated into a culture medium containing 20% FBS (Hyclone), 1% penicillin / streptomycin (Dulbecco &apos; s modified Eagle &apos; s medium (GIBCO, Sigma) supplemented with [beta] -mercaptoethanol (Sigma), 1% non-essential amino acid (GIBCO), 2 mM L- glutamine and 1000 U / ml leukemia inhibitory factor (LIF; Millipore) (Sigma-Aldrich) -treated mouse embryonic fibroblast (MEF) nutrient layer in a culture supernatant (Sigma-Aldrich, Grand Island, NY, USA)

Embryoid bodies (EBs) were formed using a hanging drop method (containing 500 cells / 20 μl per droplet) in the absence of LIF. To induce spontaneous differentiation, ES cells were cultured in 0.3% gelatin-coated plates in DMEM / 10% FBS. For in-vitro skeletal muscle differentiation, ES cells or EBs were plated in 0.3% gelatin-coated plates in DMEM / 10% FBS for a day and SkIM (Skeletal muscle Induced Media, ( 11 ); (Sigma), 1% penicillin / streptomycin (GIBCO), 1 mM non-essential amino acid (GIBCO), 0.1 mM beta-mercaptoethanol (Sigma )].

CTX (cardiotoxin) - muscle damage model

All animal experiments were conducted with the approval of the IACUC. For rotarod experiment and tissue analysis, male C57BL / 6 (8 weeks old) was anesthetized and CTX 50 μl (10 μM, Sigma) was injected into the TA tibialis muscle of both legs of each mouse to induce muscle damage It was 14. Day after, C57 EB (total cell number equivalent to about ^{5 x 10 4/50 μl PBS} ) were injected into the tibialis anterior of each mouse group. As a control, PBS was injected into the tibialis anterior. EBs were labeled with 2 μg / ml DiI (Molecular Probes Inc.) for 30 min before EB formation to track the differentiation. Immunofluorescent staining was performed after collecting the tibialis anterior to observe whether differentiation from injured muscle to skeletal muscle cells occurred.

Rota Road Experiment

The mice were examined for their ability to stay in a rotating rod (Panlab Rota-Rods LE. 8200, Harvard Apparatus) ( 14 ). The inventors investigated the motor function by measuring the latency time from when the mouse was continuously accelerated (5-40 rpm) until it fell from the rotary rod. Each mouse was tested four times for each delay time and the average delay time was calculated. Between each experiment, mice were allowed to rest for 5 minutes. Prior to CTX muscle injury and cell transplantation, the mice were placed in a stationary cylinder for 3 minutes, followed by three round-rod experiments.

Plasmid construction and transformation

MISSION ™ TRC shRNA Target Set (TRCN00000010413) or control sh-plamsid (MISSION Non-Target shRNA Control SHC002) (Sigma) was used for HDAC6 knockdown. C57 embryonic stem cells were transformed with Metafetamin (Biotex) and selected by treatment with puromycin (5 μg / ml: Sigma). For mouse Pax7 cloning, a 0.7 kb upstream sequence of the transcription initiation site of the Pax7 gene was amplified by PCR from genomic DNA and cloned into pcDNA 3.1 (Invitrogen) ( 15 ). Transformation was carried out using Metafetamin (Biotex) according to the manufacturer's instructions. The primer information used is shown in Table 1.

The primer sequence used for cloning The 5 'flanking sequence of murine Pax7 Forward 5'-AAGCTTCACAACTTACCCAGCTGATCGCTC-3 ' Reverse 3'-CTCGAGCGGGTCTCATCATCCTGGGGA-5 ' Pax7-promoter ChIP
( -582 to -338 ) Forward 5'-CACAACTTACCCAGCTGATCGC-3 ' Reverse 3'-GCGGAGTGGCTTTCTTTGGG-5 ' Pax7-promoter ChIP
( -349 to -100 Forward 5'-AAGCCACTCCGCAACCTCTGGG-3 ' Reverse 3'-AAGCTGGGCGCGGGGGGGGGTG-5 ' Pax7-promoter ChIP
( -104 to +119 Forward 5'-AGCTTCTGGAAGGGGCGC-3 ' Reverse 3'-GGCCGGGTCTCATCATCCTG-5 '

Chromatin Immunoassay (ChIP) analysis

The mouse Pax7 promoter vector was transformed into C57 cells, disrupted, and ChIP assay was performed using the ChIP assay kit (Millipore) according to the manufacturer's instructions. Chromatin was immunoprecipitated using antibodies against control Rabbit IgG, acetyl-H3, acetyl-H4 or RNA polymerase II (Millipore). ChIP (2 μl) or 1% injected DNA was quantitated by PCR using primers. PCR primers are shown in Table 1. After real-time PCR, the measurements were calculated using the following formula: 2.5 x 2 ^ ^{[CT (input) -CT (AbIP)]} ( 16 ).

RT-PCR and real-time PCR analysis

Total RNA was isolated using QIAshredder, RNeasy mini kit and RNeasy plus Mini kit (Qiagen Inc.). PrimeScript ™ 1st strand cDNA Synthesis Kit (Takara) was used to convert 1 μg of RNA into cDNA. Real-time PCR was performed with the SYBR Green PCR Master Mix (Roche) using the specific primers listed in Table 2; Oct4, Nanog, series markers. Real-time samples were run on an ABI PRISM-7500 sequence detection system (Applied Biosystems). 18S rRNA as a control group and used for standardization. End-point PCR was performed on the HDAC family and Pax7, and GAPDH was used for standardization.

Real-time PCR was performed using a specific primer (Table 2) via the miscript SYBR green PCR kit (Qiagen Inc.).

The primer sequences used in PCR primer order Size (bp) HDAC1 Forward 5'-GACCGGTTAGGTTGCTTCAA-3 ' 191 Reverse 3'-TCGTTGTAGGGCAGCTCATT-5 ' HDAC2 Forward 5'-ACGGATAGCTTGCGATGAAG-3 ' 211 Reverse 3'-TTCTGACTTGGCTCCTTTGG-5 ' HDAC3 Forward 5'-CCAGAAGCACCCAATGAGTT-3 ' 195 Reverse 3'-TTCCAAACCCTTCACCAGAG-5 ' HDAC4 Forward 5'-ACGACGCCAAAGATGACTTC-3 ' 232 Reverse 3'-ACGTTGCCAGAGCTGCTATT-5 ' HDAC5 Forward 5'-GTCGAAAGGATGGCACTGTT-3 ' 162 Reverse 3'-AGCCAGTAAAGCCGTTCTCA-5 ' HDAC6 Forward 5'-AAGTGGAAGAAGCCGTGCTA-3 ' 158 Reverse 3'-AGGGTGATGATTGTCCCAGA-5 ' HDAC7 Forward 5'-GAGCTGAAGAATGGCTTTGC-3 ' 249 Reverse 3'-GAAGAAGTTGCCGTCGTCAT-5 ' HDAC8 Forward 5'-ATGACTGTGTCCCTGCACAA-3 ' 237 Reverse 3'-CATTGGATCTCCGGCAATAG-5 ' HDAC9 Forward 5'-GCGGTCCAGGTTAAAACAGA-3 ' 203 Reverse 3'-GAGCTGAAGCCTCATTTTCG-5 ' HDAC10 Forward 5'-TCTCATTGTCGACTGGGATG-3 ' 233 Reverse 3'-GCAGAAAGGCAGCCAAATAG-5 ' HDAC11 Forward 5'-TTTTCCGCGTGGTCCGAGCC-3 ' 153 Reverse 3'-TCTGTGCCGAGACGCAGGGA-5 ' Pax3 Forward 5'-CCTCCATCGGGGCCCTCCAA-3 ' 167 Reverse 3'-GCTGACACCGTGGTCGTGGG-5 ' Pax7 Forward 5'-AGACTGGGTCCATCCGGCCC-3 ' 169 Reverse 3'-ACCGTGCTTCGGTCGCAGTG-5 ' MyoD Forward 5'-TGGAGATCCTGCGCAACGCC-3 ' 187 Reverse 3'-TGTAGTGCTCGCTGCCACGG-5 ' Myf5 Forward 5'-CCACCATGCGCGAGCGTAGA-3 ' 194 Reverse 3'-GCTCTGTCCCGGCAGGCTGTA-5 ' MyoG Forward 5'-CTGCGCAGCGCCATCCAGTA-3 ' 222 Reverse 3'-GGCGTCTGTAGGGTCAGCCG-5 ' 18s rRNA Forward 5'-GTAACCCGTTGAACCTT-3 ' 200 Reverse 3'-CCATCCAATCGGTAGTAGCG-5 '

Western blotting analysis

Cells were harvested and disrupted in a lysis buffer containing protease inhibitor (Roche). Total protein (10-30 μg) was immunobiloted with specific primary antibodies: HDAC6 (Abcam), Pax7 (DSHB), α-tubulin (Calbiochem) were used as internal controls. Band strength was quantified using TINA 2.0 (RayTest) and standardized for the strength of α-tubulin.

Immunofluorescent staining

Cells differentiated from EB or C2C12 mouse source cells were fixed in 4% PFA at 35 ^{mm high} (ibidi), blocked with blocking buffer (0.5% goat serum, 0.1% triton-x100 / 1% BSA-PBS) (R & D), and secondary antibody conjugated with fluorescent dye (Invitrogen). To analyze the tissue of CTX-injured mice, the tibialis anterior of the fourth week after cell transplantation was dissected, rinsed with PBS and then cooled with liquid nitrogen. Histologic sections (6-8 μm thickness) were prepared from rapidly cooled tissue samples, fixed with acetone, blocked with 1% BSA and incubated with anti-laminin α2 (4H8-2, Alexis Biochemicals) and fluorescent dye conjugated 2 (Invitrogen).

For quantification of regenerating myofibers, at least five fields arbitrarily selected from cross-sectional slides of three different mice were analyzed. Nuclei were stained with DAPI (Molecular Probe) and mounted on a fluorescent mounting medium (DAKO). Fluorescence images were obtained using a confocal microscope (Carl Zeiss LSM710).

Statistical analysis

The experimental results are expressed as mean ± SD. Differences in each group were compared by unpaired t-test or one-way ANOVA (analysis of variance), where P <0.05 was considered statistically significant All statistical analyzes were performed using SPSS 17.0 Inc., Chicago, US).

Experiment result

Reduction of HDAC6 and differentiation of mesenchymal lineage of embryonic stem cells

Based on the previous report ( 5-7 ) that HDAC affects the differentiation of stem cells into specific lines (neurons, bone and liver cells, etc.), HDAC is involved in muscle differentiation as well , And then examined which HDACs in a class affect muscle differentiation. Of the four HDAC groups, the present inventors have focused on HDAC1 through HDAC11 (HDAC inhibitor sensitive groups: Class I, II, and IV) for future clinical applications ( 1-2 ). The present inventors sought to select HDAC having a significant expression change in the embryoid body (EB). Expression of HDAC2 and HDAC6 was significantly decreased during EB formation of E14 ES cells among 11 HDACs. Among HDAC2 and HDAC6, the present inventors paid attention to HDAC6, whose role in stem cells was little known.

It was confirmed that HDAC6 was expressed in E14 ES cells and C57 ES cells (Fig. 1a, 1b). HDAC6 mRNA and protein were reduced by EB formation in both C57 ES cells and E14 ES cells, suggesting that HDAC6 was involved in stem cell differentiation.

The present inventors produced HDAC6 rust-down cells using shHDAC6 and conducted further experiments on stem cell differentiation. HDAC6 mRNA and protein were significantly reduced by shHDAC6 (shHD6) transformation compared to the shMock control (Figures 1c and 1d). In particular, shHDAC6 specifically inhibited HDAC6 among HDAC1 to HDAC11 (Fig. 1C).

Next, the present inventors investigated whether the deficiency of HDAC6 affects the differentiation specificity of embryonic stem cells. Interestingly, the pre-differentiation markers Oct4 and Nanog were significantly reduced in shHDAC6- cells compared to the shMock control, indicating that HDAC6 rust-down promoted differentiation of embryonic stem cells (Fig. 1e). Endoderm marker genes (Sox17, Throma-1) were not affected by HDAC6 rust-down. The ectoderm marker genes (Ncam, Nestin, Foxa) showed an ambiguous expression pattern. In contrast, the medial leaf marker (SMA, Desmin) showed a significant increase by shHDAC6

Since we wanted to observe the vascular- and / or muscle differentiation among the mesenchymal tissues, the present inventors investigated the expression of blood vessel- and muscle markers (Fig. 1F). Cells were cultured for 10 days in spontaneous differentiation conditions (DMEM / 10% FBS) and collected at different time points. The expression of the muscle marker genes (Pax3, Pax7, MyoD) in shHDAC6- cells increased with differentiation, whereas the vascular marker genes (PECAM, SMA, VE-carderine) did not. These results indicate that HDAC6 reduction not only promotes cell differentiation but also induces differentiation into the muscular system rather than the vascular system

Differentiation of HDAC6 rust-down stem cells into muscle system

Based on the foregoing results, the present inventors have confirmed Pax7 expression in various shMock and shHDAC6 cell clones in order to demonstrate the difference according to the clones. Pax7 is the most widely used embryonic muscle precursor cell marker essential for the identification of muscle precursor cells ( 9-10 ). Surprisingly, Pax7 mRNA and protein were increased in all five different shHDAC6 clones, making it clear that Pax7 has an opposite pattern of expression to HDAC6 (Figs. 2a and 2b).

As shown in Fig. 1f, the muscle marker genes of shHDAC6 cells were increased in the spontaneous differentiation condition; Thus, the present inventors further investigated the specific muscle-differentiating ability of shHDAC6 cells (Fig. 2c-2e). Cells were cultured in SkIM (Skeletal muscle Induced Media) to induce muscle-specific differentiation ( 11 ). As shown in FIG. 2d, Pax7 and muscle regulatory factors (Myf5, MyoD, Myogenin) were increased in shMock-control cells, and these genes were further increased in shHDAC6-cells than in shMock-cells.

The present inventors performed immunofluorescence staining using an anti-Max7 antibody. Pax 7 was scarcely detected in shMock-cells, whereas shHDAC6-cells stained strongly with nuclei as in C2C12 myoblasts (Fig. 2e), indicating that shHDAC6-cells were efficiently differentiated into muscle cells.

Progressive regulation of Pax7 promoter in shHDAC6 cells

Since the mRNA and protein of the muscle precursor cell marker Pax7 greatly increased in both shHDAC6 cells, the present inventors investigated whether HDAC6 represses the Pax7 promoter afterwards. Chromatin modification and binding of the transcription factor protein to the Pax7 promoter were affected by HDAC6 reduction (Fig. 3). ChIP analysis using an antibody against acetylated histone was performed. The acetylation levels of H3 and H4 core histones in the Pax7 promoter were significantly increased in shHDAC6 cells (Figures 3b and 3c). Acetylation of H3 and H4 histones is known to be an indicator that chromatin is activated or has a more open structure ( 12 ). The binding of RNA polymerase II (Pol II) to the Pax7 promoter was also increased in shHDAC6 cells (FIG. 3D), indicating that chromatin was more accessible in shHDAC6 cells, leading to increased transcription of Pax7.

Transplantation of HDAC6 rust-down cells improves rotarod movement.

Since shHDAC6 cells efficiently differentiated into inhibiotic cells in vitro , the present inventors investigated whether the muscle differentiation of HDAC6 rust-down ES cells was successfully performed in in vivo , and then CTX (cardiotoxin) A load experiment was performed (Figs. 3e-3h). The restoration of motor function was measured by the CTX injury of the tibialis anterior muscle of both legs the day before cell transplantation and the time taken to fall off weekly on the rotating rod (Fig. 3e-3g). Consistent with previous observations of superior muscle differentiation capacity, mice transplanted with HDAC6 rust-down cells (CTX + shHD6 cells) showed improved motor function (Fig. 3f). In addition, by measuring the delay time of all mice (n = 52) 2 weeks after transplantation, it was confirmed that shHDAC6 cells significantly restored motor function compared to shMock cells, and successful muscle differentiation and cell transplantation improved substantial muscle function I can see that it leads.

The regenerated muscle fibers were observed through histological analysis (Fig. 3H). The central nucleus is one of the indicators of muscle fiber regeneration ( 13 ). Cells differentiated from DiI-labeled cells exhibit red DiI-fluorescence. Regenerated muscle fibers located with DiI (? Silver) were more frequent in the shHDAC6 group than in the shMock group (Fig. 3h, 3i).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

references

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2. SC Hodawadekar, R. Marmorstein, Oncogene 26 , 5528 (Aug 13,2007).

3. M. Haberland, RL Montgomery, EN Olson, Nat Rev Genet 10 , 32 (Jan, 2009).

4. CA Dinarello, G. Fossati, P. Mascagni, Mol Med 17 , 333 (May-Jun, 2011).

5. A. Kretsovali, C. Hadjimichael, N. Charmpilas, Stem Cells Int 2012 , 184154 (2012).

6. F. Paino et al. , Stem Cells 32 , 279 (Jan, 2014).

7. X. Dong et al. , PLoSOne 8 , e63405 (2013).

8. Y. Li, D. Shin, SH Kwon, FEBSJ 280 , 775 (Feb, 2013).

9. P. Seale et al. , Cell 102 , 777 (Sep 15, 2000).

10. PS Zammit et al. , J Cell Sci 119 , 1824 (May 1, 2006).

11. Y. Mizuno et al. , FASEBJ 24 , 2245 (Jul, 2010)

12. M. Garcia-Ramirez, C. Rocchini, J. Ausio, J Biol Chem 270 , 17923 (Jul. 28, 1995).

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14. R. Darabi et al. , Nat Med 14 , 134 (Feb, 2008).

15. D. Lang et al. , Genomics 82 , 553 (Nov, 2003).

16. SR Frank, M. Schroeder, P. Fernandez, S. Taubert, B. Amati, Genes Dev 15 , 2069 (Aug 15, 2001).

<110> SNU R & DB Foundation <120> A Composition for Inducing Myogenic-lineage Differentiation of          Stem Cells <130> PN140047 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 4099 <212> DNA <213> homo sapiens <400> 1 gggcagtccc ctgaggagcg gggctggttg aaacgctagg ggcgggatct ggcggagtgg 60 aagaaccgcg gcaggggcca agcctcctca actatgacct caaccggcca ggattccacc 120 acaaccaggc agcgaagaag taggcagaac ccccagtcgc cccctcagga ctccagtgtc 180 acttcgaagc gaaatattaa aaagggagcc gttccccgct ctatccccaa tctagcggag 240 gtaaagaaga aaggcaaaat gaagaagctc ggccaagcaa tggaagaaga cctaatcgtg 300 ggactgcaag ggatggatct gaaccttgag gctgaagcac tggctggcac tggcttggtg 360 ttggatgagc agttaaatga attccattgc ctctgggatg acagcttccc ggaaggccct 420 gagcggctcc atgccatcaa ggagcaactg atccaggagg gcctcctaga tcgctgcgtg 480 tcctttcagg cccggtttgc tgaaaaggaa gagctgatgt tggttcacag cctagaatat 540 attgatctga tggaaacaac ccagtacatg aatgagggag aactccgtgt cctagcagac 600 acctacgact cagtttatct gcatccgaac tcatactcct gtgcctgcct ggcctcaggc 660 tctgtcctca ggctggtgga tgcggtcctg ggggctgaga tccggaatgg catggccatc 720 attaggcctc ctggacatca cgcccagcac agtcttatgg atggctattg catgttcaac 780 cacgtggctg tggcagcccg ctatgctcaa cagaaacacc gcatccggag ggtccttatc 840 gtagattggg atgtgcacca cggtcaagga acacagttca ccttcgacca ggaccccagt 900 gtcctctatt tctccatcca ccgctacgag cagggtaggt tctggcccca cctgaaggcc 960 tctaactggt ccaccacagg tttcggccaa ggccaaggat ataccatcaa tgtgccttgg 1020 aaccaggtgg ggatgcggga tgctgactac attgctgctt tcctgcacgt cctgctgcca 1080 gtcgccctcg agttccagcc tcagctggtc ctggtggctg ctggatttga tgccctgcaa 1140 ggggacccca agggtgagat ggccgccact ccggcagggt tcgcccagct aacccacctg 1200 ctcatgggtc tggcaggagg caagctgatc ctgtctctgg agggtggcta caacctccgc 1260 gccctggctg aaggcgtcag tgcttcgctc cacacccttc tgggagaccc ttgccccatg 1320 ctggagtcac ctggtgcccc ctgccggagt gcccaggctt cagtttcctg tgctctggaa 1380 gcccttgagc ccttctggga ggttcttgtg agatcaactg agaccgtgga gagggacaac 1440 atggaggagg acaatgtaga ggagagcgag gaggaaggac cctgggagcc ccctgtgctc 1500 ccaatcctga catggccagt gctacagtct cgcacagggc tggtctatga ccaaaatatg 1560 atgaatcact gcaacttgtg ggacagccac caccctgagg taccccagcg catcttgcgg 1620 atcatgtgcc gtctggagga gctgggcctt gccgggcgct gcctcaccct gacaccgcgc 1680 cctgccacag aggctgagct gctcacctgt cacagtgctg agtacgtggg tcatctccgg 1740 gccacagaga aaatgaaaac ccgggagctg caccgtgaga gttccaactt tgactccatc 1800 tatatctgcc ccagtacctt cgcctgtgca cagcttgcca ctggcgctgc ctgccgcctg 1860 gtggaggctg tgctctcagg agaggttctg aatggtgctg ctgtggtgcg tccccagga 1920 caccacgcag agcaggatgc agcttgcggt ttttgctttt tcaactctgt ggctgtggct 1980 gctcgccatg cccagactat cagtgggcat gccctacgga tcctgattgt ggattgggat 2040 gtccaccacg gtaatggaac tcagcacatg tttgaggatg accccagtgt gctatatgtg 2100 tccctgcacc gctatgatca tggcaccttc ttccccatgg gggatgaggg tgccagcagc 2160 cagatcggcc gggctgcggg cacaggcttc accgtcaacg tggcatggaa cgggccccgc 2220 atgggtgatg ctgactacct agctgcctgg catcgcctgg tgcttcccat tgcctacgag 2280 tttaacccag aactggtgct ggtctcagct ggctttgatg ctgcacgggg ggatccgctg 2340 gggggctgcc aggtgtcacc tgagggttat gcccacctca cccacctgct gatgggcctt 2400 gccagtggcc gcattatcct tatcctagag ggtggctata acctgacatc catctcagag 2460 tccatggctg cctgcactcg ctccctcctt ggagacccac cacccctgct gaccctgcca 2520 cggcccccac tatcaggggc cctggcctca atcactgaga ccatccaagt ccatcgcaga 2580 tactggcgca gcttacgggt catgaaggta gaagacagag aaggaccctc cagttctaag 2640 ttggtcacca agaaggcacc ccaaccagcc aaacctaggt tagctgagcg gatgaccaca 2700 cgagaaaaga aggttctgga agcaggcatg gggaaagtca cctcggcatc atttggggaa 2760 gagtccactc caggccagac taactcagag acagctgtgg tggccctcac tcaggaccag 2820 ccctcagagg cagccacagg gggagccact ctggcccaga ccatttctga ggcagccatt 2880 gggggagcca tgctgggcca gaccacctca gaggaggctg tcgggggagc cactccggac 2940 cagaccacct cagaggagac tgtgggagga gccattctgg accagaccac ctcagaggat 3000 gctgttgggg gagccacgct gggccagact acctcagagg aggctgtagg aggagctaca 3060 ctggcccaga ccacctcgga ggcagccatg gagggagcca cactggacca gactacgtca 3120 gaggaggctc cagggggcac cgagctgatc caaactcctc tagcctcgag cacagaccac 3180 cagacccccc caacctcacc tgtgcaggga actacacccc agatatctcc cagtacactg 3240 attgggagtc tcaggacctt ggagctaggc agcgaatctc agggggcctc agaatctcag 3300 gccccaggag aggagaacct actaggagag gcagctggag gtcaggacat ggctgattcg 3360 atgctgatgc agggatctag gggcctcact gatcaggcca tattttatgc tgtgacacca 3420 ctgccctggt gtccccattt ggtggcagta tgccccatac ctgcagcagg cctagacgtg 3480 acccaacctt gtggggactg tggaacaatc caagagaatt gggtgtgtct ctcttgctat 3540 caggtctact gtggtcgtta catcaatggc cacatgctcc aacaccatgg aaattctgga 3600 cacccgctgg tcctcagcta catcgacctg tcagcctggt gttactactg tcaggcctat 3660 gtccaccacc aggctctcct agatgtgaag aacatcgccc accagaacaa gtttggggag 3720 gatatgcccc acccacacta agccccagaa tacggtccct cttcaccttc tgaggcccac 3780 gatagaccag ctgtagctca ttccagcctg taccttggat gaggggtagc ctcccactgc 3840 atcccatcct gaatatcctt tgcaactccc caagagtgct tatttaagtg ttaatacttt 3900 taagagaact gcgacgatta attgtggatc tccccctgcc cattgcctgc ttgaggggca 3960 ccactactcc agcccagaag gaaagggggg cagctcagtg gccccaagag ggagctgata 4020 tcatgaggat aacattggcg ggaggggagt taactggcag gcatggcaag gttgcatatg 4080 taataaagta caagctgtt 4099 <210> 2 <211> 1215 <212> PRT <213> homo sapiens <400> 2 Met Thr Ser Thr Gly Gln Asp Ser Thr Thr Thr Arg Gln Arg Arg Ser   1 5 10 15 Arg Gln Asn Pro Gln Ser Pro Pro Gln Asp Ser Ser Val Thr Ser Lys              20 25 30 Arg Asn Ile Lys Lys Gly Ala Val Pro Arg Ser Ile Pro Asn Leu Ala          35 40 45 Glu Val Lys Lys Lys Gly Lys Met Lys Lys Leu Gly Gln Ala Met Glu      50 55 60 Glu Asp Leu Ile Val Gly Leu Gln Gly Met Asp Leu Asn Leu Glu Ala  65 70 75 80 Glu Ala Leu Ala Gly Thr Gly Leu Val Leu Asp Glu Gln Leu Asn Glu                  85 90 95 Phe His Cys Leu Trp Asp Asp Ser Phe Pro Glu Gly Pro Glu Arg Leu             100 105 110 His Ala Ile Lys Glu Gln Leu Ile Gln Glu Gly Leu Leu Asp Arg Cys         115 120 125 Val Ser Phe Gln Ala Arg Phe Ala Glu Lys Glu Glu Leu Met Leu Val     130 135 140 His Ser Leu Glu Tyr Ile Asp Leu Met Glu Thr Thr Gln Tyr Met Asn 145 150 155 160 Glu Gly Glu Leu Arg Val Leu Ala Asp Thr Tyr Asp Ser Val Tyr Leu                 165 170 175 His Pro Asn Ser Tyr Ser Cys Ala Cys Leu Ala Ser Gly Ser Val Leu             180 185 190 Arg Leu Val Asp Ala Val Leu Gly Ala Glu Ile Arg Asn Gly Met Ala         195 200 205 Ile Ile Arg Pro Pro Gly His His Ala Gln His Ser Leu Met Asp Gly     210 215 220 Tyr Cys Met Phe Asn His Val Ala Val Ala Ala Arg Tyr Ala Gln Gln 225 230 235 240 Lys His Arg Ile Arg Arg Val Leu Ile Val Asp Trp Asp Val His His                 245 250 255 Gly Gln Gly Thr Gln Phe Thr Phe Asp Gln Asp Pro Ser Val Leu Tyr             260 265 270 Phe Ser Ile His Arg Tyr Glu Gln Gly Arg Phe Trp Pro His Leu Lys         275 280 285 Ala Ser Asn Trp Ser Thr Thr Gly Phe Gly Gln Gly Gln Gly Tyr Thr     290 295 300 Ile Asn Val Pro Trp Asn Gln Val Gly Met Arg Asp Ala Asp Tyr Ile 305 310 315 320 Ala Ala Phe Leu His Val Leu Leu Pro Val Ala Leu Glu Phe Gln Pro                 325 330 335 Gln Leu Val Leu Val Ala Ala Gly Phe Asp Ala Leu Gln Gly Asp Pro             340 345 350 Lys Gly Glu Met Ala Ala Thr Pro Ala Gly Phe Ala Gln Leu Thr His         355 360 365 Leu Leu Met Gly Leu Ala Gly Gly Lys Leu Ile Leu Ser Leu Glu Gly     370 375 380 Gly Tyr Asn Leu Arg Ala Leu Ala Glu Gly Val Ser Ala Ser Leu His 385 390 395 400 Thr Leu Leu Gly Asp Pro Cys Pro Met Leu Glu Ser Pro Gly Ala Pro                 405 410 415 Cys Arg Ser Ala Gln Ala Ser Val Ser Cys Ala Leu Glu Ala Leu Glu             420 425 430 Pro Phe Trp Glu Val Leu Val Arg Ser Thr Glu Thr Val Glu Arg Asp         435 440 445 Asn Met Glu Glu Asp Asn Val Glu Glu Glu Ser Glu Glu Glu Gly Pro Trp     450 455 460 Glu Pro Pro Val Leu Pro Ile Leu Thr Trp Pro Val Leu Gln Ser Arg 465 470 475 480 Thr Gly Leu Val Tyr Asp Gln Asn Met Met Asn His Cys Asn Leu Trp                 485 490 495 Asp Ser His His Pro Glu Val Pro Gln Arg Ile Leu Arg Ile Met Cys             500 505 510 Arg Leu Glu Glu Leu Gly Leu Ala Gly Arg Cys Leu Thr Leu Thr Pro         515 520 525 Arg Pro Ala Thr Glu Ala Glu Leu Leu Thr Cys His Ser Ala Glu Tyr     530 535 540 Val Gly His Leu Arg Ala Thr Glu Lys Met Lys Thr Arg Glu Leu His 545 550 555 560 Arg Glu Ser Ser Asn Phe Asp Ser Ile Tyr Ile Cys Pro Ser Thr Phe                 565 570 575 Ala Cys Ala Gln Leu Ala Thr Gly Ala Ala Cys Arg Leu Val Glu Ala             580 585 590 Val Leu Ser Gly Glu Val Leu Asn Gly Ala Val Val Arg Pro Pro         595 600 605 Gly His His Ala Glu Gln Asp Ala Ala Cys Gly Phe Cys Phe Phe Asn     610 615 620 Ser Val Ala Val Ala Ala Arg His Ala Gln Thr Ile Ser Gly His Ala 625 630 635 640 Leu Arg Ile Leu Ile Val Asp Trp Asp Val His His Gly Asn Gly Thr                 645 650 655 Gln His Met Phe Glu Asp Asp Pro Ser Val Leu Tyr Val Ser Leu His             660 665 670 Arg Tyr Asp His Gly Thr Phe Phe Pro Met Gly Asp Glu Gly Ala Ser         675 680 685 Ser Gln Ile Gly Arg Ala Ala Gly Thr Gly Phe Thr Val Asn Val Ala     690 695 700 Trp Asn Gly Pro Arg Met Gly Asp Ala Asp Tyr Leu Ala Ala Trp His 705 710 715 720 Arg Leu Val Leu Pro Ile Ala Tyr Glu Phe Asn Pro Glu Leu Val Leu                 725 730 735 Val Ser Ala Gly Phe Asp Ala Ala Arg Gly Asp Pro Leu Gly Gly Cys             740 745 750 Gln Val Ser Pro Glu Gly Tyr Ala His Leu Thr His Leu Leu Met Gly         755 760 765 Leu Ala Ser Gly Arg Ile Ile Leu Ile Leu Glu Gly Gly Tyr Asn Leu     770 775 780 Thr Ser Ile Ser Glu Ser Ala Ala Cys Thr Arg Ser Leu Leu Gly 785 790 795 800 Asp Pro Pro Pro Leu Leu Thr Leu Pro Arg Pro Pro Leu Ser Gly Ala                 805 810 815 Leu Ala Ser Ile Thr Glu Thr Ile Gln Val His Arg Arg Tyr Trp Arg             820 825 830 Ser Leu Arg Val Met Lys Val Glu Asp Arg Glu Gly Pro Ser Ser Ser         835 840 845 Lys Leu Val Thr Lys Lys Ala Pro Gln Pro Ala Lys Pro Arg Leu Ala     850 855 860 Glu Arg Met Thr Thr Arg Glu Lys Lys Val Leu Glu Ala Gly Met Gly 865 870 875 880 Lys Val Thr Ser Ala Ser Phe Gly Glu Glu Ser Thr Pro Gly Gln Thr                 885 890 895 Asn Ser Glu Thr Ala Val Val Ala Leu Thr Gln Asp Gln Pro Ser Glu             900 905 910 Ala Ala Thr Gly Gly Ala Thr Leu Ala Gln Thr Ile Ser Glu Ala Ala         915 920 925 Ile Gly Gly Ala Met Leu Gly Gln Thr Thr Ser Glu Glu Ala Val Gly     930 935 940 Gly Ala Thr Pro Asp Gln Thr Thr Ser Glu Glu Thr Val Gly Gly Ala 945 950 955 960 Ile Leu Asp Gln Thr Thr Ser Glu Asp Ala Val Gly Gly Ala Thr Leu                 965 970 975 Gly Gln Thr Thr Ser Glu Gly Ala Val Gly Gly Ala Thr Leu Ala Gln             980 985 990 Thr Thr Ser Glu Ala Ala Met Glu Gly Ala Thr Leu Asp Gln Thr Thr         995 1000 1005 Ser Glu Glu Ala Pro Gly Gly Thr Glu Leu Ile Gln Thr Pro Leu Ala    1010 1015 1020 Ser Ser Thr Asp His Gln Thr Pro Pro Thr Ser Pro Val Gln Gly Thr 1025 1030 1035 1040 Thr Pro Gln Ile Ser Ser Thr Leu Ile Gly Ser Leu Arg Thr Leu                1045 1050 1055 Glu Leu Gly Ser Glu Ser Gln Gly Ala Ser Glu Ser Gln Ala Pro Gly            1060 1065 1070 Glu Glu Asn Leu Leu Gly Glu Ala Ala Gly Gly Gln Asp Met Ala Asp        1075 1080 1085 Ser Met Leu Met Gln Gly Ser Arg Gly Leu Thr Asp Gln Ala Ile Phe    1090 1095 1100 Tyr Ala Val Thr Pro Leu Pro Trp Cys Pro His Leu Val Ala Val Cys 1105 1110 1115 1120 Pro Ile Pro Ala Ala Gly Leu Asp Val Thr Gln Pro Cys Gly Asp Cys                1125 1130 1135 Gly Thr Ile Gln Glu Asn Trp Val Cys Leu Ser Cys Tyr Gln Val Tyr            1140 1145 1150 Cys Gly Arg Tyr Ile Asn Gly His Met Leu Gln His His Gly Asn Ser        1155 1160 1165 Gly His Pro Leu Val Leu Ser Tyr Ile Asp Leu Ser Ala Trp Cys Tyr    1170 1175 1180 Tyr Cys Gln Ala Tyr Val His His Gln Ala Leu Leu Asp Val Lys Asn 1185 1190 1195 1200 Ile Ala His Gln Asn Lys Phe Gly Glu Asp Met Pro His Pro His                1205 1210 1215

Claims (12)

A composition for inducing differentiation of stem cells into muscle cells in vitro comprising an inhibitor of HDAC6 (histone deacetylase 6) as an active ingredient.
The method of claim 1, wherein the inhibitor of HDAC6 is selected from the group consisting of a blocker of a protein consisting of the amino acid sequence of SEQ ID NO: 2, an antibody specifically binding to the protein, and an aptamer specifically binding to the protein &Lt; / RTI &gt;
2. The composition of claim 1, wherein the inhibitor of HDAC6 is a nucleic acid molecule that inhibits the expression of a nucleotide encoding the amino acid sequence of SEQ ID NO: 2.
4. The composition of claim 3, wherein the nucleotide sequence is comprised of a nucleotide sequence of SEQ ID &lt; RTI ID = 0.0 &gt; 1 &lt; / RTI &gt;
4. The composition of claim 3, wherein the nucleic acid molecule is selected from the group consisting of shRNA, siRNA, miRNA, ribozyme, PNA (peptide nucleic acids) and antisense oligonucleotides.
The composition according to claim 1, wherein the stem cell is an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC).
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