KR20130050040A - Methods for preparing multipotent cells - Google Patents

Abstract

PURPOSE: A method for preparing multipotent cells is provided to obtain myogenic lineage, adipogenic lineage, osteogenic lineage, or neurogenic lineage. CONSTITUTION: A method for preparing multipotent cells comprises: a step of obtaining differentiated cells from mammal; a step of treating the differentiated cells with a p21 downregulation inducing compound and preparing the multipotent cells. The differentiated cells are terminally differentiated cells and are selected from the group consisting of skeletal monocytes, cardiac muscle cells, smooth muscle cells, skin cells, chondrocytes, adipocytes, and bone cells.

Description

Method for preparing pluripotent cells {Methods for Preparing Multipotent Cells}

The present invention relates to a method for chemical preparation of pluripotent cells and their use.

Dedifferentiation is a phenomenon in which differentiated cells acquire 'stem cells'-like or multipotent states before they differentiate into one or more different tissue forms (1). Phenotype-based screening methods of small molecule libraries were performed to discover new regulators involved in intracellular dedifferentiation (2). Cell manipulation with small molecules has significant advantages over genetic approaches (3). For example, small molecules provide a high level of technology to temporarily regulate the function of proteins. In addition, a single small molecule has the potential to simultaneously control multiple specific targets in a cell.

European amphibians (urodele amphibians) have excellent reverse differentiation and tissue regeneration (4). For example, the whole can be regenerated after limb cutting. Moreover, this regenerative capacity is maintained throughout the animal's life (5). By studying these phenomena, one can develop an approach to improve human tissue regeneration by understanding the molecular mechanism of regeneration.

In the early stages of limb regeneration in R. amphibians, de-differentiation of skeletal muscle fibers occurs around the cleavage (6). The fiber fusion (syncytia) is cellularized to proliferate into mononuclear cells that make up the mesenchymal growth zone called blastema. Acet is essential in multipotent cells to regenerate lost limbs. However, the innate ability to regenerate these tissues is reduced in taxonomic 'high' vertebrates (7).

Other experimental approaches have been taken to induce reverse differentiation of mammalian skeletal muscle. For example, after cell cycle arrest or apoptosis in mouse myotubes, viral oncogenes were expressed to induce cellization and proliferation (8). However, some of these dedifferentiated cells show multipotency and can be re-differentiated into cells from mesenchmal lineage (8). Re-differentiation of mouse root canal can be achieved by incubating with extracts of Rhizoite amphibian limb regeneration, but cells do not have intrinsic capacity (9). Homeobox, composed of transcription inhibitory factors, and induced expression of msx1 (expressed in early body and mouse limb development) showed root canal differentiation and transdifferentiation into alternative mesenchymal systems (9, 10).

However, this approach relies on the introduction of genetic factors into the muscle nucleus, so its potential as a treatment for promoting tissue regeneration is limited.

The most recent approach to inducing root canal differentiation is an siRNA-based strategy that targets specific genes to maintain terminal differentiation. In a recent study, we performed root canal differentiation by combining the knockout of p21 (CDKN 1A, CIP1), a low-molecular and cyclin-dependent kinase inhibitor that induces canal cellularization and multidifferentiation. 11). Knockout of cyclin-dependent kinase inhibitor 2A (CDKN2A, ARF) and retinoblastoma protein (RB1, OSRC) induces cell cycle reentry in differentiated root canal nuclei, upregulates cytoplasmic machinery, and causes loss of differentiation ability (12). However, siRNA-based approaches can induce non-specific cytotoxicity and its occurrence, which may be more problematic compared to small molecule drugs as therapeutic agents (13, 14).

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 inventors have tried to produce pluripotent cells. As a result, when the differentiated cells were treated with p21 down-regulatory inducing compounds, pluripotent cells were obtained and their differentiation capacity (e.g., redifferentiation into adipose cell group, osteogenic cell group, myogenic cell group or neuronal cell group) was obtained. By confirming, this invention was completed.

Accordingly, it is an object of the present invention to provide a method for chemical preparation of multipotent or pluripotent cells.

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

Another object of the present invention is to provide pluripotent cells prepared according to the method of the present invention.

Another object of the present invention is to provide a method for regeneration of a mammalian tissue or organ.

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 invention, the invention provides a method for chemical preparation of multipotent cells from mammalian differentiated cells comprising the following steps:

(a) obtaining differentiated cells from a mammal; And

(b) treating the differentiated cells with p21 down-regulation inducing compound to obtain pluripotent cells.

The inventors have tried to produce pluripotent cells. As a result, when treated with p21 down-regulatory inducing compounds in differentiated cells, pluripotent cells were obtained and their differentiation capacity (e.g., reverse differentiation of adipose cell group, osteogenic cell group, myogenic cell group or neuronal cell group) was obtained. Confirmed.

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.

In the present specification, the term "terminal differentiated cell" refers to a mature cell having a specialized phenotype as a cell at an endpoint stage during the development process. Differentiated cells have one or more features or functions that distinguish them from other cell types and have 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 into connective tissue, nerve cells, lymph cells, vessels, kidney cells, pancreatic 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:

Step (b): Treatment of p21 Down-Regulatory Inducing Compounds to Obtain Pluripotent Cells

The differentiated cells are then treated with the p21 down-regulation inducing compound.

p21 plays an important role in regulating nuclear events due to environmental stress. p21 is induced at very high levels following 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-regulation of p21 expression induces myofiber cellularization and cell cycle re-entry to mimic the early stages of appendage regeneration of Rhesus amphibians.

The p21 down-regulation 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 reducing the stability of the p21 protein.

Preferably, the p21 down-regulation inducing compound has four kinds of compounds as follows:

First, the p21 down-regulation inducer compound used in step (b) is a compound that down-regulates p21 and inhibits the activity of glycogen synthase-3 kinase protein. Preferably, the compound corresponding to the first group is BIO (6-bromoindirubin-3'-oxaim), 3F8 (5-ethyl-7,8-dimethoxy-1H-pyrrolo [3, 4-c] isoquinoline-1,3 (2H) -dione) and AR-A 014418 (N-[(4-methoxyphenyl) methyl] -N '-(5-nitro-2-diazoyl) urea) It is one compound selected from the group consisting of. Most preferably, the compound corresponding to the first group is BIO. The chemical structure of BIO is as follows:

(Ⅰ)

(Ⅰ)

Second, the p21 down-regulatory inducing compound used in step (b) is a compound that downregulates p21 and inhibits the p38 Mitogen-Activated Protein kinase pathway. Preferably, the compound of the second group is CMPD-1 (2'-fluoro-N- (4-hydroxyphenyl)-[1,1'-biphenyl] -4-butanamide), EO 1428 ((2-methylphenyl)-[4-[(2-amino-4-bromophenyl) amino] -2-chlorophenyl] methanone) and SB203580 (4- [4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -1H-imidazol-5-yl] pyridine), and is most preferably SB203580. The chemical structure of SB203580 is as follows:

(Ⅱ)

(Ⅱ)

Third, the p21 down-regulation inducing compound used in step (b) is a compound that downregulates p21 and inhibits adenylyl cyclase. Preferably, the compound of the third group is BPIPP (5- (3-bromophenyl) -5,11-dihydro-1,3-dimethyl-1H-indeol [2 ', 1': 5 , 6] pyrido [2,3-d] pyrimidine-2,4,6 (3H) -trione), KH7 ((±) -2- (1H-benzimidazol-2-yldio) propanoic Acid 2-[(5-bromo-2-hydroxyphenyl) methylene] hydrazide) and SQ22536 (9- (tetrahydro-2-furanyl) -9H-purin-6-amine) One compound of choice, most preferably SQ22536. The chemical structure of SQ22536 is as follows:

(Ⅲ)

(Ⅲ)

Fourth, the p21 down-regulation inducing compound used in step (b) is a compound that down-regulates p21 and activates G-protein-coupled receptor-mediated signaling. Preferably it is LPA (Lysophosphatidic acid). The chemical formula of LPA is as follows:

(Ⅳ)

(Ⅳ)

More preferably, the p21 down-regulation inducing compound is IO (6-bromoindirubin-3'-oxaim), SB203580 (4- [4- (4-fluorophenyl) -2- (4-methyl Sulfinylphenyl) -1H-imidazol-5-yl] pyridine), SQ22536 (9- (tetrahydro-2-furanyl) -9H-purin-6-amine) or LPA (Lysophosphatidic acid) It is one compound.

According to a preferred embodiment of the present invention, down-regulation of p21 expression in the present invention induces proliferation of cellized root canal, re-entry and differentiation into the cell cycle.

According to a preferred embodiment of the invention, the intrinsic myogenic potential is maintained even after down-regulation of p21 expression.

As described above, the present invention can obtain pluripotent cells from differentiated cells through a very simple process.

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, when 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) are pluripotent cells capable of differentiating into skeletal muscle cells, adipocytes or bone cells. to be.

On the other hand, according to the present invention, it is very interesting to obtain pluripotent cells through simple treatment from differentiated cells. Preferably, after treatment with the p21 down-regulation inducing compound of step (b), the method further comprises the step of treating the cells obtained in step (b) to obtain pluripotent cells, wherein the pluripotent cells are prepared in step have the ability to differentiate into differentiated cells of (a) and cells of other germ lineages.

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, antibodies, antisense oligonucleotides, RNAi cons Includes truck and ribozymes. Various reverse differentiation agents can induce reverse differentiation by contacting the cells in vivo or in vitro with one or more reverse differentiation factors for a time sufficient to induce reverse differentiation in fully differentiated mammalian cells.

The three germ layers are composed of endoderm, mesoderm, and ectoderm, and in endoderm, cells constituting the stomach, colon, liver, pancreas, bladder, inner side of the urethra, epithelial part of the airway, lungs, pharynx, thyroid, parathyroid and small intestine tissues are generated. In mesoderm, cells that make up skeletal muscle, bone, dermis, connective tissue, genitourinary system, heart, blood (lymph cells), kidney and spleen are developed. In ectoderm, the mesoderm nervous system, lens of the eye, skull and sensory organs, ganglion and The cells that make up nerves, pigment cells, head connective tissues, epidermis, hair and mammary gland develop.

According to the method of the present invention, the differentiated cells of step (a) can be differentiated into other trioderm lineages following the treatment of the p21 down-regulation inducing compound followed by treatment of the dedifferentiating agent.

According to a preferred embodiment, C2C12 cells derived from skeletal muscle are characterized by differentiation into pluripotent cells by differentiation into neurons when cultured with reversin, retinoic acid and N2 additives after the low molecule treatment of the present invention.

More preferably, the derivatizer is reversin (N6-cyclohexyl-N2- (4-morpholin-4-yl-phenyl) -9H-purine-2,6-diamine), trichostatin A ), Valproic acid, buphenyl and 5-azacytidine.

Preferably, after treatment with the p21 down-regulatory inducer compound of step (b) of the present invention, the differentiation inducing agent is treated with the cells obtained in step (b) to form other cell lineages of the same triplet germ line as the differentiated cells of step (b) ( cell lineage).

In the present specification, 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 stem cells obtained in step (b) is insulin, dexamethasone, rosiglitazone, isobutylmethylxanthine (IBMX) or mixtures thereof.

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

According to a preferred embodiment of the invention, the pluripotent or pluripotent cells of the invention are myogenic lineage (myogenic lineage), adipogenic cell lineage (adipogenic lineage), osteogenic lineage (osteogenic lineage) or neurogenic cell lineage ( neurogenic lineage).

According to another aspect of the invention, the invention provides a pluripotent cell prepared according to the method of the invention.

According to another aspect of the invention, the invention provides a pluripotent cell prepared according to the method of the invention.

It is possible to provide a composition for treating myopathy comprising muscle forming cell lineage or muscle cells differentiated from pluripotent or 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, injury or muscular dystrophy caused by accident or surgery, and the term 'muscular dystrophy' refers to Becker's muscular dystrophy, congenital muscular dystrophy, and Duchenne muscular dystrophy. (Duchenne muscular dystrophy), distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facial shoulder dystrophy, dystrophic muscular dystrophy, myotonic muscular dystrophy, oropharyngeal dystrophy, muscular dystrophy Oral complications, spinal muscular atrophy or Brown-Vialetto-Van Laere syndrome (BVVL).

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, 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.

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

As used herein, the term 'neuropathy' is a neurological disease resulting from physical, metabolic, toxic, chemical or immunological damage of the central nervous system, preferably amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, traumatic brain injury, Reversible or metabolic encephalopathy, stroke, ischemic encephalopathy, hepatic encephalopathy and hypoxia, epilepsy, head trauma, Parkinson's disease, Alzheimer's disease, Huntington's disease, tertiary neuralgia, Yeti neuralgia, bell palsy, severe sclerosis, muscular dystrophy, progressive Peripheral neuropathy caused by muscular atrophy, progressive atrophy-induced muscle atrophy, herniated, destroyed or escaped invertebrate disc syndrome, cervical spondylosis, congestive disorder, thoracic outlet destruction syndrome, lead, dap, teak, porphyrinosis or Guillain-Barré syndrome It includes, but is not limited to.

The compositions of the present invention comprise (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” 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 compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. 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.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Typical dosages of the pharmaceutical compositions of the invention are 10 ² -10 ¹⁰ cells per day on an adult basis.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, 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 of a mammalian tissue or organ comprising the following steps:

(a) obtaining differentiated cells from a mammal;

(b) treating the differentiated cells with a p21 down-regulatory inducing compound to obtain pluripotent cells; And

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

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

Step (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).

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

(a) The present invention provides a method for producing pluripotent or pluripotent cells using p21 down-regulation inducing compounds.

(b) myogenic lineage, adipogenic lineage, osteogenic lineage or neurogenic cell lineage through the pluripotent or pluripotent cell preparation method of the present invention. Can be provided.

(c) The chemical-based approach of the present invention is important for the development of strategies for inducing limb regeneration in mammals and suggests a new approach for the development of pluripotent or pluripotent cells from terminal differentiated cells.

Figure 1 shows the structure of the small molecules used for skeletal muscle dedifferentiation or differentiation (in alphabetical order).
Figure 2a shows a schematic diagram of the experimental procedure to investigate the cell proliferation by treating small molecules in the C2C12 differentiated mouse root canal. Figure 2b shows 2.5 μM BIO (6-bromoindirubin-3'-oxime), 10 μM SB203580 (4- [4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -1H-imidazol-5 in C2C12 muscle cells -IT] pyridine), 300 μM SQ22536 (9- (Tetrahydro-2-furanyl) -9H-purin-6-amine) or 30 μM Lysophosphatidic acid (LPA) for 48 hours to induce DNA synthesis -BrdU signal was confirmed to be increased. Treatment of C2C12 muscle cells with 50 μM MLB or 5 μM MPc11 for 48 hours shows no increase in DNA synthesis. FIG. 2C shows that 7-AAD staining did not induce cytotoxicity after treatment of C2C12 muscle cells with 10 μM SB203580, 300 μM SQ22536, 30 μM LPA, 50 μM MLB or 5 μM MPc11 for 48 hours. 0.1% Triton X-100 was treated as a positive control of cytotoxicity confirmation. Figure 2d shows the results confirmed by MTT analysis that the cytotoxicity was not induced by treating the C2C12 muscle cells with 2.5 μM BIO for 48 hours. 2E shows cell cycle re-entry induced by treatment with 2.5 μM BIO, 10 μM SB203580, 300 μM SQ22536 or 30 μM LPA for 48 hours in C2C12 root canal via propidium iodide (PI) staining. The cell content of the G ₂ -M phase in the flow cytometry is shown. C2C12 myoblasts were used as positive controls.
FIG. 3A shows Western blot results confirming reduced p57 expression of C2C12 muscle cells treated with 2.5 μM BIO or 10 μM SB203580 for 48 hours. C2C12 muscle cells treated with 300 μM SQ22536 or 30 μM LPA do not reduce the expression of p57. However, when C2C12 muscle cells were treated with BIO, SB203580, SQ22536 or LPA, it showed no effect on the expression of p27. In contrast, when C2C12 muscle cells were treated with BIO, SB203580, SQ22536 or LPA, p21 expression was reduced. GAPDH expression was used as a loading control. The numbers below the band images show the intensity of the band compared to untreated cells. FIG. 3B shows that C2C12 muscle cells were treated with 2.5 μM BIO, 10 μM SB203580 or 30 μM LPA for 48 hours to reduce expression of differentiated muscle-specific acetylcholine receptors (detection of FITC-binding α-Bungarotoxin). This is the result of confirming reverse differentiation. Treatment with 300 μM SQ22536, 50 μM MLB or 5 μM MPc11 for 48 hours shows no decrease in acetylcholine receptor expression. Figure 3c induces reverse differentiation by treating B2, SB203580, or LPA to C2C12 muscle cells, and confirmed the reduction of FITC-binding α- bogarotoxin labeling by fluorescence microplate reader analysis. Reliability is shown in p <0.05 compared to untreated cells. 3D shows fusion indices (eg, multiple cell nuclei are multinucleated) when C2C12 muscle cells were treated with 2.5 μM BIO, 10 μM SB203580, 300 μM SQ22536 or 30 μM LPA for 48 hours after incubation for 5 days in differentiation medium. Results in no effect on sexual root canal fusion). In contrast, after 5 days of incubation in differentiation media, the fusion index decreased when 1 μM reversin was treated for 48 hours. Reliability is shown in p <0.05 compared to untreated cells.
Figure 4 shows that C2C12 muscle cells were treated with 2.5 μM BIO, 10 μM SB203580, 300 μM SQ22536 or 30 μM LPA for 48 hours and adipocyte-forming factor for 7 days to induce lipid accumulation, which is characteristic of adipocyte formation. Indicates. Lipids were visualized by Oil Red O staining. Treatment with adipocyte-forming factors in C2C12 muscle cells treated with untreated C2C12 cells or 50 μM MLB or 5 μM MPc11 for 48 hours showed no lipid accumulation. Lipid accumulation was observed as previously reported paper when C2C12 myoblasts were treated with 250 nM reversin and incubated in adipocyte culture medium for 48 hours (33). Lipid accumulation of 3T3-L1 adipocytes is shown for comparison. 4bi shows lipid accumulation as measured by AdipoRed ^™ labeling. Lipid accumulation increased significantly when C2C12 muscle cells were treated with 2.5 μM BIO, 10 μM SB203580, 300 μM SQ22536 or 30 μM LPA for 48 hours and then treated with adipocyte-forming factors. In contrast, treatment with C2C12 muscle cells treated with untreated C2C12 cells or 50 μM MLB or 5 μM MPc11 for 48 hours showed no increase in lipid accumulation. The results show a reliability of p <0.05 compared to untreated cells. Figure 4bii is a C2C12 muscle cells treated with BIO, SB203580, SQ22536 or LPA for 48 hours and treated with 250 nM reversin, and then treated with adipocyte-forming factor cultures signal from the Adipored ^TM probe (probe) It was confirmed that lipid accumulation increased by increasing. C2C12 muscle cells were treated with MLB or MPc11 for 48 hours, treated with 250 nM reversin, and then cultured with adipocyte-forming factors, resulting in no increase in lipid accumulation. The results show a reliability of p <0.05 compared to untreated cells. FIG. 4C shows that C2C12 muscle cells were treated with BIO, SB203580, SQ22536 or LPA for 48 hours and treated with adipocyte-forming factors, and showed insulin-path mediated glucose uptake ability as an adipocyte-specific function. When incubated with adipocyte-forming factor in C2C12 muscle cells treated with untreated C2C12 cells or 50 μM MLB or 5 μM MPc11 for 48 hours, they show no insulin-path mediated glucose uptake. The results have a reliability of p <0.05 compared to the cell material that did not stimulate the insulin-path.
FIG. 5A shows the results of inducing mineralization, which is characteristic of bone formation conversion, by treating C2C12 muscle cells with 2.5 μM BIO, 10 μM SB203580, 300 μM SQ22536 or 30 μM LPA for 48 hours and treating bone formation factors. Indicates. Mineralization was not seen when incubated with osteogenic factors in C2C12 muscle cells treated with untreated cells or 50 μM MLB or 5 μM MPc11 for 48 hours. Mineralization was formed when C2C12 myoblasts were treated with 250 nM reversin and incubated in osteogenic medium for 48 hours, consistent with previous reports (33). Comparison was made to mineralization of ATDC5 osteogenic cells. 5bi shows significant increase in mineralization when C2C12 muscle cells were treated with BIO, SB203580, SQ22536 or LPA for 48 hours and treated with osteogenic factors. In contrast, when osteoblasts were treated with untreated C2C12 cells or C2C12 muscle cells treated with MLB or MPc11 for 48 hours, there was no increase in mineralization compared to untreated cells (p <0.05). Figure 5bii is treated with BIO, SB203580, SQ22536 or LPA for 48 hours and treated with 250 nM reversin 48 hours in C2C12 muscle cells, after incubation with the addition of osteogenic factor, via alizarin red staining It shows an increase in mineralization. In addition, the difference for reversin treatment between the four treatment groups was reduced. When C2C12 muscle cells treated with MLB or MPc11 for 48 hours were treated with osteogenic factor for 48 hours after 250 nM reversin treatment, it was shown that mineralization did not increase when cultured with osteogenic factor. The results have a reliability of p <0.05 compared to untreated cells. 5Ci shows that increased alkaline phosphatase activity is one feature of bone formation inversion. After 48 hours of treatment with BIO, SB203580, SQ22536 or LPA in C2C12 cell water and treatment with osteogenic factors, alkaline phosphatase activity was significantly increased. In contrast, treatment with osteogenic factors in untreated C2C12 muscle cells, or C2C12 muscle cells treated with MLB or MPc11 for 48 hours, shows no increase in alkaline phosphatase activity. Untreated, MLB or MPc11 treated C2C12 muscle cells showed similar assays. Figure 5cii shows the difference in alkaline phosphatase activity between the four treatment groups when C2C12 muscle cells were treated with BIO, SB203580, SQ22536 or LPA for 48 hours and 250 nM reversin for 48 hours and then cultured with osteogenic factor. Shows a decrease. Conversely, alkaline phosphatase when C2C12 muscle cells treated with untreated C2C12 cells, MLB or MPc11 for 48 hours were treated with osteogenic factor and treated with 250 nM reversin for 48 hours and then cultured with osteogenic factor. It does not increase activity. Untreated, MLB or MPc11 treated C2C12 muscle cells showed similar assays.
FIG. 6A shows that C2C12 muscle cells were treated with 2.5 μM BIO, 10 μM SB203580 or 300 μM SQ22536 for 48 hours, treated with neuronal factors, treated with 250 nM reversin for 48 hours, and then cultured in neuronal media to form neuronal transcription factors. Induced expression of Hes-6. Conversely, 30 μM LPA, 50 μM MLB or 5 μM MPc11 was treated for 48 hours in C2C12 muscle cell water and 250 nM reversin for 48 hours and then cultured in neuronal medium did not induce Hes-6 expression. Shows. FIG. 6B shows that morphological changes appearing as neurogenesis were observed in C2C12 muscle cells after treatment with BIO and treatment with 250 nM reversin for 48 hours. 6C shows depolarization-dependent vesicle recycling, which is a neuro-specific feature when cultured with neurogenic factors after 48 hours treatment with BIO, SB203580 or SQ22536 and 48 hours treatment with 250 nM reversin in C2C12 muscle cells dependent vesicle recycling). Vesicle recycling is fluorescently labeled. Visualized by FM 1-43. Conversely, when treated with C2C12 muscle cells for 48 hours and 250 nM reversin for 48 hours, MLB or MPc11 did not show depolarization-dependent vesicle recycling when cultured in media containing neurogenic factors. Figure 6d is treated with BIO, SB203580 or SQ22536 for 48 hours and 250 nM reversin 48 hours in C2C12 muscle cells, and then cultured with neuronal factors FM 1-43 by depolarization using high concentration extracellular potassium Absorption increased. On the contrary, C1C12 muscle cells were treated with MLB or MPc11 for 48 hours and 250 nM reversin for 48 hours, and then cultured with neuronal factor showed no FM 1-43 fluorescence signal. It has a reliability of p <0.05 compared to the control with low extracellular potassium. P19 neurons were used to compare the depolarization-dependent increase in FM 1-43 fluorescence signal. FIG. 6E shows that glutamate-dependent calcium influx, which is a neuro-specific characteristic, was cultured with CIO cells, which were treated with BIO, SB203580 or SQ22536 for 48 hours, and treated with 250 nM reversin for 48 hours, followed by culture with neuronal factors. This is the result of checking. Glutamate-dependent calcium influx was visualized with the fluorescent label, Fluo-3. In contrast, glutamate-dependent calcium influx was not observed when C2C12 muscle cells were treated with MLB or MPc11 for 48 hours and 250 nM reversin for 48 hours and then cultured with neuronal factors.
7 shows a schematic of a low molecular method for obtaining pluripotent cells from fully differentiated skeletal muscle tissue. Recently published results show that the redifferentiation of muscle tissue and the formation of new muscle tissue in essentially new limbs (eg monopotent cells) during limb regeneration of Rhesus amphibians such as salamanders (31). The low molecular method of the present invention can broaden the developmental plasticity of cells to mammalian muscle tissue by dedifferentiating them into cells with pluripotent capacity, as shown by the transition to cells that characterize neurons.

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

C2C12 mice ( mus musculus ) myoblasts were cultured according to previously reported methods (11). To differentiate into the root canal, myoblasts were cultured in collagen-coated tissue culture plates (Sigma, USA). Differentiation was induced by culturing confluent myoblasts in differentiation medium (DMEM with 5% horse serum, 50 units / ml penicillin and 50 μg / ml streptomycin) for 10 days. To enhance differentiation and remove mononuclear myoblasts, 50 μM AraC3 is treated for 72 hours after incubation for 24 hours in differentiation medium.

To obtain a purified culture of the root canal, the differentiated root canal culture was trypsinized, centrifuged at 800 rpm for 10 minutes, and re-suspended in a 10 ml differentiation medium, followed by 2 to 40 µm mesh (BD sciences, USA). Separated once. The mesh was washed with 20 ml culture medium to obtain root canal. In general, to obtain a root canal of one 10 cm 2 dish, a differentiated C2C12 culture of five confluent 10 cm 2 dishes is required.

3T3-L1 mouse ( mus musculus ) embryonic fibroblasts were cultured and differentiated into adipocytes by the reported method (11). Osteo-chondrogenic precursor cells from ATDC5 mice (mus musculus) were cultured and differentiated into osteoblasts by the previously reported method (11).

Skeletal Muscle Root Canal Cellization

C2C12 root canal cultures were treated with 20 μM myoseber for 48 hours and harvested by the previously reported method (11).

Induction of Lipid Formation in Cellized Skeletal Muscle Root Canal

Cells were incubated for 48 hours in DMEM with 10% FBS, 100 nM insulin, 5 μM dexamethasone, 1 μM rosiglitazone and 0.5 mM IBMX (Isobutylmethylxanthine), and 100 nM insulin and 1 μM rosiglitazone Were further incubated for 7 days. Culture medium was changed every 48 hours.

Induction of bone formation in cellized skeletal muscle root canal

Induction of bone formation in the cellized skeletal muscle root canal was performed by the previously reported reversin-treatment method (33).

Induction of neurogenesis in the cellized skeletal muscle root canal

The method of inducing neurogenesis was based on reports of neurons forming in reversin-treated C2C12 myoblasts (28). Cells were incubated at 1.5 × 10 ⁶ cells / 9.6 cm 2 in laminin-coated culture dishes. After small molecule treatment, cells were treated with 250 nM reversin for 48 hours, neural induction medium (DMEM / F12 with N2, invitrogen, USA) and 1.5 μM all-trans retinoic acid 11 Incubated for 7 days.

Statistical analysis

The mean value of three independent experiments was determined by Mann-Whitney U test (TalkStats software; Jelsoft Enterprises). p values showed significance below 0.05. All error ranges are the standard deviation of the median.

result

In chemical biology research, a chemical approach to manipulating cells is the most important area. Attempts have been made to develop small molecules to induce the early stages of amphibian limb regeneration in mammalian muscle but have not succeeded in complete dedifferentiation (15, 16). Therefore, there is much interest in the field of chemical biology in the demonstration of mammalian muscle dedifferentiation using small molecules.

DNA synthesis and cell cycle re-entry induction of BIO, SB203580, SQ22536 and LPA

The low molecule used for this invention is shown in FIG. 2A shows the extent of the proliferation of muscle cells. In the present invention, the concentration of each small molecule was performed at a concentration having activity with reference to the previously reported literature (17, 20, 21). 2.5 μM BIO (6-bromoindirubin-3'-oxime), 10 μM SB203580 (4- [4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -1H-imidazol-5-yl] pyridine), 300 μM Cells treated with SQ22536 (9- (Tetrahydro-2-furanyl) -9H-purin-6-amine) or 30 μM Lysophosphatidic acid (LPA) for 48 hours were treated with BrdU (5'-bromo-2 ') in the cells. -Deoxyuridine) shows increased DNA synthesis due to increased incorporation (FIG. 2B). BrdU binding was relatively low in cells treated with SQ22536. MPc11 (inhibitor of synthesis of F1F0 mitochondrial ATP synthase) 9 and 50 μM MLB B (magnesium lithospermate B, natural antioxidant) 8 were used as negative controls (22, 23). C2Cl2 muscle cells treated with 50 μM MLB or 5 μM MPc11 for 48 hours did not increase proliferation and induced cytotoxicity (FIGS. 2B and 2C). Due to cell staining by BIO, BIO treated cells cannot be stained with 7-AAD. Thus, MTT assay shows that BIO does not induce cytotoxicity (FIG. 2D). Nuclear staining showed that cell cycle re-entry to increase cells in G ₂ -M phase upon treatment with BIO, SB203580, SQ22536 or LPA (FIG. 2E).

Regulation of CDKN Expression by BIO, SB203580, SQ22536, or LPA

Numerous Cyclin-Dependent Kinase Inhibitors (CDKN), including p21 (CIP1 / CDKN1A), p27 (CDKN1B / KIP1) and p57 (CDKN1C / KIP2), show persistent differentiation of skeletal muscle (18, 24). ). Treatment of BIO or SB203580 in muscle cell water reduced p57 expression (FIG. 3A). However, low molecular weight treatment does not significantly affect p27 expression (FIG. 3A). On the other hand, low molecular weight treatment reduces p21 expression (FIG. 3A).

Induced differentiation of BIO, SB203580, SQ22536 or LPA

When muscle cells were treated with BIO, SB203580, SQ22536 or LPA, FITC-linked α-bungarotoxin labeling indicating dedifferentiated cells was reduced (FIG. 3B). In contrast, SQ22536, MLB or MPc11 did not reduce FITC-linked α-bungarotoxin labeling (FIG. 3B). It was confirmed by fluorescence plate reader assay that BIO, SB203580, SQ22536 or LPA attenuated the FITC-bound α-Bungarotoxin label (FIG. 3C).

Myogenic potential effects of BIO, SB203580, SQ22536 or LPA

The ability to form muscle (myoelectric potential, myogenic potential) of muscle cells was analyzed using a fusion index (11). After 5 days of incubation in differentiation medium, BIO, SB203580, SQ22536 or LPA did not affect the fusion index (FIG. 3D). In contrast, treatment with reversine reduced the cell fusion index.

BIO , SB203580 , SQ22536  or LPA Fat cell induction

C2C12 muscle cells treated with BIO, SB203580, SQ22536 or LPA were treated with a cocktail of adipogenic factors and cultured to induce lipid accumulation (FIG. 4A). Treatment of C2C12 muscle cells with MLB or MPc11 and adipogenic factors did not induce lipid accumulation. The conversion of adipocyte formation using p21 knockout in our previous study was dependent on reversin treatment (11). However, after treatment with BIO, SB203580, SQ22536 or LPA in C2C12 muscle cells, adipocyte-forming factors were confirmed to accumulate lipid without treatment with reversin (FIG. 4B). Furthermore, after treatment with BIO, SB203580, SQ22536 or LPA, the degree of lipid accumulation increased in the experimental group treated with reversin and adipocyte-forming factors (FIG. 4bii).

The uptake of glucose by insulin-stimulation, which is characteristic of adipocytes, is attributed to the fluorescent glucose analogue 2-NBDG (2- [N- (7-nitrobenz-2-oxa-1,3-diaxol-4-yl) amino ] -2-deoxyglucose) (25). Treatment with BIO, SB203580, SQ22536 or LPA for 48 hours followed by incubation with a cocktail of adipocyte-forming factors to induce insulin-stimulated glucose uptake (FIG. 4ci). MLB or MPc11 and adipocyte-forming factors were not insulin sensitive. In addition, treatment with reversin and adipocyte-forming factors for 48 hours and treatment with BIO, SB203580, SQ22536 or LPA increased 2-NBDG uptake. However, differences in 2-NBDG uptake in insulin-stimulated and non-insulin-stimulated cells appeared similar (FIG. 4Cii).

BIO , SB203580 , SQ22536  or LPA of Osteogenic ( osteogenic ) transform

Increased mineralization is a major feature of osteoogenesis (26). C2C12 muscle cells were treated with BIO, SB203580, SQ22536 or LPA and incubated with a bone formation factor cocktail that induces mineralization (FIG. 5A). No mineralization was observed in the experimental groups treated with MLB or MPc11 and osteogenic factors. In the experimental group treated with BIO or SB203580, more mineralization was induced compared to the experimental group treated with LPA or SQ22536 after incubation with osteogenic factors (FIG. 5bii). In addition, treatment with BIO, SB203580, SQ22536 or LPA and reversin and osteogenic factors reduced the difference in mineralization between the experimental groups.

BIO, SB203580, SQ22536 or LPA and osteogenic factors were treated to induce alkaline phosphatase activity indicating osteogenic conversion (FIG. 5C). Alkaline phosphatase activity was not induced in C2C12 muscle cells treated with MLB or MPc11 and osteogenic factors. Induction of alkaline phosphatase activity was higher in BIO and SB203580 than in the group treated with LPA and SQ22536. In addition, the experimental group treated with BIO, SB203580, SQ22536 or LPA, and reversin and bone formation factor increased the degree of alkaline phosphatase activity (Fig. 5cii). In addition, when reversin was added during the treatment, the difference in alkaline phosphatase activity between the experimental groups was reduced.

BIO , SB203580  And SQ22536 Neurogenesis of neurogenic ) Judo

RT-PCR analysis of Hes-6 (genes up-regulated in C2C12 myoblasts neurized as transcription factors that promote neuronal differentiation) after treatment of C2C12 cells with small molecules It was measured (FIG. 6A). Hes-6 expression was not upregulated when incubated with neural reversin and neurogenic factors following treatment with LPA, MLB or MPc11. Upregulated Hes-6 expression involves morphological changes in which the neurons of the cell are formed (FIGS. 5B, 28). The repolarization of the depolarization-dependent vesicles is a physiological characteristic specific to neurons (FIG. 6C). The occurrence of depolarization-dependent vesicle recirculation was confirmed by microplate reader assay (FIG. 6D). Glutamate-dependent calcium influx is also a neuronal specific physiological feature (29). When treated with BIO, SB203580 or SQ22536 and incubated with reversin and neuronal factors, glutamate-dependent calcium influx was observed (FIG. 6E). In contrast, there was no glutamate-dependent calcium influx in the experimental group treated with MLB or MPc11 and incubated with reversin and neuronal factors for 7 days.

Review

In the animal kingdom, limb regeneration of Rhesus amphibians is a special example of tissue regeneration (7). The early stages of limb regeneration are associated with muscle cellularization and cell cycle re-entry (4, 6). Recently, siRNA-based approaches to induce similar actions in mammalian muscle have been studied (11, 12). However, because of the problems of non-specific cytotoxicity and poor biological contribution, the low molecule-based approach is better than the siRNA-based approach.

The focus of the present invention is on the differentiation of purified population of the root canal excluding 'contaminating' undifferentiated myoblasts. To achieve the object of the present invention, the inventors used previously reported sequential cell seiving (11, 16). The inventors previously confirmed that the method is suitable for obtaining purified myotube cultures (11). The inventors induced cellularization using myoseberg, a low molecular molecule with relatively low cytotoxicity (11,15). Inhibition of glycogen-3 kinase with low molecular weight BIO, activation of G-protein-binding receptor-mediated signaling with LPA, inhibition of adenyryl cyclase with SQ22536 or inhibition of p38 MAP kinase pathway with SB203580 The cells of the muscle cell were re-entered into the cell cycle (FIG. 2B). This shows that complex biochemical pathway regulation can be regulated to induce cell proliferation.

In previous studies, expression of siRNA-mediated gene knockdown or viral oncogenic genes in the root canal induced proliferation and apoptosis (8, 13, 14). However, BIO, LPA, SB203580 and SQ22536 did not induce cytotoxicity (FIG. 2C). In addition, the cell cycle of the proliferating cell mass was similar to the proliferating muscle myoblasts and normal cells (FIG. 2D). Therefore, small molecules of muscle cells, BIO. Proliferation introduction by LPA, SB203580 or SQ22536 has shown to be suitable for developing therapeutics to improve tissue regeneration.

Induction of mammalian muscle cell proliferation is associated with knockout of p21 expression (11,18). Treatment with the small molecules BIO, LPA, SB203580 or SQ22536 reduced p21 expression of muscle cells (FIG. 3A). p27 expression is associated with the conversion of cell cycle retraction in differentiated cardiomyocytes treated with BIO (21). However, our results show that the molecular weight of mammalian muscle cells does not dramatically affect the expression of p27. This result may be due to biochemical differences between the miyoverine-derived cell and differentiated cardiomyocytes. In addition, p21 expression in cardiomyocytes was not measured (21). In addition, the results of the present inventors show that the treatment of low molecular weight BIO and SB203580 in the cell water reduced the expression of p57 (FIG. 3A). p57 is not an important regulator of cell cycle withdrawal in muscle cells, but downregulation by BIO and SB203580 is suitable for inducing differentiation of other tissues where small molecules play an important role in maintaining p57 at rest (cell cycle withdrawal). Tell the way.

Acetylcholine receptor expression is considered a marker of muscle differentiation (30). Results of the present invention suggest that inhibition of glycogen synthase-3 kinase using BIO, activation of G-protein-coupled receptor-mediated signaling using LPA, or inhibition of the p38 MAP kinase pathway using SB203580 may be associated with acetylcholine in mammalian muscle cells. It shows a significant decrease in the expression of receptors (FIGS. 3B-3C). The results of the present invention indicate that the introduction of cell cycle reentry in the cell material is a major factor of reverse differentiation. Inhibition of adenyryl cyclase with SQ22536 does not affect the expression of the acetylcholine receptor and the cells have a multipotency to form mast cells and osteogenic cells (FIGS. 3B-3C, 4 and 5). . This result would be characteristic of myoseborn-derived muscle cells. Previous studies have shown that myocerin monotreated cells did not reverse differentiation but could reduce the expression of acetylcholine receptors (16, 30). Therefore, myoseborn-derived muscle cells will be 'less differentiated' compared to the original root canal culture.

De-differentiated muscle cells of the limbic amphibian limb remodel new muscle tissue (4). The results of the present inventors show that BIO, LPA, SB203580 or SQ22536 do not inhibit the reversed differentiation of cells into the muscle root canal (FIG. 3D). In addition, the inventors show differentiation into adipocytes and osteogenic cells, showing the state of multipotency by treatment with BIO, LPA, SB203580 or SQ22536 (FIGS. 4 and 5). The results of the present invention induced the differentiation of the cells using siRNA-mediated p21 knockdown with reversin, a myoblast dedifferentiation agent (11). Only p21 knockdown was insufficient to produce multipotent cells (11). In the present invention, a multipotent state was induced by treating BIO, LPA, SB203580 or SQ22536 without reversin treatment. The results of the induction of cell differentiation by the low molecular weight method show that the low molecular weight that regulates the complex biochemical reaction is superior to the siRNA method.

A pluripotent state is defined as a cell population that has the ability to differentiate into trioderm (endoderm, mesoderm and ectoderm) and its derived cells (2). In the present invention, the inventors show that the mammalian muscle cells are treated with BIO, SB203580 or SQ22536 and treated with reversin to differentiate into cells having neuronal characteristics (FIG. 6). Cells treated with small molecules showed morphological changes to neurons. This is thought to be neurogenesis in muscle cells because it induces 'typical' neurons and other morphological changes derived from cellular stress by other factors, such as cytochalasin B or dimethyl sulfoxide (DMSO) treatment (28 ). The inventors conducted a functional test to confirm the conversion to neurons (FIGS. 6D-6E). Previously, this test was used to show neuronal cell turnover in muscle cells that introduced REST-VP16 constructs that activate neuronal genes inhibited by transcriptional inhibitors and neuro-restrictive silencer factors. (29). LPA treatment did not exhibit neuronal cell turnover, indicating that activation of G-protein-coupled receptors is required for muscle cells to have multipotents rather than pluripotents.

In the amphibian limb regeneration process, recent studies show that dedifferentiated root canals form new muscle tissue in lineage-restricted regeneration limbs (31). Therefore, the small molecule-based approach for inducing mammalian muscle dedifferentiation of the present invention is permitted in adult mammalian skeletal muscle to obtain greater embryological plasticity than the partial regeneration observed in nature. A schematic diagram of the small molecule-based approach of the present invention is shown in FIG. Fibroblasts of the skin are thought to form a multipotent group during the regeneration process of limpidane amphibians (4, 31). Therefore, the low molecular method in the present invention explains that it acts in a similar way to amphibian dermal fibroblasts to induce dedifferentiated mammalian muscle cells.

The small molecule-based method for inducing dedifferentiated muscle of the present invention offers numerous possibilities for further study. For example, human muscle biopsies can be utilized to produce patient-specific multipotent cells. This method is also applicable to mammalian models of wound healing. The small molecules used in the present invention (BIO, LPA, SB203580 and SQ22536) downregulate p21 expression. Murphy Roths Large mice show improved wound healing capacity associated with reduced p21 expression (32). Therefore, these small molecules can improve wound healing and reduce scarring, thus forming the basis of topical medical application. The results of the present invention provide for the development of a method for inducing limb regeneration of stunts and suggests a novel approach to pluripotent stem cells of fully differentiated mammalian tissue.

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. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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Claims (12)

Chemical preparation of multipotent or pluripotent cells from mammalian differentiated cells comprising the following steps:
(a) obtaining differentiated cells from a mammal; And
(b) treating the differentiated cells with p21 down-regulation inducing compound to obtain pluripotent cells.
The method of claim 1, wherein the differentiated cells are terminally differentiated cells.
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.
The method of claim 1, wherein the differentiated cells are connective tissue, nerve cells, lymph cells, vasculature cells, kidney cells, pancreatic 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.
The method of claim 1, wherein the p21 down-regulation inducing compound is BIO (6-bromoindirubin-3'-oxime), SB203580 (4- [4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -1 H-imidazol -5-yl] pyridine), SQ22536 (9- (Tetrahydro-2-furanyl) -9H-purin-6-amine) or LPA (Lysophosphatidic acid).
The method of claim 1, further comprising the step of treating the cells obtained in step (b) with a dedifferentiator after treatment with the p21 down-regulation inducing compound of step (b) to obtain pluripotent cells, The pluripotent cells are characterized in that they have the ability to differentiate into differentiation cells of step (a) and cells of the other germ line.
7. The method of claim 6, wherein the derivatizer is reversin (N6-cyclohexyl-N2- (4-morpholin-4-yl-phenyl) -9H-purine-2,6-diamine), trichostatin A A), valproic acid, buphenyl and 5-azacytidine. The method of claim 1, wherein the compound is selected from the group consisting of 5-azacytidine.
The method according to claim 1, wherein the method comprises treating the cells obtained in step (b) with a differentiation inducing agent after treatment with the p21 down-regulation inducing compound of step (b), and the other cells of the same germ line as the differentiated cells of step (b). And further comprising differentiating into lineage.
The method of claim 3, wherein the differentiated cells are skeletal muscle cells.
Pluripotent cells prepared according to the method of claim 1.
Omnipotent cells prepared according to the method of claim 6.
Regeneration of a mammalian tissue or organ comprising the following steps:
(a) obtaining differentiated cells from a mammal;
(b) treating the differentiated cells with a p21 down-regulatory inducing compound to obtain pluripotent cells; And
(c) differentiating said pluripotent cells into a desired tissue or organ.

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