WO2012064090A2 - Chemical preparation method of skeletal muscle-derived multipotent cells and use thereof - Google Patents

Abstract

The present invention provides a chemical preparation method of skeletal muscle-derived multipotent cells comprising: (a) a step for obtaining differentiated cells from mammals; and (b) a step for obtaining multipotent cells by treating the differentiated cells with a dedifferentiation agent after inducing the down-regulation of p21 expression of the differentiated cells. The multipotent cells of the invention enable not only dedifferentiation into a musculogenesis cell population but also the transdifferentiation into an adipogenesis cell population or an osteogenesis cell population. Therefore, the chemical-based approach method of the invention is important in the development of strategies for inducing limb regeneration in mammals. In addition, the invention discloses a new approach method for the development of induced pluripotent stem cells (iPSCs) from completely differentiated refractory tissues.

Description

 【Specification】

 [Name of invention]

 Chemical preparation of skeletal muscle-derived pluripotent cells

[Technical field]

 The present invention provides a method for chemical preparation of skeletal muscle-derived pluripotent cells.

It is about a use.

[Background technology]

Dedifferentiation is a phenomenon in which differentiated cells acquire 'stem cells'-like or multipotent state before they differentiate into one or more different tissue forms [1]. All species have natural differentiation as part of their ability to regenerate tissues [2]. For example, acinar cells or duct eel is present in the pancreas of mammals can be reversed to produce insulin-expressing beta-cells [3]. However, retrodifferentiation varies among species, and systematically primitive vertebrates have greater retrodifferentiation. For example, the cells of the central nervous system of amphibians and fish can dedifferentiate [4]. Moreover, the retina [5], lens [6] and limbs [groups] of the urodele amphibians undergo differentiation during the entire regeneration of the tissues. However, the greatest retrodifferentiation is found in plants. For example, tobacco / coi / aa

_MeS0 phyll protoplasts cause reverse differentiation as part of the reprogramming process to generate whole plants [8].

In the 1990s, advances in the field of combinatorial chemistry have facilitated large-scale production of small molecular libraries based on molecular scaffolds of known bioactive compounds [9]. Recently, we and other researchers have used this approach to identify novel regulators of various biological processes. [1] 12 The use of small molecules in regulating intracellular processes has Has numerous advantages [13]. Typically, small molecules provide temporary regulation of protein function and are often vice versa, and their effects are small. It can be precisely controlled by changing the concentration of the molecule. In addition, a single small molecule has the potential to simultaneously regulate various specific targets in the cell. The above approach can produce the desired phenotype through desirable synergies.

 Phenotype-based screening methods for small molecular libraries have been carried out to find novel regulators associated with intracellular dedifferentiation [14, 15]. An excellent example is the discovery of myoseverin, a tubulin-binding molecule identified from a 2,6,9-trisubstituted purines library [14, 16]. The molecular scaffold of myosevers is based on olomoucine, a known inhibitor of cyclin-dependent kinase 2, a cell cycle regulatory protein. Myose verbin has been reported to promote reverse differentiation in a C2C12 mouse myoblast model for skeletal muscle differentiation. The above results were significant because proliferation of monocytes through fragmentation of multinucleated myofibers is an important feature of limb regeneration in Rhesus amphibians [17-19]. However, a later study in the pmi28 mouse myoblast model for skeletal muscle differentiation suggested that only myoseber can induce cellularization of muscle fibers [20]. Cellized monocytes remained unproliferated and could not cause reverse differentiation.

During limb regeneration in Rhesus amphibians, the nuclei of multinucleated skeletal muscle fibers reenter the cell cycle [21]. In contrast, the nuclei of finally differentiated mammalian skeletal muscle cells are relatively difficult to experimentally induce cell cycle reactivation [22]. Many cycl in-dependent kinase inhibitors (CDKNs) are involved in the maintenance of skeletal muscle final differentiation, for example pl8 (also known as CDKN2C or INK4C), p21 (also known as CIPl or CDK 1A), p27 (also known as CDKNlB or KIP1) and P57 (also known as CDK 1C or KIP2) [23-25]. Recent studies have shown that in the absence of exogenous growth factors, inhibition of p21 CDKN alone is sufficient to reactivate the cell cycle in mammalian skeletal muscle [24]. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

[Content of invention]

 [Problem to solve]

 The inventors have tried to produce pluripotent cells (nrnltipotent eel Is). As a result, the present inventors induced down-regulation of p21 expression in differentiated cells obtained from skeletal muscle tissue having a very high refractory to obtain pluripotent cells and their differentiation capacity (eg, back-differentiation into muscle cells). The present invention was completed by finally confirming dedifferentiation, transdifferentiation into adipocytes or bone cells.

 Accordingly, it is an object of the present invention to provide a method for chemically preparing pluripotent cells.

 Another object of the present invention is to provide a pluripotent cell.

 Another object of the present invention is to provide a method for regenerating a mammalian tissue or organ. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

[Measures of problem]

According to one aspect of the present invention, the present invention provides a method for chemically preparing a pluripotent cell (mult i potent eel Is) from mammalian differentiated cells comprising the steps of: a) obtaining differentiated cells from a mammal; ; And (b) inducing down-regulation of p21 expression in the differentiated cells to treat pluripotent cells after treatment with a reverse differentiation agent ((16 £ ^ 6! 1 3 ^ 011 agent) after induction of p21 low expression. The present inventors have tried to prepare multipotent cells. As a result, the present inventors induced down-regulation of p21 expression in differentiated cells obtained from skeletal muscle tissue having a very high refractory to obtain pluripotent cells and their differentiation capacity (eg, back-differentiation into muscle cells). , Heterologous differentiation into adipocytes or bone cells) was finally confirmed.

 The present invention describes for the first time a relatively simple and reversible chemical-based method that can induce reverse differentiation of mammalian skeletal muscle (eg, cell cycle re-entry and differentiation), and is a fully differentiated fire in mammals. We present a new approach for the development of iPSCs from male tissues. According to the present invention the present invention obtains mononuclear cells that proliferate through the continuous treatment of small molecules (chemicals) and transient p21 suppression (suppress ions) in mammalian skeletal muscle tissue having very high refractory properties. Thereafter, pluripotent cells can be obtained through step-wise treatment of reversin, a small molecule, to proliferating mononuclear cells derived from muscle fibers. The chemical preparation of the pluripotent cells of the present invention will be described in detail by dividing each step as follows: Step (a): Obtaining differentiated cells from a mammal

 According to the present invention, differentiated cells are obtained from a mammal.

 As used herein, the term 'mammal' includes, but is not limited to, humans, mice, rats, guinea pigs, rabbits, monkeys, pigs, horses, cattle, sheep, antelopes, dogs, and cats.

 Preferably, the differentiated cells are terminally differentiated cells,

As used herein, the term 'terminal differentiated cells' refers to cells at the endpoint stage during development, and has a mature phenotype with a characterized phenotype. Says a cell. Differentiated cells are distinguished from other cell types but have a fishery characteristic or function and have a limited ability to proliferate.

 Preferably, the differentiated cells are one cell selected from the group consisting of skeletal muscle cells, cardiomyocytes, smooth muscle cells, skin cells, chondrocytes, adipocytes and osteocytes, more preferably the differentiated cells are skeletal muscle cells, One cell selected from the group consisting of cardiomyocytes and smooth muscle cells, most preferably said differentiated cells are skeletal muscle cells.

 Preferably, the differentiated cells are connective tissue, nerve cells, lymph cells, vasculature cells, kidney cells, pancreas cells, lung cells, urethral cells, bladder cells, gastric cells, liver cells, small intestine cells, colon cells and esophageal cells. One cell selected from the group consisting of:

Preferably, step ( _a ) of the present invention comprises a method of incorporating a cell-derived agent into the differentiated fusion to segment the syncytia derived from the muscle cells of the mammal and obtain mononuclear cells or eel lulates. This is done by treating cellularization-inducing agent.

 According to a preferred embodiment of the present invention, a mononuclear cell or a cell material is prepared by treating a skeletal muscle fusion with a cellization-inducing agent.

 As used herein, the term "syncytia" refers to a multinuclear cell having a large cell-like structure filled with cytoplasm containing many nuclei, and in eukaryotes, most cells have a single nucleus. In that sense the fusion is a very unusual form.

The cellularization-inducing formulations of the present invention can use any chemical known in the art that can inhibit microtubule assembly. According to a preferred embodiment of the present invention, the cell-derived agent of the present invention is myosebine, myosebine B, colchicine, nocodazole, allocolchin taxane, grapephylolock, combretastatin, steg Nacin, dihydroxy-pentamethoxy ananone, 2- methoxyestradiol (2-ME) and its analogues, benzimidazole carbamate, ansamitocin, TN16, vinbla Stin, vincristine, dolastatin, curacin A, etoposide, teniposide, sanguinarine and Chelidonineol, more preferably, myosebergin, myosebergin B, colchicine and nocodazole, even more preferably myosebergin and myosebergin B; And most preferably is myosebourne.

 The present invention identifies myosebers identified using a purine-based compound library to identify novel regulators associated with intracellular dedifferentiation.

 Schultz and co-workers identified myoseber, a potent microtubule-degrading compound, using a 2,6,9-alternative purine library constructed by Combinatorial Chemistry [14]. The low cytotoxicity of myoseberg and its analogs allows it to be applied as a cytostatic antitumor agent. Myoserine used in the present invention is a small chemical molecule identified from the 2,6,9-trisubstituted purines library, and has the following chemical structure (Π):

 Myoserine is based on olomoucine, a purine derivative known as an inhibitor of cyclin-dependent kinase (CDK) 2 in cell cycle regulatory proteins, whose chemical structure (ΙΠ) is as follows:

6

Alternative Site (Article 26)

Myosebern not only restrains the cell cycle in the G ₂ / M transit ion by disrupting spindle assembly, but also reverses fully differentiated muscle cells to environmental factors, resulting in muscle regeneration and enjoyable cell differentiation. Can be applied.

 The present invention induces cellularization of the multinucleated skeletal muscle root canal by treating the skeletal muscle fusion described above with a cytolation-inducing agent. More specifically, root canal cultures were treated with 10 uM myosebine, 15 iiM myosevery B, 1 yM colchicine, 1 nocodazole or 10 μΜ Taxol for 24 hours to obtain a cellized root canal. To remove relatively large amounts of intracellular debris in addition to the cells, they were centrifuged after trypsinization and filtered through a 70 m mesh. As a result, treatment with 10 μΜ myoseberg produced the largest number of monocytes (see Figure lc).

 According to a preferred embodiment of the invention, the myoseberg of the invention is treated with 5-50 μΜ, more preferably 5-30 μΜ, even more preferably 5 × 20 μΜ, most preferably 10-15 μΜ do. Step (b): Induce down-regulation of p21 expression

 The differentiated cells are then treated with p21 down-regulatory inducing compounds.

p21 plays an important role in regulating nuclear events due to environmental stress, p21 is induced at very high levels due to oxidative stress and DNA damage. The p21 protein acts as an inhibitor of cyclin-dependent kinases and effectively blocks cell cycle progression, so it is overexpressed in aging or fully differentiated cells. According to the present invention, down-p21 expression Regulation induces myofibrillarization and cell cycle reentry to mimic the early stages of adnexal regeneration of Rhesus amphibians.

 The p21 down-regulatory inducing compounds of the present invention are compounds that induce down-regulation by inhibiting the expression of the p21 gene, inhibiting the activity of the p21 protein or decreasing the stability of the p21 protein.

 According to a preferred embodiment of the present invention, down-regulation of p21 expression of the present invention can be carried out using any method known in the art, for example using antisense oligonucleotides, siRNA, miRNA, or natural product extracts. It may be carried out by, but is not limited thereto.

 The term "siRNA" in the present invention means a nucleic acid molecule capable of mediating RNA interference or gene silencing (see TO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99 / 07409 and W0 00/44914). siRNA is provided as an efficient gene knock-down method or gene therapy method because it can inhibit the expression of the target gene. siRNA was first discovered in plants, worms, fruit flies and parasites, but has recently been used in mammalian cell research by developing / using siRNAs (Degot S, et al. 2002; Degot S, et al. 2004; Ballut L, et al. 2005).

 The siRNA molecule of the present invention may have a structure in which a sense strand (corresponding sequence corresponding to a p21 m NA sequence) and an antisense strand (a sequence complementary to a p21 mRNA sequence) are positioned opposite to each other to form a double strand. Further, according to another embodiment, siRNA molecules of the present invention may have a single chain structure having self-complementary sense and antisense strands.

 siRNAs are not limited to fully paired double-stranded RNA portions of RNA, but are paired by mismatches (bases not complementary), bulges (no bases on one chain), and the like. May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases.

The siRNA terminal structure is capable of both blunt terminal black and cohesive terminal as long as the expression of the p21 gene can be inhibited by the R Ai effect. Cohesive end structures are possible with both 3'-end protrusion structures and 5'-end protrusion structures. The siRNA molecules of the present invention may have a form in which a short nucleotide sequence (eg, about 5-15 nt) is inserted into a self-complementary sense and antisense strand, wherein the siRNA molecule formed by expression of the nucleotide sequence is a molecule Due to the internalization, the hairpin structure is formed, and the stem-and-loop structure as a whole is formed. This stem-and-loop structure can be processed in vitro or in ^ to produce an active siRNA molecule capable of mediating RNAi.

As used herein, the term "antisense oligonucleotide" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, and binds to a complementary sequence in the mRNA to inhibit translation of the mRNA into a protein. It works. An antisense sequence of the invention refers to a DNA or RNA sequence that is complementary to _P 21 and capable of binding to p21 mRNA, and is responsible for the translation of p21 mRNA, translocation into the cytoplasm, maturation or any other overall biological function. May inhibit essential activity. The antisense nucleic acid has a length of 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases. As used herein, the term “miRNA (microRNA)” refers to a single-stranded RNA molecule of 21-25 nucleotides, and is a modulator that controls gene expression in eukaryotes through inhibition of the disruption or translation of target mRNAs. This miRNA consists of two stages of processing. The first miRNA transcript (primary miRNA) is made into the stem-loop structure of 70-90 bases, or pre-miRNA, by the RNaseEI type enzyme called Drosha in the nucleus, and then migrates into the cytoplasm and is called Dicer. Cleaved to make up to 21-25 bases of mature miRNA. The miRNA thus produced complementarily binds to the target mRNA and acts as a post-transcript ional gene suppressor, inducing translation inhibition and mRNA destabilization. miRNAs are involved in a variety of physiological phenomena and diseases.

According to a preferred embodiment of the present invention, down-regulation of p21 expression in the present invention induces the proliferation of cellized root canal or re-entry and differentiation into the cell cycle. According to a preferred embodiment of the present invention, the cellized root canal of the present invention maintains its inherent myogenic potential even after down-regulation of p21 expression.

 According to one aspect of the invention, the pluripotent cells obtained by the method of the invention are cells of the same lineage or germ layer as the differentiated cells used in step (a). For example, in the case of using skeletal muscle cells as differentiated cells, the pluripotent cells obtained by the present invention are cells of the same germ cells of skeletal muscle, such as skeletal muscle, bone, dermis, connective tissue, urogenital system, heart, blood (lymph cells), kidney and It is a pluripotent cell that can differentiate into the cells that make up the spleen.

 According to a preferred embodiment of the present invention, the differentiated cells used in step (a) are skeletal muscle cells, and the pluripotent cells obtained in step (b) can be differentiated into skeletal muscle cells, adipocytes or osteocytes. to be.

 According to the preparation method of the present invention, p21 down-regulated differentiated cells are treated with a reverse differentiation agent.

 As used herein, the term “dedifferentiation agent” refers to an agent that promotes the dedifferentiation of one or more cell types, and includes chemicals, nucleic acids, peptides, polypeptides, small organic molecules, antibody antisense oligonucleotides, RNAi constructs. And ribozymes. Various retrodifferentiating agents can induce retrodifferentiation by contacting one or more retrodifferentiating factors with cells in vivo or int ^ for a time sufficient to induce retrodifferentiation in fully differentiated mammalian cells.

 According to a preferred embodiment of the present invention, the reverse differentiation agent which can be used in the present invention is reversin (N6-cyclonucleus-N2- -morpholin-4-yl-phenyl) -9H-purine-2 6-diamine) Trichostatin A, valproic acid, buphenyl, and 5-azacytidine (5_azacyt idine), but are not limited thereto. Most preferably, it is reversin.

Reversin is a 2,6-replaced purine compound with the following chemical structure (IV) and capable of proliferating and re-differentiating myogenic cells (eg, proliferation into bone and fat). And re-differentiation).

 As described above, differentiation from skeletal muscle fusion to myogenic cells by culturing the pluripotent cells of the invention obtained according to the chemically defined approaches of the present invention in chemically well-established culture conditions. Not only was possible but also heterologous differentiation into chondrogenic and adipogenic cells (see Figures 3 to 6).

 According to a preferred embodiment of the present invention, the pluripotent cells of the present invention can differentiate into myogenic lineage, adipogenic lineage or osteogenic lineage.

 Yamanaka and co-workers produced embryonic stem cells similar to embryonic stem cells in 2006 and named them iPSCs (Kazutoshi Takahashi and Shiny a Yamanaka, Cell, 126: 663-676 (2006)). The study provided the basis for producing patient- and disease-specific customized pluripotent pluripotent cells without ethical problems such as embryo destruction or egg donation, but the stability and differentiation ability of established pluripotent stem cells were significantly reduced. It is true. In order to overcome the above problems, it is urgent to develop a method that can increase the stabilization and differentiation capacity of pluripotent stem cells using small molecules (eg, myose verbine of the present invention). Thus, the chemical-based approach of the present invention is important for the development of strategies for inducing limb regeneration in mammals, and a novel approach for the development of iPSCs from fully differentiated afferent tissues. present.

Preferably, the method is followed by treatment of the differentiation inducing agent to the cells obtained in step (b) after treatment with the dedifferentiation agent of step (b) to another cell lineage of the same three germ lineage as the differentiated cells of step (a). And further comprising the step of differentiating. The three germ layers are composed of mesoderm mesoderm and ectoderm, endothelial cells in the stomach, colon, liver pancreas, bladder, the inside of the urethra, the epithelial part of the airway, lung, pharynx, thyroid gland, parathyroid gland and small intestine tissues, The mesoderm develops the cells that make up skeletal muscle, bone, dermis, connective tissue, urogenital system, heart, blood (lymph cells), kidneys and spleen. Pigment cells, head connective tissues, epidermis, hair and cells that make up the mammary gland develop.

 As used herein, the term 'differentiation inducing agent' refers to growth factors, hormones, and the like included in the culture medium to differentiate into the desired cells.

 According to a preferred embodiment, the differentiation inducing agent used to obtain the lipogenic cell lineage from the pluripotent pleasant cells obtained in step (b) is inslin, texamethasone, rosiglitazone,

Isobutylmethylxanthine (IBMX) or a combination thereof.

 According to a preferred embodiment, the differentiation inducer used to obtain osteogenic cell lineage from the pluripotent stem cells obtained in step (b) is ascorbic acid-2-phosphate, dexamethasone, β-glycerophosphate or a combination thereof. .

 According to a preferred embodiment of the present invention, the pluripotent cells of the present invention can differentiate into myogenic lineage, adipogenic lineage or osteogenic lineage. According to another aspect of the invention, the invention provides a pluripotent cell prepared according to the method described above.

 It is possible to provide a composition for treating myopathy comprising muscle forming cell lineage or muscle cells differentiated from pluripotent cells prepared according to the method of the present invention as an active ingredient.

In the present specification, the term 'muscle disease' refers to muscle fatigue, muscular dystrophy, accidental or surgical wounds or muscular dystrophy, and the term 'muscular dystrophy' refers to Becker's muscul r dystrophy, congenital muscular dystrophy (congenital) muscular dystrophy), Duc ^' henne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facial shoulder dystrophic muscular dystrophy, muscular dystrophy, muscular dystrophy Muscular dystrophy, myotonic muscular dystrophy, sarcoglycanism, spinal muscular atrophy or Brown-Vial etto-Van Laere syndrome (BL).

 It is possible to provide a composition for treating bone diseases comprising osteogenic cell lineage differentiated from pluripotent cells prepared according to the method of the present invention as an active ingredient.

As used herein, the term 'bone disease' refers to osteoporosis, juvenile osteoporosis, osteogenic imperfecta, hyperosclerosis, hypercalcemia, hyperparathyroidism, osteomalacia, soluble bone disease, osteonecrosis, Paget's disease of bone, bone development Diseases, bone fractures, bone loss due to rheumatoid arthritis, inflammatory rheumatoid arthritis, osteomyelitis, metastatic bone disease, periodontal bone loss, rickets, bone loss by cancer or senile loss of bone. The composition of the present invention comprises ( _a ) a pharmaceutically effective amount of cells differentiated from the pluripotent or pluripotent cells described above (eg, myogenic cell groups, osteogenic cell groups); And (b) a pharmaceutically acceptable carrier.

 The term "pharmaceutically effective amount" as used herein means an amount sufficient to achieve the efficacy or activity of cells differentiated from the pluripotent cells described above.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in the preparation, lactose, dextrose, sucrose, sorbbi, manny, starch, acacia rubber, phosphate chame, alginate, Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyridone, cellulose, water, syrup, metal cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Including, but not limited to. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. in addition to the above components. have. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention may be administered parenterally, for example, by subcutaneous injection, intramuscular injection, transdermal administration, intraarticular injection, and the like. 5 Appropriate dosages of the pharmaceutical compositions of the present invention will vary depending on factors such as formulation method, mode of administration, age, weight, sex, morbidity of food, time of administration, route of administration, rate of excretion and response sensitivity. Can be prescribed. Typical dosages of the pharmaceutical compositions of the invention are 10 ² -10 ¹⁰ cells per day on an adult basis.

 10 The pharmaceutical composition of the present invention is known in the art

— Manufactured in unit dose form or incorporated into a multi-dose container by formulating with pharmaceutically acceptable carriers and / or excipients—according to how conventionally readily available ^ones can be practiced. Can be prepared. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oil or aqueous media, or may be in the form of axes, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers. According to another aspect of the invention, the invention provides a method of regeneration 20 of a mammalian tissue or organ comprising the following steps:

 (a) obtaining differentiated cells from a mammal;

 (b) treating the differentiated cells with p21 down-regulation followed by dedifferentiation agent to obtain pluripotent cells; And

 25 (C) differentiating the pluripotent cells into the desired tissue or organ.

 Since the method of the present invention uses the above manufacturing method, the common content between the two is omitted in order to avoid excessive complexity of the present specification,

Step 30 (c): Differentiate pluripotent cells into desired tissues or organs The pluripotent cells obtained in step (b) are then differentiated into the desired tissues or organs. ·

 The differentiation is performed by adding a differentiation inducing agent of a desired tissue or organ to the culture medium of the pluripotent cells obtained in step (b).

【Effects of the Invention】

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

 (a) The present invention relates to a method for chemical preparation of muscle-derived pluripotent cells and uses thereof.

 (b) The method of the present invention obtains pluripotent mononuclear cells or cells proliferating from differentiated mammalian skeletal muscle through continuous treatment of small molecules and transient p21 suppression, followed by stepwise treatment of reversin. It is a very simple and efficient way of obtaining cells.

 (c) The pluripotent cells of the present invention are capable of heterodifferentiation into adipogenic or bone forming cell groups as well as dedifferentiation into muscle forming cell groups.

 (d) Therefore, the chemical-based approach of the present invention is important for the development of strategies for inducing limb regeneration in mammals, and is novel for the development of iPSCs from fully differentiated afferent tissue. Present your approach.

[Brief Description of Drawings]

1 is a result showing the effects of AraC, 5-FU, and myoserine on C2C12 myoblasts. FIG. La is a cell fusion index of the culture medium that differentiates after 50 μM AraC for 72 hours in differentiating C2C12 myoblast culture. increase the fusion index (ie, the number of cell nuclei inserted into the multinucleated root canal fusion). Treatment of different C2C12 myoblast cultures with another anti-mitotic drug 5-FI 10 μΜ) for 72 hours did not cause an increase in the cell fusion index. Error bars = standard deviation; *, Ρ = <0.05 compared to untreated differentiated C2C12 culture. The data is representative of three independent experiments. FIG. Lb shows the differentiation of 50 μΜ AraC for 72 hours in differentiating C2C12 myoblast cultures. This results in increasing the twitch count of the culture (ie, root canal causing spontaneous contraction at least one rate per second). Treatment of 10 μΜ 5-FU in differentiated C2C12 myoblast cultures for 72 hours did not cause an increase in twitch counts. Error bars = standard deviation; *, Compared to untreated differentiated C2C12 culture = <0.05. The data is representative of three independent experiments. FIG. Lc is the result showing that root canal culture causes cellularization by treatment of 10 μM myosebergin, 15 μM myosebergin B, 1 yM colchicine, 1 μM nocodazole or 10 μM taxol for 24 hours. Living cells were classified as cells not stained with trypan blue die. Myosevery has been found to have the least cytotoxicity and induce cellularization. -Error bars 편 standard deviation; *, Ρ = <0.05 compared with colchicine treatment **, Δ = <0.05;#, compared with myocerine treated β and Ρ = <0.05 compared with treatment with nocazoleazole. The data is representative of three independent experiments. 2 shows the effect of ρ21 down-regulation in the cellized root canal. 2A shows a schematic of an experimental protocol for observing the proliferative response of cellized root canals following p21 down-regulation. FIG. 2B is a flow cytometric analysis of PKpropidium iodide staining showing that p21 down-regulation for 48 hours can induce proliferation in cellularized, male muscle fibers. That is, the shift to the G ₂ / M phase of the cell cycle was observed. 2C is a graph showing that p21 down-regulation for 48 hours induces short term cell division of the cellized C2C12 root canal. The data is representative of three independent experiments. FIG. 2D shows the flow cytometry for BrdU insertion in the cellized C2C12 root canal, indicating that p21 downregulation for 48 hours induces DNA synthesis. FIG. 2E shows that treatment of p21 down-regulation or reversin induces differentiation in cellized C2C12 root canal, as a reduced label (fluorescence) of FTTC-α-Burorogroxine, a ligand for acetylcholine receptor Detected. FIG. 2F shows the results of a microplate reader assay that quantifies the signal of FITC-a-Bungaro in cellized C2C12 root canals. Treatment with p21 down-regulation or reversin induced reverse differentiation in the cellized C2C12 root canal. Error bar = Standard Deviation; *, P = <0.05 compared to myoblasts; **, 7 ° = <0.05 compared to C2C12 cells treated with control siRNA. Data is representative of three independent experiments.

 3 is a result showing that p21 down-regulation reverses the EMG decreased by reversin treatment. FIG. 3A shows that reversin treatment reduces the ETP of C2C12 myoblasts and C2C12 cells, resulting in a decrease in cell fusion index after incubation for 10 days in differentiation medium. Error bars = standard deviation; *, P 0.05 compared to untreated myoblasts; **, P = <0.05 compared to C2C12 cells treated with myoserine alone. 3B is a graph showing that p21 knockdown reverses the inhibitory effect of reversin in C2C12 cell water. Error bars = standard deviation; *, P 0.05 compared to C2C12 cells treated with myoserine alone; **, P 0.05 compared to C2C12 cells treated with myoserine and reversin. The data is representative of three independent experiments. 3C is an optical microscopic analysis of muscle formation in C2C12 cultures derived from cellized root canals treated with 50 nM reversin or 50 nM reversin and 80 pmol p21 siRNA. Hemafocillin staining shows that more nuclei are located in the root canal fusions in cultures derived from C2C12 cells treated with p21 siRNA. Exemplary nuclei are indicated by arrows in the continuous tract of the root canal cytoplasm.

4 shows that p21 down-regulation promotes the conversion of cellularized root canal to adipocytes. FIG. 4A shows C2C12 cell water treated with p21 knock-down and reversin and cultured in adipose media can accumulate lipids. As previously described, C2C12 myoblasts treated with reversin and cultured in lipogenic medium also accumulated lipids [15]. However, C2C12 cell water treated with reversin and cultured in lipogenic medium did not accumulate lipid. FIG. 4B is a microscopic analysis of lipid accumulation in C2C12 cells treated with p21 knock-down, reversin and lipogenic cocktails, similar to cytoplasmic staining in C2C12 myoblasts treated with reversin and lipogenic cocktails. Showed. FIG. 4C shows C2C12 cells treated with p21 knock-down, reversin and lipogenesis cocktails after insulin stimulation (adipocyte-specific function). The results show an increased uptake of 6-NBDG, a fluorescent glucose analogue. Replacement of p21 siRNA with control siRNA failed to produce cells capable of ingesting insulin-stimulated glucose. C2C12 myoblasts treated with reversin (500 nM) and lipogenic cocktails also showed increased uptake of 6-NBDG after insulin stimulation. 6-NBDG intake was less than that observed in 3T3-L1 adipocytes after insulin stimulation. Error bars = standard deviation; *, 尸 = <0.05 compared to group treated with 6-NBDG alone. The data is representative of three independent experiments. 4D shows the results of visualizing fluorescence microscopy of 6-NBDG uptake after insulin stimulation. 6-NBDG was found to accumulate in the membranes of C2C12 myoblasts and cell-derived adipocytes. 4E shows C2C12 cells treated with p21 knock-down reversin (500 nM) and an lipogenesis cocktail showed insulin-sensitive free fatty acid release after epinephrine-stimulated (fat cell-specific function). Replacement of p21 siRNA with control siRNA failed to produce cells with the adipocyte-specific function. C2C12 myoblasts treated with reversin (500 nM) and lipogenic cocktails also showed insulin-sensitive free fatty acid release following epinephrine-stimulation. The amount of free fatty acids released and insulin sensitivity were lower than those observed in 3Τ3-ίΓ adipocytes. Error bars = standard deviation; *, P = <0.05 compared to the group treated with epinephrine alone. The data is representative of three independent experiments.

FIG. 5 shows that p21 down-regulation promotes bone formation in cellized root canals. 5A is a graph showing increased alkaline phosphatase activity in C2C12 cell cultures cultured in bone formation conditions after reversin treatment and p21 knock-down. Replacing p21 siRNA with control siRNA resulted in a significant decrease in alkaline phosphatase activity. ATCD5 cells, which were cartilage progenitor cells cultured under osteogenic conditions, were used as positive controls. Figure 5b is added to the cell lysate, and AttoPhos substrate solution ^® picture obtained after 300 seconds. Experimental groups are described in wells (numbers) floating under microplates. FIG. 5C shows Alizarin Red S staining visualizing mineralization in ATDC5 cells and C2C12 cells after incubation for 10 days in osteogenic medium. C2C12 cells treated with reversin (500 nM) and p21 siRNA were 500 nil Reversin induced a degree of mineralization similar to C2C12 myoblasts treated. Substitution of p21 siRNA with control siRNA reduced mineralization. 5D is the semi-quantitative result of mineralization via the color assay. C2C12 cells treated with reversin (500 nM) and p21 siRNA induced mineralization after addition of a bone formation cocktail. The degree of mineralization was similar to C2C12 myoblasts treated with 500 nM reversin and bone formation cocktails. However, the mineralization of these cells was lower than that observed in ATCD5 cells, which are cartilage progenitor cells cultured under the bone formation cocktail. Error bars = standard deviation; *, P = <0.05 compared to C2C12 cell water + bone formation cocktail. Data is the overall mean for three independent experiments.

 FIG. 6 shows a schematic diagram of a chemical approach for pluripotent cells to pluripotency from fully differentiated skeletal muscle tissue. Proliferating myoblasts can be treated with AraC to enhance segmentation into multinucleated root canal fusions. [Specific contents to carry out invention]

 Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . Example

Experimental Materials and Methods

Antibodies and Reagents

Myose verbine, myose verbine B, colchicine, nocodazole, neurodazine, cytosine β-D—arabinofuranoside (AraC), 5-fluorouracil (5-FU), 5-bromo- 2'-deoxyuridine (BrdU), human transferrin, sodium salenite, dexamethasone, 3-isobutyl- 1-methyl xanthine (IBMX) and insulin were purchased from Sigma (M0, USA). Reversin was guipped from Santa Cruz Biotechnology (CA, USA). p21 siRNA and control siRNA were purchased from Santa Cruz Biotechnology (CA, USA), α-bungarotoxin and 6- (Ν— Nitrobenz-2-oxa-1, 3-diazol-4-yl) amino) -6-deoxyglucose (6-NBDG) was purchased from Molecular Probes (OR, USA). Cell culture

5 C2C12 mouse Oros musculus myoblasts contained 10% fetal bovine serum (FBS; Life Technologies, CA, USA), 50 units / mL penicillin (Pen; Life Technologies., CA, USA) and 50 jug / ml strepto Mycin (Strep; Life Technologies., CA, USA) was maintained in a growth medium consisting of DMEM (Dulbecco's Modified Eagle's Medium; Life Technologies., CA, 0 USA) supplemented. Myoblasts are 5% horse serum, --- 50 units / mL ^. Penicillin and -50- ^ g / mL -streptomycin supplemented with DMEM

Differentiation of skeletal muscle into myotubes was induced by incubation for 8 days in the growth medium. Differentiation medium was changed every 48 hours. For the differentiation step, myoblasts were cultured in collagen-coated 6-well 5 tissue culture plates (Sigma, M0, USA). To enhance differentiation and remove contaminants, myoblast cultures were treated with 50 μΜ AraC [26] for 72 hours or 10 μΜ 5-FU [2 phases for 48 hours as previously reported. AraC or 5-FU treatment was performed 18 hours after the addition of differentiation culture medium. To obtain pure root canal culture, trypsinized the differentiated root canal culture and centrifuged at 800 rpm for 10 minutes, then gently resuspend in 10 mL differentiation medium and pass through a 70 m mesh (BD bioscience, CA, USA) Mononuclear cells were isolated. The mesh was then washed with 20 mL differentiation medium to obtain root canal.

3T3-L1 mouse (/ s musculus) embryonic fibroblasts were maintained in proliferation medium consisting of DMEM supplemented with 10% FBS, 505 units / mL penicillin and 50 ^ g / mL straptomycin. Differentiation of 3T3-L1 fibroblasts into adipocytes was induced as follows: Post-confluent cells at 48 hours (considered day 0) were treated with 10% FBS, 0.5 mM 3-isobutyl-1-methyl-xanthine. , 2 // g / mL dexamethasone, 1 // g / mL insulin, 50 units / mL penicillin and 50 / g / mL0 straptomycin were incubated for 2 days in DMEM. After that, the cell Every 2 days incubated in fresh DMEM supplemented with 10% FBS and 1 ^ g / mL insulin. 8 days after induction of differentiation, 3T3-L1 adipocytes were used for the experiment.

ATDC5 cells were provided by Professor Jeon Jang-soo from the School of Life Sciences, Gwangju Institute of Science and Technology. ATDC5 cells were cultured in a maintenance medium consisting of a 1: 1 complex of 5¾> FBS, 10 / g / mL human transferrin and 3Χ1 (DMEM with Γ ⁸ M sodium selenite and Ham's F-12 medium). To induce chondrogenesis, 10 // g / mL bovine insulin was added to the cell culture medium, the batch was changed every 48 hours and chondrocytes were used for the experiment 8 days later.

 P19 mouse embryonic carcinoma cells were provided by Professor Song Mi-Ryung, School of Life Sciences, Gwangju Institute of Technology. As reported previously [28], neurogenesis of P19 cells was performed through cell ball formation and treatment of 500 nM retinoic acid in non-adherent culture dishes. . To inhibit dividing growth into glial-like cells, 50 μΜ AraC was complemented 48 hours after plating in culture medium. Skeletal Muscle Root Canal Cellization

Root canal cultures were treated with 10 μΜ myoserine, 15 μΜ myoserine Β, 1 yM colchicine, 1 μΜ nocodazole or 10 μΜ taxol for 24 hours (treatment as reported in IC ₅₀ mice for root canal degradation). Method [16]). Cellization was found to produce a relatively large amount of intracellular debris in addition to monocytes. Trypsin was treated to the cell monolayer and gently centrifuged for 5 min at 750 rpm. The cell pellet was gently resuspended in 10 mL growth medium and passed through a 70 IM cell strainer (BD Biosciences, CA, USA) to remove debris. To determine the viability of monocytes, the obtained cells were resuspended in DMEM at a density of 1 × 10 ⁶ cells AnL and stained with 0.4% trypan blue die (Sigma ᅳ Aldrich, Mo, USA). 100 cells were counted with a hemocytometer (Mariefeld GmbH, Germany) and cells not stained with trypan blue were considered viable cells. siRNA-mediated gene silencing Mononuclear cells were transfected with 50 pmol of the target siRNA or control siRNA according to the manufacturer's protocol (Santa Cruz Biotechnology, CA, USA). Monocytes 48 hours after transfection were used for the experiment. Quantification of Myoblast Differentiation Using Fusion Fusion Index

 Cell fusion index was calculated as previously reported [29]. Cultures were fixed by successive reactions in 70 (v / v) and 96% (v / v) ethanol and then stained with hemafecillin solution (Sigma, M0, USA) to visualize nuclei. Images of the stained culture were obtained using a CKX41 inverted microscope (Olympus, Japan) and recorded with a DigiEye 330 digital camera (Dewinter, India) and Biowizard 4.3 software (Dewinter, India). The cell fusion index was determined in five microscopic fields and calculated as follows: cell fusion index = number of nuclei located in the skeletal muscle root canal / total number of nuclei. The root canal was limited to a syncytia containing three or more nuclei. Flow cytometer

Analysis of cell cycle state:

Trypsin-treated cells or cells were centrifuged at 1,000 rpm for 5 minutes and then 2.5 × 10 ⁶ cells were resuspended in 1 mL PBS. The cells were carefully transferred to 2.5 mL ethanol via pipetting and fixed by reaction for 15 minutes under ice. Cells were centrifuged at 1,500 rpm for 5 minutes and a PI solution of 500, 50 g / m propidium iodide (Sigma, MO, USA, 0.1 mg / mL RNase A (Sigma, M0, USA), 0.05% Triton After resuspension in X-100 (Sigma MO, USA), the reaction was allowed to react for 40 minutes under ice, after which PBS (3 mL) was treated and the cells were centrifuged at 1,500 rpm for 5 minutes Flow cytometry (Beckman Coulter EPICS). ® XL-MCL), supernatants were removed and cells were resuspended to a density of 1 × 10 ⁶ cells / 500 ≠ PBS complete solution.

Analysis of DNA Synthesis:

The cells or cells were reacted with 10 μΜ BrdU for 24 hours and then obtained with trypsin / EDTA. . Afterwards, cells were allowed to spin at 1,000 rpm for 5 minutes. After centrifugation and washing with PBS, 1 × 10 ⁶ cells were resuspended in 500 ^ 0.5% paraformaldehyde (Sigma, MO, USA) (in PBS). The cells were reacted for 20 minutes under ice, washed once with PBS, and then centrifuged at 1500 rpm for 5 minutes. The obtained cells were resuspended in 1 mL of freshly prepared 3N HCl / 0.5% Tween 20 (Sigma, MO, USA) and allowed to permeate at 25 ^° C. for 20 minutes. The cells were washed and then resuspended in 1 mL of 0.1 M disodium tetraborate (Sigma, MO, USA). Cells were then centrifuged at 1,500 rpm for 5 minutes and washed with FACS buffer (PBS + 0.5% FBS + 5 mM EDTA). Cells were stained by treating them with FACS buffer (500) containing a mouse anti-BrdU antibody (Sigma, MO, USA) conjugated with anti-mouse FITC (1: 100 lime) and reacting for 20 minutes on ice. . Cells were washed twice with FACS complete solution and then resuspended in 500 ιΛ FACS buffer at IX 10 ⁶ cell density. Cells were analyzed by flow cytometry within 20 minutes. Α-Bungarotoxin Labeling of Acetylcholine Receptors (AChRs)

As previously described in [14], and mononuclear cells were banung in particular god 5) C0 _2/95 air, 30 ^° C to the inside of the 20 / g / mL culture medium FITC- conjugated α- Bungah for 30 minutes. Cells were fixed with 2% paraformaldehyde (in PBS) and washed, then mounted with fluorescence mounting medium (Sigma-Aldrich, M0, USA) to fluorescence with MetaMorph 7.5 image capture software (Molecular Devices, CA, USA) Visualization was carried out under a microscope (Olympus 1X81, Japan). The obtained images were analyzed with Photoshop CS4 software (Adobe Systems Incorporated, CA, USA). In order to quantify the alpha-bogaroroxine label, [30] cells were washed with PBS and fluorescence was fluorescence microplate reader (SpectraMax GeminiXS, Molecular Devices, CA, USA; excitation wavelength / fluorescence emission λ: 495 as previously described). nm / 521 nm). De-differentiation of monocytes derived from the cellization of skeletal muscle root canal

The initial report of reversin used a dose of 5 μΜ for 4 days [15]. However, the present inventors have found that the above-mentioned concentration is derived from the root canal by It has been found that in derived monocytes results in a wide range of cytotoxicity (more than 30% of the cell monolayers are destroyed). Thus, to reduce cytotoxicity, mononuclear cells were treated with low concentrations of reversin for a short time (500 nM for 3 days). After 3 days of reaction, reversin was removed and monocytes were induced to undergo adipogenesis, osteogenic or neurogenesis processes (as described below). Induction of Bone Formation in Cellized Skeletal Muscle Root Canal

Induction of bone formation in skeletal muscle root canals that were cellized in reversin-treated myoblasts was performed as previously described [15, 31]. Cells were 10 in DMEM + 10% FBS with 50 βg / mL ascorbic acid-2-phosphate (Sigma, M0, USA), 0.1 μΜ dexamethasone and 10 mM β-glycerophosphate (Sigma, M0, USA) Incubated for days. Then, the cartilage culture conditions in ATDC5 cellular (endochondral) was switch to mimic bone formation: 5% FBS, 10 g / mL of human transferrin, 3X10- ⁸ M sodium saelre nitro and 10 / mL bovine including Insular Lin Incubated for an additional 10 days in a-MEM (mini ¾ m essential medium) under 97% air / 3¾> C0 ₂ . Culture medium was changed every 48 hours. Induction of Adipocyte Formation in Cellized Skeletal Muscle Root Canal

 Induction of adipocyte formation in the cellized skeletal muscle canal was performed as previously described in reversin-treated myoblasts [15, 31]. Cells were incubated for 7 days in DMEM + 10% bovine serum with 1.7 μΜ insulin, 5 μΜ dexamethasone and 0.5 mM isobutyl methyl xanthine. Oil Red 0 Dye

Cell differentiation in adipocytes was assessed by oil red (XSigrna, M0, USA) staining. 3T3-L1 cells were washed with PBS, fixed with 4% paraformaldehyde for 1 hour at room temperature, and then twice with PBS. Washed. Cells were then stained for 5 minutes with 60% isopropanol containing 2% oil red 0 dissolved. Insulin—Stimulated Glucose Uptake Analysis

3T3-L1 adipocytes or muscle-derived adipocytes were dispensed into 24-well plates (BD Bioscience, CA, USA) at a concentration of 2 × 10 ⁵ cells / well. After 48 hours, cells were incubated for 3 hours in serum-free low glucose DMEM. After addition of 100 nM insulin and 100 μΜ 6- (Ν_ (7_nitrobenzium 2-oxa-1,3-diazol-4-yl) amino) -2-deoxyglucose (6—NBDG) to the cells ， Reacted at 37 ^° C for 30 minutes. Cells were then washed with PBS and lysed with 100 μΐ potassium phosphate buffer (ρΗ 10) containing 1% Triton X-100. 50 ≠ DMS0 was added to the dissolved solution, then a portion (120) was transferred to a 96-well microtiter plate (BD Bioscience, CA, USA).-Fluorescence was measured using a fluorescence microplate reader (Spec traMAX GeminiXS and SoftMax Pro). V5 software, Molecular Devices, CA, USA; _ex -466 nm, X _em = 540 nm)

 For microscopic analysis of NBDG uptake, muscle-derived adipocytes were

Aliquots were placed in 6-well plates (BD Bioscience, CA, USA) at a concentration of 2.5 × 10 ⁵ cells / well and incubated for 24 hours. Thereafter, cells were treated with 100 μΜ 6-NBDG together with the desired drug (drug). Cells were washed three times with PBS and fixed with 3.7% (w / v) formaldehyde (in PBS) (Sigma, MO, USA). Cells were washed three times with PBS and then mounted with Fluormount (Sigma, MO, USA). NBDG uptake was immediately visualized using a fluorescence microscope (Olympus 1X81, Japan) equipped with MetaMorph 7.5 image capture software (Molecular Devices, CA, USA). The obtained images were analyzed with Photoshop CS4 software (Adobe Systems Incorporated, CA, USA). Free Fatty Acid Release Assays

According to the previously described method, epinephrine-stimulated free fatty acid (FFA) release from adipocytes was measured using the free fatty acid quantification kit (Biovision, CA, USA). 3T3-L1 adipocytes grown until reaching confluence in 24-well plates were incubated for 3 hours in serum-free culture medium. Then, 10 μΜ dissolved in salt in fat cells Epinephrine (Sigma, MO, USA) was treated to induce FFA release. The desired drug was added 10 minutes before the epinephrine treatment. Cell lysates were obtained and a 50 ^ organic phase was used for the chromogenic assay (VERSA Max microplate reader, Molecular Devices, Calif., USA). Palmitic acid was used to make a standard curve. Alkaline phosphatase assays

Alkaline phosphatase activity was measured using the fluorogenic substrate AttoPhos ^® AP system (Promega, WI, USA). Cells were washed twice with PBS and lysed at 250! Λ Cel Lytic ™ M (Sigma, MO, USA). For the assay, 100 / g / 100 ^ cell lysate was used, with 100 ^ AttoPhos substrate added immediately using a multi-channel pipette. Fluorescence was measured using a microplate reader (SpectraMAX GeminiXS, Molecular Devices, CA, USA; λ _6Χ = 420 nm; X _em = 560 nm). Alizarin Red Dyeing

 Cells were washed once with PBS and fixed for 20 minutes with phosphate-completed formalin (Sigma, M0, USA). The fixed cells were washed once with distilled water and then stained with 1% alizarin red S (Sigma, M0, USA) for 5 minutes dissolved in distilled water. The remaining die was removed with distilled water and the cells washed once more. Finally, after the cells were dried in air, images of the stained cells were obtained under an optical microscope (CKX41, Olympus, Japan) and analyzed by software (Leica). Quantification of mineralization using alizarin red

Alizarin red staining was quantified as described previously [33]. Briefly, after Alizarin Red staining, 800 ^ of 10% (v / v) acetic acid was added to each well and the plates were reacted with stirring for 30 minutes at room temperature. A monolayer was obtained from the plate using a cell scraper and vortexed by transferring to a 1.5-mL microcentr i fuge tube with 10 (v / v) acetic acid. Load 500 μί mineral oil into the slurry and for 10 minutes After heating to 85 ^° C, it cooled rapidly on ice. The slurry was then centrifuged at 20,000 g for 15 minutes and the 500 ^ supernatant was transferred to a new microcentrifuge tube. Add 200 ^ 10% (v / v) ammonium hydroxide to the supernatant and aliquot (150) of the supernatant in three pairs on plates with opaque walls and transparent bottoms in 96-well format at 405 nm. (triplicate) Read. Statistical analysis of the data

 Mann-Whitney U test (TalkStats software; Jelsoft Enterprises Ltd., UK) was used to determine statistical significance. P values below 0.05 (both-tail test) were considered significant. Experiment result and additional discussion

Establishment of Cell Culture Conditions for Induction of Root Canal Cellization: AraC treatment enhances muscle differentiation; Myosevery is a preferred agent in the induction of muscle cellularization

 The list of small molecules used in this study is shown in Table 1. Table 1

The structure of the small molecules used in this study to promote skeletal muscle dedifferentiation and re-differentiation,

In order to effectively study the process of root canal cellization, a high degree of muscle differentiation must be achieved. However, contamination of the culture medium containing undifferentiated myoblasts is a major problem, which results from the cultured root canal culture. Contradictory results have resulted [14, 20]. In order to increase the muscle formation process in C2C12 myoblast cultures, it was tested to remove the myoblasts proliferating phase 2 anti-mitotic drugs AraC [26] and 5-FU [2]. Treatment of AraC during the differentiation process was found to significantly increase the fusion index (indicator la), which is an indicator of the number of nuclei inserted into the root canal (Fig. La). On the other hand, another measure of the muscle formation process is the presence of spontaneous 'twitching' root canals in culture. In addition, AraC treatment on C2C12 cells that differentiate when cells are cultured in collagen-coated dishes increased the presence of spontaneously contracting 'twitching' root canals (FIG. Lb). Therefore, treatment of AraC in this study was used to increase C2C12 differentiation.

 In order to effectively study the root canal cellularization process, an optimal cellularization-inducing agent has been identified. Four other small molecules were tested to induce cellularization with relatively low cytotoxicity: induction of cellularization by colchicine, nocodazole, myosebourne and myosebourne B. myosebourne It was shown to produce the largest number of monocytes, which could rule out 0.4% trypan blue die, a marker for cell viability (FIG. Lc). Thus, myoseburin was used for C2C12 cellization and heterodifferentiation 0 ^ 313 ^ £ ^ 611 ^ first place studies. p21 down-regulation can induce proliferation, re-entry into the cell cycle and differentiation into the cellized root canal:

The scrambled siRNA and p21 siRNA were treated 24 hours after the monocytes generated by C2C12 root canal cell division were dispensed (FIG. 2A). PKpropidium iodide) staining on the cell nuclei showed that p21 down-regulation induced a shift from refractory G ₀ phase to G ₂ -M phase of the cell cycle (FIG. 2B). This effect was also confirmed in counting the number of cells observed at 40 × magnification with p21 down-regulation leading to a small, but significant increase in cell number (FIG. 2C). The above results indicate that p21 down-regulation It was again confirmed by the observation that BrdU, an indicator of increased DNA synthesis, caused an increase in the site of the inserted cells (FIG. 2D).

 Previous studies have shown that myotube cellularization does not cause a change in monocyte differentiation compared to parental myotubes [20]. For example, monocytes do not express late myogenic markers. Keep it. The effect of p21 down-regulation on the differentiated state of monocytes derived from the cellized root canals was evaluated using FITC-conjugated α-bogaroroxine that binds to the acetylcholine receptor, a marker of root canal formation. ρ21 down-regulation reduced the specific markers by α-unga of monocytes (FIGS. 2E and 2F), which shifted from the root canal ('away') to myoblasts ('towards') to differentiation It means that it happened. The effect of p21 down-regulation was similar to that by reversin, which is known to increase the developmental plasticity of muscle cells. The cellized root canal maintains its inherent myogenic potential after p21 down-regulation:

A fascinating feature of limb regeneration in Rhesus amphibians is that early canalization occurs followed by proliferation and differentiation to rebuild muscles in growing limbs [17]. In light of the fact that p21 down-regulation can induce a proliferative response in the transplanted root canal, the ability of monocytes to re-enter myogenic differentiation has been investigated. As a first test, we investigated the effects of reversin on myocyte differentiation, known to increase the developmental plasticity of myocytes [34]. Reversin treatment was found to reduce muscle cells and cellized root canal and ability to re-enter the muscle building process (FIG. 3A). Interestingly, the inhibitory effect of reversin on the muscle formation of cellized monocytes could be overcome by knock-down of p21. Thus, the treatment of p21 induces proliferation of the cellized root canal without a decrease in myoelectric potential. As a result, the root canal showed a higher cell fusion index (p. 3b and 3c) after p21 knock-down. p21 down-regulation promotes the conversion of cellularized root canal to adipocytes:

It is known that reversin induces higher plasticity in myoblasts and then leads to differentiation into adipocytes through cultivation under a cocktail of adiogenic factors [15]. As can be seen from the decrease in oil red 0 staining that binds to lipid droplets accumulating in the adipocytes that differentiate (FIG. 4A), the root canal cellized by control siRNA, reversin and lipogenic factors Cannot differentiate into adipocytes. However, as can be seen in the increased oil red 0 staining (FIG. 4A), down-regulation of p21 in the cellized root canal prior to reversin treatment promoted the conversion to adipogenic lineage. Accumulation of lipids could be observed by light microscopy (FIG. 4B). Ingestion of insulin-stimulated glucose is characteristic of adipogenic cells [ ₃₅ ]. Root canal cellized by reversin and lipogenic factors did not show insulin-stimulated uptake of fluorescence-tagged glucose analogs (FIG. 4C). Moreover, down-regulation of p21 in the root canal that was cellized prior to reversin treatment promoted the conversion of cells to shift glucose after insulin stimulation (FIG. 4C). However, the level of 6-NBDG intake was lower than that observed in the 3T3-L1 adipocyte line. Microscopic analysis showed increased binding of fluorescence-tagged glucose to cells reflecting lipid accumulation, which is characteristic of adipocyte morphology (FIG. 4D). An additional test of insulin sensitivity in adipocytes is to test the inhibition of insulin-mediated free fatty acid release following epinephrine stimulation [36, 37]. Root canal cellized by control siRNA, reversin and lipogenic factors did not show insulin-stimulated inhibition of free fatty acid release after epinephrine stimulation (FIG. 4E). However, downregulation of p21 in the cell root canal prior to reversin treatment promoted the conversion of cells with increased amounts of intracellular free fatty acids to cells with reduced amounts of free fatty acids after exposure to epinephrine (FIG. 4e). Furthermore insulin treatment inhibited the release of epinephrine-stimulated free fatty acids in the cell population, similar to that observed in myoblasts treated with reversin and lipogenic factors (FIG. 4E). However, the degree of fat phase accumulation and release was lower than that observed in the 3T3-L1 adipocyte line. p21 down-regulation promotes bone formation in the cellized root canal:

 Reversin-treated myoblasts can be differentiated into osteoblasts by culturing under a cocktail of osteogenic factors [15]. Gaining alkaline phosphatase activity results in conversion of bone formation in C2C12 myoblasts transfected with regulators of the bone formation process such as Wnt-1-induced secreted protein 1 and Bone morphogenet ic protein 2 Has been used as an indicator [38]. As shown in the absence of alkaline phosphatase activity, culture of C2C12 root canal cellized under osteogenic factors did not induce conversion to osteogenic cells (FIGS. 5A and 5B). However, p21 downregulation in the root canal treated with a cocktail of reversin and osteogenic factors after cellularization promoted differentiation into cells with markedly increased alkaline phosphatase activity (FIGS. 5A and 5B). Beating Although alkaline phosphatase activity was shown to be relatively small but significantly increased compared to untreated C2C12 cells in the experimental group of the present study, the acquisition of the alkaline phosphatase activity was achieved by control of the cultivated root canal treated with scrambled siRNA. It is even more markedly increased than the activity seen in.

Mineralization is a feature of ossification that occurs during bone formation [33]. Calcification (calci f icat ion) was confirmed by alizarin Red S staining in C2C12 cells after p21 knock-down, reversin treatment and incubation in osteogenic medium. ATDC5 cells induced to cause endochondral bone formation were used as controls. Optical microscopy analysis showed that mineralization occurred in the cell water after incubation in p21 knock-down, reversin treatment and bone formation medium (FIG. 5C). Mineralization was quantified by microplate reader-based assays (FIG. 5D) [33]. C2C12 cells after culture in _P 21 knock-down, reversin treatment and bone formation medium showed an increase in mineralization to a similar extent as C2C12 myoblasts cultured under the same conditions. Mineralization levels were lower than observed in ATDC5 cells after osteogenic culture. Moreover, C2C12 cells treated with control siRNA instead of p21 siRNA resulted in a significantly reduced level of mineralization after bone formation culture.

The results presented in this study are the first description of a chemical-based protocol that mimics the early stages of limb regeneration in amphibians. Previously Experimental approaches have been developed to induce adult cells to form into pluripotent or stem cells. Examples include somatic cell nuclear transfer and transfection of transcription factors associated with omnipotence [45]. However, the above approaches did not utilize fully differentiated adult cells, which accounted for the majority of the eel hilar population in humans [46]. In addition, the above approaches currently face technical problems such as low efficiency. The chemical approach to obtaining pluripotency described in the present invention can de-differentiate and cell lineage conversion ^-in skeletal muscle tissue, which is considered to have a very high atrophy [22].

We considered the cellularization of the multinucleated skeletal muscle fusion as the first and most important step in the induction of pluripotency, since similar phenomena occur in chylotomy skeletal muscle dedifferentiation and subsequent limb cleavage [47]. Our comparisons to other compounds that induce skeletal muscle cellization are useful because the effects of chemicals vary from tissue to tissue [22, 39, 40]. An optimized protocol for differentiation of myoblasts (FIG. 2A) was used to find that the chemical myosevers were more effective at producing live mononuclear cells from muscle syncytia. Although this study was the first report of myoseberge activity in myotubes derived from myoblasts treated with anti-mitotic agents AraC or 5-FU, the relatively low cytotoxicity of myoseberge was reported [14]. ] Cells derived from myosin-treated muscle fusions were still found to have atrophy (FIGS. 2B and 2C). The above findings are consistent with the reports by Duckmanton et al. [2] and in contrast to the initial reports of myosebergine activity [14, 16], suggesting that there is a need to obtain high-purity root canals before induction of cellularization. It means. Knock-down of p21, a cyclin-dependent kinase inhibitor (CDKN), induced proliferative reaction in the cellized root canal (FIGS. 2B-2D), which again reiterated the recent finding that p21 is a key regulator of final differentiation in skeletal muscle [24]. Check it once. Combination of myoserine and p21 knock-down resulted in myofiber cellular and cell cycle re- Mammalian muscles are induced to mimic the critical early stages of appendage regeneration in Rhesus amphibians such as entry [21, 41]. Recent studies on adnexal regeneration in Rhesus amphibians show that muscle cells form a group of progenitor cells that later re-differentiate into new muscle tissue. In other words, there is no contribution of other tissue types in the regenerating limb [41]. The study confirmed that muscle cells obtained by treatment of small molecules can also be re-differentiated into muscle myotubes. The inhibitory effect of reversin treatment on re-differentiation may be associated with down-regulation of myogenic regulator expression [42]. Thus, we believe that the action of p21 knock-down on the expression of anabolic regulators is a faint area to be investigated later, which p21 knock-down reverses the inhibitory effect of reversin on re-differentiation. (FIG. 3B and FIG. 3C). Moreover, muscle cells treated with p21 knock-down and reversin induced the formation of progenitor cells that could have various fates (FIGS. 4 and 5). Similar situations are also found in dermal-derived fibroblasts. It occurs in the regenerate of chylopods known to induce the formation of progenitor cells that form cartilage, connective tissue and tendons. Thus, the chemical approach described in this study allows mammalian adult skeletal muscle to have a greater degree of pluripotency than those found in the natural example of adnexal regeneration. A schematic diagram of the chemical approach of the present invention is presented in FIG. 6.

The ability of reversin-treated myoblasts to re-differentiate into adipocytes and osteocytes has been previously reported [34, 42]. However, our data indicate that the above findings should be interpreted with caution. Previous studies of reversin identified phenotypic markers such as expression of lineage-specific markers or lipid accumulation during adipocyte formation. This study attempted to monitor adipocyte and bone cell formation and compare them with known cell types using more powerful and functional assays. For example, we have found that reversin-treated muscle cells accumulate lipids. However, their functional 'performance' is significantly reduced compared to lineage-specific adipocytes (FIGS. 4C and 4E). In reversin-treated muscle cells It may be possible to additionally 'push' the adipocyte-forming process by modifying the cocktail of lipogenic factors, e.g. to thiazolidine, which activates the nuclear chamber adipogenic factor, peroxysome growth factor-activated receptor. Treatment of triazolidinediones [48].

The chemically defined method of the present invention for producing pluripotent cells from differentiated muscle has numerous viable fields for future research. For example, it has been reported that myoblasts can form neuronal cells by reversin treatment and culture in neuronal differentiation medium [43]. Similar 'jumps' from mesodermal lineage cells (eg, adipocytes and bone cells) to neuroectodermal lineage cells could be possible in cellized muscle fibers. Optionally… Muscle biopsies can be performed according to the chemical-based protocols described in this study in an attempt to produce patient-specific pluripotent cells. For example, lateral myoskeletal muscle strips can be obtained from a patient under local anesthesia. [44L skeletal muscle is a very attractive tissue form for the approach of the present invention because of the residual muscle precursor eel Is This is because by activating this function, the lost temper can be reproduced [44]. Furthermore, the chemical approach described in this study mimics the early events of adnexal regeneration of Rhesus amphibians, and demonstrates the present invention in mammalian models of wound healing in attempts to obtain functional progenitor cells prior to signs of inflammation and wound closure. It may be more desirable to apply the method. Thus, the results of this study are closely related to the development of a method for inducing limb regeneration in mammals and suggest a future approach for the development of omnipotent stem cells derived from afferent, fully differentiated mammalian tissue. present. Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof. references

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Claims

[Patent Claims]

[Claim 1]

 Chemical preparation of pluripotent cells (mult i potent or pluri otent eel Is) from mammalian differentiated cells comprising the following steps:

 (a) obtaining differentiated cells from a mammal; And

 (b) treating the differentiated cells with a dedifferentiation agent after down-regulation induction of p21 expression to obtain pluripotent cells.

[Claim 2]

 The method of claim 1, wherein the differentiated cells are terminally differentiated cells.

[Claim 3]

 The method of claim 1, wherein the differentiated cells are one cell selected from the group consisting of skeletal muscle cells, cardiomyocytes, smooth muscle cells, skin cells, chondrocytes, adipocytes and osteocytes.

[Claim 4]

 The method of claim 1, wherein the differentiated cells are connective tissue, nerve cells, lymph cells, vasculature cells, kidney cells, pancreas cells, lung cells, urethral cells, bladder cells, gastric cells, liver cells, small intestine cells, colon cells and esophagus And one cell selected from the group consisting of cells.

[Claim 5]

The method according to claim 1, wherein step (a) comprises the step of a cell-derived agent in the differentiated fusion to segment the syncytia derived from the muscle cells of the mammal and to obtain mononuclear cells or eel lulates. Method characterized in that carried out by processing the cellularization-inducing agent.

[Claim 6]

 6. The method of claim 5, wherein said cellularization-inducing agent is myoseverin, myoseverin B, colchicine or nocodazole.

[Claim 7]

 7. The method of claim 6, wherein said cellularization-inducing agent is myoseborn.

[Claim 8]

The method of claim 1, wherein down-regulation of p21 expression is performed using antisense oligonucleotides, siRNAs or miRNAs.

[Claim 9]

 The process of claim 1 wherein step (b), said derivatizer comprises reversine;

2- (4-mor pho 1 i noan i 1 i no) -6 ^to cyc 1 ohexy 1 am i nopur i ne) ¾.

[Claim 10]

 The method of claim 1, wherein down-regulation of p21 expression induces proliferation of cellularized root canals, re-entry and differentiation into the cell cycle.

[Claim 11]

 The method of claim 1, wherein the pluripotent cells are capable of differentiating into myogenic lineage, adipogenic lineage, or osteogenic lineage.

[Claim 12]

The method according to claim 1, wherein the method is followed by treatment of a differentiation inducing agent to the cells obtained in step (b) after treatment with the reverse differentiation agent of step (b), to be identical to the differentiated cells of step (a). 3 differentiating into other cell lineages of the germ line.

[Claim 13]

 The method of claim 3, wherein the differentiated cells are skeletal muscle cells.

[Claim 14]

 Pluripotent cells prepared according to the method of claim 1.

[Claim 15]

 Regeneration of a mammalian tissue or organ comprising the following steps:

 (a) obtaining differentiated cells from a mammal;

(b) treating the differentiated cells with _P 21 down-regulation induction and then treating with a differentiation agent ^ 6 (^ £ ^^ 11 1011 agent) to obtain pluripotent cells; And

 (c) differentiating said pluripotent cells into a desired tissue or organ.