WO2017026878A1 - Culture medium composition for inducing musculoskeletal progenitor cell, and pharmaceutical composition comprising musculoskeletal progenitor cell for preventing or treating musculoskeletal diseases - Google Patents

Abstract

The present invention relates to: a culture medium composition for inducing differentiation of a musculoskeletal progenitor cell from a stem cell; a method for inducing differentiation of a musculoskeletal progenitor cell from a stem cell using the culture medium composition; a musculoskeletal progenitor cell produced by the method; and a pharmaceutical composition and a cell therapeutic agent containing the cell for preventing or treating musculoskeletal diseases. The culture medium composition according to the present invention contains an LIF, and is capable efficiently inducing differentiation of a musculoskeletal progenitor cell from a stem cell by activating FGF2 signal transfer, inhibiting TGF-β/activin/nodal signal transfer, activating a Wnt signal, and inhibiting an ERK signal. The musculoskeletal progenitor cell obtained thereby can be differentiated into a bone through endochondral ossification, and also into cartilage, tendon, and muscle, and therefore can be useful in preventing or treating a variety of musculoskeletal diseases.

Description

A pharmaceutical composition for preventing or treating musculoskeletal diseases, comprising a culture medium composition for inducing musculoskeletal precursor cells and musculoskeletal precursor cells

The present invention relates to a medium composition for inducing differentiation from stem cells to a musculoskeletal progenitor cell, a method for inducing differentiation from stem cells to musculoskeletal precursor cells using the medium composition, and a method for inducing differentiation into musculoskeletal progenitor cells, To a pharmaceutical composition and a cell treatment agent for preventing or treating musculoskeletal diseases.

Osteification is a process of bone formation, which is known to be due to two methods of intramedullary or intramedullary ossification. Intramural ossification is a direct process that is converted into mesenchymal bone, which occurs within the skull bone. On the other hand, ossification of cartilage occurs by the process of cartilage tissue being converted into bone after the process of cartilage tissue formation from agglomerated mesenchymal cells. This process of ossification is essential for most bone formation in vertebrate animals.

On the other hand, human embryonic stem cells (hESCs) are totally differentiable cells, which can grow without limitation and can differentiate into all cell types. hESC is a very useful tool for studying embryonic development at the cellular level and is a useful tool for cell replacement therapy. hESCs can be differentiated into specific tissues including, for example, skeletal tissues including bone and cartilage, and can be used for skeletal tissue repair.

Human induced lpuripotent stem cells (hiPSCs) are known as pluripotent stem cells capable of differentiating into any cell type. hiPSC is a cell that is useful for studying embryonic development at the cellular level and is attracting attention as a cell therapy agent. Since these cells can be differentiated into bone structures such as bone and cartilage by transplantation, they can be usefully used for repairing and repairing damaged skeletal tissues.

Mesenchymal stem cells (MSCs) are self-renewing cells that can differentiate into mesenchymal-type cells such as osteoblasts, adipocytes and chondrocytes. MSC has been applied to clinical trials under various conditions, and it has been attempted in trauma, skeletal diseases, graft versus host disease, cardiovascular disease, autoimmune disease, liver disease and the like. However, there is a limitation in that it is very difficult to obtain a sufficient amount of MSC necessary for therapeutic application.

In bone tissue engineering, the developmental process of intramedullary ossification has been used to form bone structures. However, the probability of osteoprosthesis using mesenchymal stem cells is very low, less than 5% even in animal models. Thus, recent studies have focused on the method of inducing osteogenesis by mimicking osteochondral ossification using mesenchymal stem cells, but the research is insufficient.

In this regard, it has recently been found that mouse skeletal stem / progenitor cells (SSCs) are distinct from mesenchymal stem cells, and are highly likely to be specifically differentiated into bone and cartilage. Therefore, there is an increasing need for studies on cells capable of differentiating into bone and cartilage through osteoarthritis while overcoming the limitations of mesenchymal stem cells.

Thus, the present inventors have been able to induce musculoskeletal precursor cells from human embryonic stem cells or human induced pluripotent stem cells, and that the musculoskeletal precursor cells can be differentiated into bone through osteoarthritis and differentiated into cartilage, The present invention has been completed.

It is an object of the present invention to provide a medium composition for inducing differentiation from stem cells to a musculoskeletal progenitor cell, and a method for inducing differentiation from stem cells into musculoskeletal progenitor cells using the medium composition.

It is still another object of the present invention to provide a musculoskeletal precursor cell produced by the above method, a pharmaceutical composition for preventing or treating musculoskeletal diseases including the cell, and a cell treatment agent.

In order to achieve the above object, the present invention provides a pharmaceutical composition comprising a FGF2 (Fibroblast Growth Factor 2) signaling activator, a TGF-β / activin / nodal signal transduction inhibitor, a Wnt signal activator, an extracellular signal-regulated kinase signaling inhibitor, and a leukemia inhibitory factor (LIF). The present invention also provides a medium composition for inducing differentiation of stem cells into musculoskeletal progenitor cells.

The present invention also provides a method for inducing differentiation of stem cells into musculoskeletal progenitor cells, comprising culturing stem cells in the culture medium.

In addition, the present invention provides a mucsloskeletal progenitor cell produced by the above method.

The present invention also provides a pharmaceutical composition for preventing or treating musculoskeletal diseases, which comprises the above musculoskeletal precursor cells.

The present invention also provides a cell therapy agent for treating musculoskeletal diseases, which comprises the above musculoskeletal precursor cells.

The culture medium composition of the present invention comprises LIF and is useful for stimulating stem cells by activating FGF2 signaling, inhibiting TGF-beta / activin / nodal signaling, activating Wnt signaling, and inhibiting ERK signaling Can induce the differentiation of musculoskeletal precursor cells efficiently, and the musculoskeletal progenitor cells obtained through this can differentiate into bone through ossification of cartilage and can be differentiated into cartilage, tendon, and muscle so that prevention of various musculoskeletal diseases Or may be useful for treatment.

Figure 1A shows the result of observing the morphology of hMSPC induced differentiation from hESC.

FIG. 1B is a graph showing the results of immunofluorescence detection of the expression of a fully differentiable marker in hMSPC.

FIG. 1C shows the results of RT-PCR analysis of the expression of the differentiation-ability marker in hMSPC.

FIG. 1D is a graph showing the results of immunofluorescence for expression of ectoderm, mesoderm, and endoderm markers in hMSPC.

FIG. 1E shows the results of RT-PCR for expression of ectoderm, mesoderm, and endoderm markers in hMSPC.

2A shows the results of flow cytometry analysis of expression of mesenchymal stem cell markers in hMSC and hMSPC.

FIG. 2B is a graph showing the results of comparing bone formation, cartilage formation, and fat formation in hMSC and hMSPC.

FIG. 2C is a graph showing the change in bone cell marker expression during the bone formation process of hMSC and hMSPC for 9 days. FIG.

FIG. 2D shows the results of comparing changes in expression of cartilage markers before and after hESC-induced differentiation of hMSPC into chondrocytes.

Fig. 3A shows the results of immunohistochemical staining of expression of SM22a, a smooth muscle marker, in hMSPC.

FIG. 3B is a graph showing the expression of SM-MHC, a smooth muscle marker, in hMSPC by immunofluorescence.

FIG. 3C is a graph showing the results of immunofluorescence for the expression of CD31, an endothelial cell marker, in hMSPC.

FIG. 3D is a graph showing the results of immunofluorescence for the expression of VE-cadherin, an endothelial cell marker, in hMSPC.

FIG. 3E is a graph showing the expression of MAP2, a neuronal cell marker in hMSPC, by immunofluorescence.

FIG. 4A is a graph showing the results of immunofluorescence detection of the expression of the differentiation-ability markers in hMSPC derived from hiPSC.

FIG. 4B shows the results of RT-PCR analysis of the expression of the fully differentiable marker in hMSC derived from hiPSC.

4C is a graph showing the results of immunofluorescence for expression of ectoderm, mesoderm, and endodermic markers in hMSC derived from hiPSC.

FIG. 4D shows the results of RT-PCR analysis of the expression of ectoderm, mesoderm, and endodermic markers in hMSC derived from hiPSC.

FIG. 4E shows the results of flow cytometry analysis of the expression of the mesenchymal stem cell marker in hMSC derived from hiPSC.

FIG. 4F shows the results of comparing bone formation, cartilage formation, and fat formation in hMSC derived from hiPSC.

FIG. 4G shows the expression of smooth muscle marker in hMSC derived from hiPSC by immunofluorescence.

FIG. 5A is a graph showing the result of transplanting hMSPC subcutaneously and confirming its differentiation. FIG.

FIG. 5B is a diagram showing the results of transplantation of hMSPC into the fascia and confirmation of its differentiation.

FIG. 5C is a diagram showing the results of transplantation of hMSPC under the fascia around the tendon and confirmation of its differentiation.

6A shows the result of transplantation of hMSPCs under the kidney capsules of immunodeficient mice and histological examination by H & E staining 5 weeks later.

Figure 6B shows the results of transplantation of hMSPCs under the kidney capsules of immunodeficient mice and tissue validation by Movat ' s pentachrome staining 5 weeks later.

FIG. 6C is a graph showing the results of confirming the relationship between ossification of osteochondral and angiogenesis.

7A is an experimental design diagram for a fracture study.

FIG. 7B is a photograph showing a fracture study.

FIG. 7C is a view showing results of fracture studies for hMSPC. FIG.

FIG. 7D is a view showing results of fracture studies on hMSPC. FIG.

FIG. 7E is a view showing a result of a fracture study on hMSPC.

FIG. 7F is a graph showing the result of confirming the bone formation effect of hMSPC.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition comprising an FGF2 (Fibroblast Growth Factor 2) signaling activator, a TGF-beta / activin / nodal signal transduction inhibitor, a Wnt signal activator, an extracellular signal- An inhibitor, and a leukemia inhibitory factor (LIF). The present invention also provides a medium composition for inducing differentiation of a stem cell into a musculoskeletal progenitor cell.

The term " stem cell " in the present invention is an undifferentiated cell having an ability to differentiate into various body tissues, including a totipotent stem cell, a pluripotent stem cell, a multipotent stem cell, cell. The stem cells may be used in combination with terms such as precursor cells, progenitor cells, and the like.

In the present invention, the stem cell may be an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC). That is, the culture medium composition of the present invention can induce the differentiation of musculoskeletal precursor cells from embryonic stem cells or induced pluripotent stem cells.

The embryonic stem cell refers to a cell having a starch-like ability, and it is a cell of embryonic stem cell including the ability to develop into any cell derived from transplant without proliferation, infinite proliferation, autoregulation and all three embryonic stages , But is not limited thereto.

The term " mucosloskeletal progenitor cell " in the present invention refers, without limitation, to cells that can differentiate into bone, cartilage, tendons, and muscles.

The term "differentiation" refers to a phenomenon in which the structure or function of a cell is specialized during the growth of a cell by the proliferation and proliferation, that is, the cell or tissue of the organism is changed in shape or function to perform a task given to each. In general, a relatively simple system is separated into two or more qualitatively different systems. For example, there may be qualitative or quantitative differences between parts of a biological system that were initially homogeneous, such as head or trunk distinction between eggs that were initially homogeneous in origin, or distinctions of cells such as muscle cells or neurons The state in which a difference occurs or as a result is divided into qualitatively distinguishable parts or partial systems is called eruption.

The embryonic stem cell or induced pluripotent stem cell used in the present invention is derived from human, bovine, horse, goat, sheep, dog, cat, mouse, rat or alga, preferably human.

The stem cells used in the method of the present invention may be autologous or allogenic to the subject to be derived.

In the present invention, the FGF2 signaling activator may include, but is not limited to, bFGF (basic FGF).

In the present invention, the inhibitor of TGF-beta / activin / nodal signal transduction is E-616452 (2- [3- (6-methyl-2-pyridinyl) Yl) -1,5-naphthyridine), A-83-01 (3- (6-methyl-2-pyridinyl) -1-carbothioamide) or SB431542 (4- [4- (1,3-benzodioxol-5-yl) -5- (2- pyridinyl) But is not limited thereto.

In the present invention, the Wnt signal activator is SB216763 (3- (2,4-dichlorophenyl) -4- (1 -methyl-1 H-indol- (3-chloro-4-hydroxyphenyl) amino] -4- (2-nitrophenyl) -lH- pyrrole-2,5-dione), Kenpaullone Dihydro-pyrido [3,2-d] - [1] benzazepin-6 (5H) -one), CHIR99021 (9- , 2 ': 2,3] azepino [4,5-b] indol-6 (5H) -one), CP21R7 (3- Yl) -pyrrole-2,5-dione), SB203580 (4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -5- ), H-89 (5-isoquinolinesulfonamide), Purmorphamine (2- (1-naphthoxy) -6- (4-morpholinoanilino) -9-cyclohexylpurine) -1 (2- (4-acetyl-phenylazo) -2- [3,3-dimethyl-3,4-dihydro- But are not limited thereto.

In the present invention, the ERK signal inhibitor is selected from the group consisting of AS703026 (N - [(2S) -2,3-dihydroxypropyl] -3 - [(2-fluoro-4-iodophenyl) amino ]- isonicotinamide) (4-bromo-2-chloroanilino) -7-fluoro-N- (2-hydroxyethoxy) -3-methylbenzimidazole-5-carboxamide, PD0325901 - [(2-fluoro-4-iodophenyl) amino] -benzamide), ARRY-438162 (2R) -2,3-dihydroxypropoxy] -3,4-difluoro-2- Fluoro-N- (2-hydroxyethoxy) -1-methyl-1H-benzimidazole-6-carboxamide ), RDEA119 ((S) -N- (3,4-difluoro-2 - ((2- fluoro-4-iodophenyl) amino) -6-methoxyphenyl) -1- -Dihydroxypropyl) cyclopropane-1-sulfonamide), GDC0973 ([3,4-difluoro-2- (2- fluoro-4-iodoamylino) phenyl] TAK-733 ((R) -3- (2,3-dihydroxypropyl) -6-fluoro-pyridin- -5 (3H, 8H) -dione), RO5126766 (3 - [[3- (4-fluorophenyl) Methyl-7-pyrimidin-2-yloxychromen-2-one), or XL-518 ([3,4 (3-hydroxy-3 - [(2S) -2-piperidinyl] -1-azetidinyl] Methanone), but is not limited thereto.

The culture medium composition of the present invention may preferably include N2B27 medium and KOSR medium.

The N2B27 medium contains neural basal medium (Neurobasal, Gibco), DMEM / F12 (Gibco), N2 (Gibco, catalog number: 17502048), and B27 (Gibco, catalog number: 12587010) The cells were cultured in RPMI 1640 medium supplemented with 48% DMEM / F12, 1% N2, 2% B27, 1 mM glutamine, 1% nonessential amino acid, 0.1 mM? -Mercaptoethanol, 0.1% penicillin-streptomycin, Serum albumin.

The KOSR medium can be prepared by replacing DMEM / F12 with Knockout DMEM (Life Technologies) in complete medium and may be adjusted accordingly. The complete medium consisted of 20% KnockOut Serum Replacement (Invitrogen), 1 mM glutamine (Invitrogen), 1% nonessential amino acid (Invitrogen), 0.1 mM? -Mercaptoethanol (Invitrogen), and 0.1% penicillin / streptomycin , And DMEM / F12 (Invitrogen) supplemented with 15 ng / ml bFGF (R & D Systems).

The present invention also provides a method for inducing differentiation of stem cells into musculoskeletal progenitor cells, comprising culturing stem cells in the culture medium.

The stem cells that can be induced into musculoskeletal precursor cells by culturing in the medium composition are preferably embryonic stem cells or inducible pluripotent stem cells.

In addition, the present invention provides a mucsloskeletal progenitor cell produced by the above method.

The musculoskeletal precursor cells of the present invention can differentiate into bone, cartilage, muscle, or tendon.

The musculoskeletal precursor cells of the present invention can be differentiated into ectoderm or mesoderm.

The musculoskeletal precursor cells of the present invention are positive for CD73, CD105, CD146 or CD166 among the mesenchymal stem cell markers and negative for CD90.

The musculoskeletal precursor cells of the present invention are negative for Oct4, Nanog, Sox2 or Gdf3 among the differentiation-inducing markers and positive for Lin28.

The present invention also provides a pharmaceutical composition for preventing or treating musculoskeletal diseases, which comprises the above musculoskeletal precursor cells.

The present invention also provides a cell therapy agent for treating musculoskeletal diseases, which comprises the above musculoskeletal precursor cells.

The pharmaceutical composition and the cell treatment agent can be applied to all diseases to which stem cells can be applied, but most preferably they can be used for prevention or treatment of musculoskeletal diseases.

The pharmaceutical composition or cell treatment agent comprising the musculoskeletal precursor cells of the present invention can be used for osteoporosis, osteogenesis, osteogenesis imperfecta, osteopetrosis, osteosclerosis, Paget's disease, , Osteoarthritis, rickets, fracture, periodontal disease, segmental bone defect, osteolytic bone disease, primary and secondary hyperparathyroidism, hyperostosis, degenerative arthritis, degenerative arthritis, deformity arthropathy, deformity ankle arthritis, deformed arthropathy, deformed dog Arthritis, arthrosis of the patella, osteoarthritis of the patella, osteoarthritis of the simple humerus, osteochondroma of the humerus, lateral humeral condylar hyperhidrosis, humeral medial rectal hyperplasia, Hevadyn's nodule, Bushard's nodule, deformed moyamoya CM arthropathy, meniscus injury, disc disc degeneration, Biceps tendon tendon, ligament injuries, dry injured frozen shoulder, rotator cuff tear, calcific tendinitis, shoulder impulse syndrome, recurrent dislocation, There is an effect that can prevent or treat a disease selected from the group consisting of.

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

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and intrathecal administration. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on such factors as formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, . The preferred dosage of the composition is within the adult reference 10 ² -10 ⁸ cells / kg range. The term pharmaceutically effective amount refers to an amount sufficient to prevent or treat musculoskeletal disorders.

The composition of the present invention may be prepared in unit dose form by incorporating into a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art, or may be prepared by inserting it into a multi-dose container . The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents. In addition, the composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. It may also be administered once or, if necessary, further.

The term "cell therapeutic agent" used in the present invention refers to a medicament (US FDA regulation) used for the purpose of treatment, diagnosis and prevention of cells and tissues produced by separation, culture and special manipulation from human, Refers to drugs used for therapeutic, diagnostic and prophylactic purposes through a series of actions, such as living, proliferating, screening, or otherwise altering the biological characteristics of a cell in vitro or in vitro.

The term " prevention " in the present invention means all the actions of inhibiting or delaying the progress of musculoskeletal diseases by administration of the composition or cell therapy of the present invention.

The term " treatment " used in the present invention means all the actions of improving or alleviating musculoskeletal diseases by administration of the composition or cell therapy of the present invention.

The pharmaceutical composition or cell treatment agent of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of musculoskeletal diseases.

Terms not otherwise defined herein have meanings as commonly used in the art to which the present invention belongs.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1: Experimental Materials and Methods

1.1 Preparation of mouse

Balb / c-nude All mice (20-24 g) in the background at 7-10 weeks of age were purchased from Orient bio (seongnam, Korea). All animal-related experiments were conducted in accordance with the Guidelines of the Animal Care and Use Committee of Chonbuk National University. The animals were maintained at a controlled temperature (21-24 ° C) and a 12:12 h light / dark cycle environment and allowed free access to water and food.

1.2 hESC (human embryonic stem cells) and hiPCS (human induces lpuripotent  stem cells) into hMSPC (human muscloskeletal progenitor cells)

H9 hESCs were purchased from WiCell (Madison, MI, USA). hESC and hiPSC were transferred into a mitomycin C-treated CF1 monolayer on plates prepared 1 day before and cultured. The culture medium (complete medium) contained 20% KnockOut Serum Replacement (Invitrogen), 1 mM glutamine (Invitrogen), 1% nonessential amino acid (Invitrogen), 0.1 mM? -Mercaptoethanol (Invitrogen), and 0.1% penicillin / streptomycin (Invitrogen) supplemented with 10 ng / ml bovine serum albumin (Invitrogen) and 15 ng / ml bFGF (R & D Systems).

The medium was exchanged with differentiation induction medium (N2B27: adjusted KOSR = 1: 1) to induce hESC and human induced pluripotent stem cells (hiPSC) to differentiate into hMSPCs. The differentiation induction medium contained 20 ng / ml human LIF (Life Technologies), 15 ng / ml basic FGF, 3 μM CHIR99021 (Calbiochem), 1 μM PD0325901 (Calbiochem), 10 μM SB431542 (Sigma). At this time, the N2B27 medium contained 48% Neurobasal (Life Technologies), 48% DMEM / F12, 1% N2 (Life Technologies), 2% B27 (Life Technologies), 1 mM glutamine, 0.1% penicillin-streptomycin, and 5 mg / ml bovine serum albumin (Sigma). KOSR medium was prepared by replacing DMEM / F12 with Knockout DMEM (Life Technologies) in complete medium. The adjusted KOSR was prepared by culturing CF1 mouse embryonic fibroblasts in KOSR for 24 hours and collecting culture broth. In order to increase single cell viability, ROCK (Rho-associated coiled-coil kinase) inhibitor (Y-27632, 10 μM, Calbiochem) and PKC (protein kinase C) inhibitor (G, 2.5 μM, Sigma) were added to hECS and hiPSC Lt; / RTI >

Differentiated cells were induced by culturing trypsinized hESCs or hiPSCs with TrypLE (Life technology) in induction medium on induction medium on bitronectin + gelatin (1 ng / ml, Sigma). Cells were transfected every 2-3 days with trypsinization without ROCK inhibitor and PKC inhibitor.

1.3 Histological analysis

The digested samples were fixed overnight at 4 ° C in 2% paraformaldehyde (PFA) (Wako) and lime was removed with 0.4 M EDTA in PBS (pH 7.2) for 2 weeks at 4 ° C. The samples were then dehydrated in alcohol or xylene, embedded in paraffin or embedded in OCT by cryoprotection in sucrose and cut. Representative sections were stained with H & E, and modified Movat pentachrome (Cosmobio).

1.4 Immunofluorescence

Cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and resuspended in 10% normal goat, normal rabbit or fetal bovine serum in phosphate buffer (PBS) Respectively. Samples were obtained from Tuj1 (Covance), Nestin (Chemicon), Desmin (Chemicon), Foxa2 (Millipore), Sox17 (R & D), α-SMA, Sigma, Nanog (Santa Cruz) (R & D), HNK-1 (Santa Cruz), MAP (R & D), CD31 (DAKO), vascular endothelial-cadherin -2 (Santa Cruz), and Gremlin-1 (Santa Cruz) overnight at 4 ° C. The cells were then stained with secondary antibody Alexa Fluor 488-goat anti-mouse IgG, Alexa Fluor 594-donkey anti-rabbit IgG, Alexa Fluor 488-donkey anti-rabbit IgG, and Alexa Fluor 594-donkey anti-mouse IgG Invitrogen) . The nuclei were stained with DAPI (4,6-diamidino-2-phenylindole). Images were acquired using an Olympus IX71 fluorescence microscope and MetaMorph software (Molecular Devices).

To stain tissue with immunofluorescence, explants and severed femurs were fixed with 4% PFA (Wako) in PBS overnight at 4 ° C. Lime was removed from all samples with Morse solution. After paraffin embedding (Leica Biosystems), the samples were cut to a thickness of 5 μm. The slides were stained with H & E and Movat pentachrome. Tissue sections were blocked with 3% hydrogen peroxide for 15 minutes and then incubated overnight at 4 ° C with the primary antibody. The primary antibodies treated at the cross-sections were: a mouse monoclonal antibody (Abcam) for HLA class I, a goat polyclonal antibody (Santacruz) for Collagen Type II, a rabbit polyclonal antibody (Santacruz) for osteocalcin, , Osterix (Abcam), p-myosin light chain (Abcam), Scleraxis (antibodies-online), Runx2 (Novus), and Sclerostin (Santacruz). The secondary antibodies used are Alexa 555 (Invitrogen) and Alexa 488 (Invitrogen) IgG. Immunostained sections were stained with TO-PRO3 (Invitrogen) to stain nuclei. Fluorescence labeled tissue sections with a Leica DM 5000 microscope (Leica Microsystems) or a confocal microscope (LSM510; Carl Zeiss) were captured and validated with Zen software.

1.5 RNA isolation and RT-PCR

Total RNA was extracted using the RNeasy Plus mini kit (Qiagen) according to the manufacturer's instructions and treated with DNase I (Promega) for 15 minutes to eliminate the possibility of contamination by genomic DNA. CDNA was synthesized from 1 μg of RNA in 20 μl volume using PrimeScript 1st strand cDNA Synthesis Kit (TaKaRa). PCR amplification was then performed using a 50 μl volume of reaction solution containing a general reaction mixture for PCR (TaKaRa) kit and 1 μM primer set and cDNA (<500 ng) in a PCR machine (MyCycler, BIORAD). GAPDH was used as an internal control group. Table 1 lists the primers used.

1.6 Flow cytometry

The cultured cells were treated with trypsin / EDTA to separate into a single cell suspension, blocked with 2% BSA in PBS, and then stained with CD73, CD90, and CD90 in buffer solution [1XPBS, 1% BSA, and 0.01% sodium azide] 0.0 &gt; CD205, &lt; / RTI &gt; CD146, CD166 (BD Biosciences). Cells were then incubated with Alexa Fluor 488 secondary mouse IgGs (Invitrogen, Carlsbad, Calif.) And analyzed using a flow cytometer (FACStar Plus Flow Cytometer, BD Biosciences). Normal mouse IgGs (BD Biosciences) were used as negative control.

1.7  in vitro in Mesenchymal stem cells ( MSC ) And hMSPC Osteoblast , Adipocyte and chondrocyte differentiation

To differentiate mesenchymal stem cells (MSC) and hMSPC into osteoblasts and adipocytes, cells were isolated with trypsin / EDTA, centrifuged at 100 x g for 5 min and seeded at 5 x 10 ³ cells / cm ² in a culture vessel Respectively. Cells were cultured in induction medium at 37 ° C, 5% CO ₂ for one day. The medium was replaced with a pre-warmed complete osteogenesis differentiation kit (Life Technology) or a StemPro adipogenesis Differentiation Kit (Life Technology). The cultures were re-fed every 3-4 days. After 14 days, the cells were stained with alkaline phosphatase staining (Roche) or alizarin red S (Sigma) for observation of osteogenesis, and oil red O) (Sigma).

In order to differentiate into chondrocytes, the cells were separated with trypsin / EDTA, centrifuged at 100 × g for 5 minutes, and reconstituted with 1 ml of StemPro chondrogenesis differentiation kit (Life Technology) After cloudy, the cells were centrifuged again. For the formation of micromass, the pellet was resuspended in 1 x 105 viable cells / l in the differentiation medium and ⁵ ul of the cell solution was inoculated at the center of the unsubstantiated 96-well plate. After culturing the micromass for 2 hours under high humidity conditions, the cartilage formation medium heated in the culture vessel was added and cultured in a 5% CO ₂ incubator at 37 ° C. The cultures were re-fed every 3-4 days. After 14 days, the cartilage-producing pellet was stained with Alcian blue, and gene expression analysis was performed.

1.8 in vitro Induction of hMSPC into endothelial cells and smooth muscle cells

hMSPC and HUVEC were differentiated into endothelial cells (ECs) or smooth muscle cells (SMCs). The cells were treated with 50 ng / ml vascular endothelial growth factor (ProSpec, Rehovot, Israel) and 10 ng / ml bFGF (basic fibroblast growth factor) in EC differentiation medium (EGM) -2 (Lonza, Walkersville, MD) ml), 2.5 ng / ml TGF-β1 (5 μg / ml) in SMC differentiation medium (SMCM: ScienCell Research Laboratories, Carlsbad, Calif. (transforming growth factor beta 1, ProSpec) for 6 days.

1.9 in vitro in hNSC human neuronal stem cells &lt; RTI ID = 0.0 &gt; and hMSPC  Induction of differentiation into neurons

Human neural stem cells (hNSCs) differentiated from ready-to-use H9 hESCs were purchased from GIBCO. hNSCs were maintained in knockout DMEM / F12 (GIBCO) containing 2 mM GlutaMAX, 20 ng / ml bFGF, 20 ng / ml EGF and 2% StemPro neural supplements. To differentiate into neurons, hMSPC and hNSC were plated in polyornithine and laminin-coated culture dishes. Two days later, the medium was replaced with neural differentiation medium (Neurobasal medium containing 2% B27, 2 mM GlutaMAX and antibiotics). On the 7th day of differentiation, 0.5 mM dibutyl cAMP (Sigma) was added daily for 3 days.

1.10 in vivo Induces differentiation of hMSPC into muscle, ligament, cartilage and bone

hMSPC (10 ⁶ -10 ⁷ cells in Matrigel) was transplanted into the subcutaneous and fascia of Balb / c nude mice to determine the differentiability of hMSPC. hMSPC aggregates (4 x 10 ⁵ ) were implanted under the kidney capsules of Balb / c nude mice. After 2-6 weeks of transplantation, the transplanted cells were removed and the tissue was analyzed.

1.11 fracture studies

Collagen cell carriers (CCC, 500042933, Viscofan-bioengineering, Weinheim, Germany) were placed in phosphate buffered saline (PBS) for 30 minutes to analyze the bone formation of hMSPCs in the long bone fracture model. After washing the PBS, the CCC was left overnight to dry to make it slightly opaque. SPC was inoculated into CCC and cultured. In one 6-week-old Balb / c-nude mouse, unilateral femoral osteotomy was pinned. The SPC supported by the CCC was then inserted into the fracture site of the mouse. The fractured bone was placed for 6 weeks. Images of the fracture site were obtained using an X-ray (Kodak DXS 4000 pro system, Rochester, USA).

To analyze the bone formation of hMSPC in a skull fracture model, a 7-week-old Balb / c-nude mouse was made with a 5-mm bony skull of the right parietal region. SPC cells were primed for 7 days in StemPro Osteogenesis Differentiation Kit (Life technology). After priming SPC, 1x10 ⁴ cells were inoculated into a scaffold made of hyaluronic acid-loaded poly (lactic-co-glycolic acid (HA-PLGA) for 24 hours and transplanted into empty areas.

1.12 micro CT (microCT)

Using a Micro-CT (Skyscan 1076, Antwerp, Belgium), three-dimensional reconstructed computed tomography (CT) images were obtained by scanning the hMSPC-generated bone site with lime removed. The data were then digitized using a frame grabber, and the resulting images were transferred to a computer using the Comprehensive TeX Archive Network (CTAN) topographic reconstruction software.

Example 2: Experimental results

2.1 Identification of induction of differentiation of hMSPCs derived from hESCs

The differentiation of hMSPCs was induced from hESCs as shown in Example 1.2 above, and the morphological changes of induced hMSPCs were observed, and the results are shown in Fig. 1A.

As shown in FIG. 1A, it was confirmed that the single celled undifferentiated H9 hESCs changed into a single population within the 5th passage. After 5 passages, the cells showed a shape similar to that of fibroblasts, and morphological changes of the cells were not observed even after 20 passages.

In addition, the expression of the differentiation marker was observed by immunofluorescence in the hMSPC after passage from the hESC to the passage of 10 or more passages, and the observation result is shown in Fig. 1B.

As shown in FIG. 1B, H9 hESC was positive for both OCT4, NANOG, SOX2, and LIN28, confirming the ability to differentiate. On the other hand, hMSPC derived from H9 hESC showed negative for OCT4, NANOG and SOX2, but positive for LIN28.

1C shows the results of RT-PCR of the expression of the differentiation-ability markers identified by immunofluorescence in the hMSPC of 5, 10, and 15 passages, respectively.

As shown in FIG. 1C, H9 while hESC the identified mRNA expression in all around the multipotential markers and hMSPC Prior multipotential marker of oct4, nanog, sox2, gdf3 expression derived from H9 hESC are Although not observed, lin28 expression was observed And lin28 expression was higher than that of H9 hESC. This result is consistent with the result of FIG. 1B.

In addition, the expression of ectoderm, mesoderm, and endoderm markers in hMSPC derived from hESC of passage number 10 was confirmed by immunofluorescence, and the results are shown in Fig. 1D.

As shown in FIG. 1D, the ectoderm markers Tubulin and Nestin, the mesoderm markers Desmin and a-SMA were positive in hMSPC, and the endoderm markers Foxa2 and Sox17 were negative. Therefore, it can be seen that hMSPC derived from H9 hESC is likely to develop into ectoderm or mesodermal lobe.

In addition, the expression of ectoderm, mesoderm, and endoderm markers identified by immunofluorescence in FIG. 1D in hMSPC of 5th passage was confirmed by RT-PCR. FIG. 1E shows the results.

As shown in FIG. 1E, the expression of mesodermal markers such as α- SMA and desmin was observed in hMSPC , while the expression of tubulin and nestin as ectoderm markers was observed, whereas the expression of foxa2 and sox17 as endoderm markers was not observed. These results are consistent with the results of FIG. 1D.

2.2 in vitro Of hMSPC and mesenchymal stem cells

To compare the characteristics of hMSPC and mesenchymal stem cells, expression of surface antigens was compared as follows.

Flow cytometry analysis was performed on the mesenchymal stem cell markers CD73, CD90, CD105, CD146 and CD166 in hMSC and the hMSPC of Example 2.1 above. The results of flow cytometry analysis in which the mesenchymal stem cell markers are indicated by orange lines and the control group is indicated by blue lines are shown in Fig. 2A.

As shown in Fig. 2A, CD73, CD105, CD146, and CD166 among mesenchymal stem cell markers were found in both hMSC and hMSPC, whereas CD90 of mesenchymal stem cell markers was found in hMSC, but not in hMSPC Respectively. Therefore, it was confirmed that hMSPC has similar potential for differentiation to mesenchymal stem cells.

In addition, bone formation, cartilage formation and fat formation were compared in hMSC and hMSPC of Example 2.1, and the results are shown in Fig. 2B.

As shown in FIG. 2B, it was confirmed that hMSCs were differentiated into bone, cartilage and fat, and hMSPC was differentiated into bone and cartilage but not into fat.

In addition, the expression of bone cell markers osteocalcin, ALP and RUNX2 during 9 days of osteogenic process of hMSC and hMSPC of Example 2.1 is shown in Fig. 2C.

As shown in FIG. 2C, the bone cell markers ALP and RUNX2 were expressed in the bone formation process of hMSPC, and it was confirmed that the markers were expressed at a higher level in the bone formation process of hMSPC than hMSC. Therefore, it was confirmed that hMSPC could be differentiated into bone and cartilage.

FIG. 2D shows the results of comparing the expression of the cartilage markers AGC, SOX9, COL1A1, COL1A2 and catrigen2 before and after differentiation of hMSPC derived from hESC of 2.1 above into chondrocytes.

As shown in FIG. 2D, the cartilage markers AGC, catrigen2, COL1A1, and COL1A2 were expressed more in hMSPC than in hMSC, and the cartilage markers were expressed at a higher level after hMSPC was differentiated into cartilage cells.

These results indicate that hMSPC is more likely to differentiate into bone cells and cartilage cells than hMSC, and hMSPC is not differentiated into adipocytes.

2.3 in vitro Of potential hMSPCs for differentiation into smooth muscle and skeletal muscle

In order to evaluate the ability of the hMSPC of 2.1 above to differentiate into two major vascular system cells, smooth muscle and endothelial cells,

Immunofluorescence was performed on SM22α and SM-MHC (smooth muscle myosin heavy chain) as markers of smooth muscle in hMSPC, and the results are shown in FIGS. 3A and 3B. Human atrial smooth muscle cells (HASMC) were used as a positive control for SM22α and SM-MHC.

As shown in Figs. 3A and 3B, it was confirmed that hMSPC had the potential to differentiate into smooth muscle.

The results of immunofluorescence for the endothelial cell markers CD31 and VE-cadherin are shown in FIGS. 3C and 3D. HUVEC was used as a positive control for endothelial cell differentiation.

As shown in FIGS. 3C and 3D, no expression of CD31 and VE-cadherin was observed in hMSPC, confirming that hMSPC had no potential to differentiate into endothelial cells. On the other hand, the control group HUVEC confirmed that the marker was expressed.

FIG. 3E shows the results of performing immunofluorescence on MAP2 as a neural cell differentiation marker. NSC (neuronal stem cells) were used as a positive control for neuronal differentiation.

As shown in FIG. 3E, it was confirmed that hMSPC had no potential to differentiate into neurons.

Taken together, the hMSPC is not capable of differentiating into endothelial cells. In addition, as confirmed in Example 2.1 above, it was confirmed that hMSPC is likely to develop into ectoderm but not into neurons. Therefore, hMSPC can be differentiated into endoderm, more specifically bone, cartilage, and muscle.

2.4 hiPSC human induced pluripotent  stem cells) hMSPC  Identification of induction of differentiation and induced hiPSC

hiPSC was prepared by reprogramming IMR90 fetal fibroblasts by overexpression of sendai virus-mediated OCT4, KLF4, SOX2, and MYC according to the protocol developed by Hasegawa et al. (Fusaki et al., 2009).

HMSPC was derived from hiPSC in the same manner as in Example 1.2 to obtain hMSPC. The expression levels of Oct4, Nanog, Sox2, and Gdf3 as hmPSC-derived hMSPCs were confirmed by immunofluorescence and RT-PCR, respectively, and are shown in Figs. 4A and 4B, respectively.

As shown in FIG. 4A, iPS cells were positive for both OCT4, NANOG, SOX2, and LIN28, indicating that they were capable of differentiating. On the other hand, hMSPC derived from hiPSC was negative for OCT4, NANOG and SOX2, but positive for LIN28.

As shown in Figure 4B, whereas the identified mRNA expression in all around the multipotential markers in iPS cells, in the hMSPC derived from hiPSC former of multipotential marker oct4, nanog, sox2, gdf3 expression did not observed, lin28 expression was observed , And this result is consistent with the result of Fig. 4A.

In addition, the expression of markers for ectoderm, mesoderm, and endoderm in hMSPC derived from hiPSC was confirmed by immunofluorescence and the results are shown in Fig. 4C, respectively.

As shown in FIG. 4C, the ectoderm markers Tubulin and Nestin, the mesoderm markers Desmin and a-SMA were positive in hMSPC, and the endoderm markers Foxa2 and Sox17 were negative. Therefore, it can be seen that the hMSC derived from hiPSC is likely to develop into ectoderm or mesodermal lobe.

In addition, the expression of the ectoderm, mesoderm, and endoderm markers identified by immunofluorescence in FIG. 4C in hMSPC of the fifth passage was confirmed by RT-PCR, and is shown in FIG. 4D.

As shown in Fig. 4D, in hMSPC, α- SMA , desmin And ectodermal markers tubulin and nestin , while the expression of endoderm markers foxa2 and sox17 was not observed. This result is consistent with the result of FIG. 4C.

In order to compare the characteristics of hMSC-derived hMSCs and mesenchymal stem cells, expression of surface antigens was compared as follows.

Flow cytometric analysis of mesenchymal stem cell markers CD73, CD90, CD105, CD146 and CD166 was performed in hMSPC derived from hiPSC. The results of flow cytometry analysis in which the mesenchymal stem cell markers are indicated by orange lines and the control group is indicated by blue lines are shown in Fig. 4E.

As shown in FIG. 4E, CD73, CD105, CD146, and CD166 among the mesenchymal stem cell markers were all found in hMSPC, whereas CD90 of mesenchymal stem cell markers was not found in hMSPC. Therefore, it was confirmed that hMSCs derived from hiPSC had similar potential for differentiation to mesenchymal stem cells.

In addition, hMSPC bone formation, cartilage formation, and fat formation induced by hiPSC were compared, and the results are shown in FIG. 4F.

As shown in FIG. 4F, hMSC derived from hiPSC was differentiated into bone and cartilage but not into fat.

4G shows the result of performing immunofluorescence on SM22α, SM-MHC (smooth muscle myosin heavy chain) as markers of smooth muscle in hMSPC derived from hiPSC.

As shown in Fig. 4G, it was confirmed that the hMSPC derived from hiPSC had the potential to differentiate into smooth muscle.

Taken together, these results confirm that hMSPC derived from hiPSC has the same characteristics as hMSPC derived from hECS, and hMSC can be obtained using hiPSC as an alternative to hECS.

2.5 in vivo Of differentiation of hMSPC into cartilage, muscle and tendon

In order to measure the differentiation potential of hMSPC derived in the same manner as in Example 1.2 in vivo , hMSPC was transplanted into the subcutaneous and fascia of immunodeficient mice. Five weeks after transplantation of hMSPCs onto the site, the tissues were stained with H & E and Movat's pentachrome and stained for comparison with TO-PRO3. The results are shown in FIGS. 5A to 5C.

As shown in FIG. 5A, it was confirmed that hMSPC formed mineralized cartilage tissue (green) and non-mineralized cartilage tissue (yellow) under the skin (FIGS. 5Aa and 5Ab). In addition, it was confirmed that a typical cartilage-like structure was formed by H & E staining, and thus it can be seen that hMSPC formed chondrocytes. In addition, immunohistochemical analysis showed that collagen type II (Col2), a cartilage marker, and hLA (human leukocyte antigen), a human cell marker, were positive, indicating that the transplanted hMSPCs differentiated into chondrocytes. [Scale bar: 1 mm (a), 100 μm (b, c), 500 μm (i), 100 μm (ii, iii, iv)].

As shown in Fig. 5B, it was confirmed that hMSPC was differentiated into a typical muscle form in the fascia as a result of Movat's Pentachrome staining (Fig. 5Ba and Fig. 5Bb) and H & E (Fig. 5Bc) staining. Thus, have. Immunohistochemical analysis showed that the transfected hMSPCs were differentiated into muscle cells because it was confirmed that p-MLC (phosphorylated myosin light chain), which is a muscle marker, and hLA (human leukocyte antigen), a human cell marker were positive . [Scale bar: 1 mm (a), 100 μm (b, c), 500 μm (i), 100 μm (ii, iii, iv)].

As shown in Fig. 5C, it was confirmed that hMSPC was differentiated into a typical muscle form under the fascia around the tendon as a result of Movat's Pentachrome staining (Figs. 5Ca and 5Cb) and H & E (Fig. 5Cc) staining. . In addition, immunohistochemical analysis revealed that the transfected hMSPCs were differentiated into dry cells because it was confirmed that Scm (scleraxis), which is a key marker, and hLA (human leukocyte antigen), a human cell marker, were positive. [Scale bar: 1 mm (a), 100 μm (b, c), 500 μm (i), 100 μm (ii, iii, iv)].

Based on the above results, it was confirmed that the hMSPC of the present invention can be differentiated into cartilage, muscle, tendon and endochondral bone depending on the transplantation site, and excellent in the differentiation ability.

2.6 Confirmation of intra-cartilage osteogenesis of hMSPC by ossification in cartilage

Example 1.2 The same induction hMSPC (4 x 10 ⁵ cells) and the rats are implanted under the kidney capsule of immunodeficient mice. After hMSPC was transplanted to the site, the transplanted cells were removed, and the tissues were stained with H & E and Movat's pentachrome, and stained with TO-PRO3. The results are shown in FIGS. 6A and 6B.

As shown in Fig. 6A, it was confirmed that hard tissue, that is, bone was formed at the implantation site of hMSPC (Fig. 6Aa and Fig. 6Ab). It was confirmed that bone and cartilage were formed together with H & E staining and pentachrome staining. It was confirmed that the tissue was composed of mineralized cartilage tissue (green), non-mineralized cartilage tissue (yellow ) And mineralized bone tissue (red) (Fig. 6Ac and Fig. 6Ad). Therefore, it can be seen that the transplanted hMSPC is differentiated into cartilage and bone under the kidney capsule. In addition, immunohistochemical analysis revealed the expression of hLA (human leukocyte antigen) and Col II, a cartilage marker, in the cells inside the tissue. In the cells outside the tissue, human leukocyte antigen (hLA) It was confirmed that OsM (osterix), a bone marker, and OCN (osteocalin), a bone marker, were differentiated into bone and cartilage because the transplanted hMSPC was positive. [Scale bar: 500 μm (b, c), 100 μm (i, ii, iii, iv)]. In addition, in order to confirm the presence of intra-ossicular ossification and angiogenesis, the bone marrow section was stained with anti-vWF (von Willebrand factor) antibody. The result of the staining is shown in FIG. 6C, and as shown in FIG. 6C, vWF-positive cells were found at the junction of kidney and bone marrow. Since vWF-positive cells are derived from mice, it can be seen that angiogenesis is necessary for intra-cartilage differentiation.

When the differentiation was further promoted, it was confirmed that cartilage disappeared and a medullary cavity was formed at the transplantation site of hMSPC as shown in Fig. 6B (Fig. 6Ba and Fig. 6Bb). It was confirmed that a marrow cavity was formed similarly by H & E staining and pentachrome staining (Figs. 6Bc and 6Bd). As a result of immunohistochemical analysis, it was confirmed that hLA (human leukocyte antigen) as a human cell marker, Osx as a bone marker, OCN as a bone marker, and Sclerostin as a bone marker were positive, so that the transplanted hMSPC was differentiated into a bone . [Scale bar: 1 mm (b, c), 100 μm (i, ii, iii, iv)].

When the hMSPC is implanted under the kidney capsule, the hMSPC is differentiated into bone and cartilage, and the process of forming the lecture is completed and the cartilage disappears. Thus, it can be understood that the hMSPC is differentiated into the bone through ossification of the cartilage.

2.7 Confirmation of the effect of hMSPC on fracture recovery

In order to confirm the effect of the hMSPC induced recovery in the same manner as in Example 1.2 on fracture healing, a fracture study was conducted as shown in Examples 1.11, 7A and 7B, and the results are shown in Figs. 7C to 7E .

As shown in FIGS. 7C to 7E, cells expressing hLA and Runx2 simultaneously were found in the fracture site, indicating that the differentiation of the transplanted cells was differentiated into chondrocytes and bone cells.

In addition, bone formation of hMSPC was confirmed in vivo by injecting HA-PLGA and cells into the infected skull of immunodeficient mice. After priming of hMSPC with osteogenic medium for 7 days, cells were inoculated into HA-PLGA graft. As shown in FIG. 7F, Runx2, a bone formation marker, was expressed in chondrocytes and osteocytes of tissues, and it was confirmed that transplantation of HA-PLGA-loaded hMSPC completely cures skull bone defects. Therefore, it was confirmed that hMSPC could be involved in intramural ossification.

The results of the above experiments show that the FGF2 (Fibroblast Growth Factor 2) signaling activator, TGF-β / activin / nodal signal transduction inhibitor, Wnt signal activator, ERK (extracellular such as embryonic stem cells or inducible pluripotent stem cells, by using a medium composition for inducing the differentiation of stem cells into mucosal bone marrow progenitor cells, which comprises a signal-regulated kinase signaling inhibitor and a leukemia inhibitory factor (LIF) It is possible to differentiate into hMSPC from cells, and since hMSPC having differentiation ability into bone, muscle, cartilage, and tendon in vivo can be used effectively for various musculoskeletal diseases.

Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.

Claims (14)

1. (TGF-beta / activin / nodal) signal transduction inhibitor, a Wnt signal activator, an extracellular signal-regulated kinase (ERK) signal inhibitor, and A medium composition for inducing differentiation from stem cells to a musculoskeletal progenitor cell, comprising a leukemia inhibitory factor (LIF).
2. 2. The composition of claim 1, wherein the FGF2 signaling activator is bFGF (basic FGF).
3. 2. The method of claim 1, wherein the TGF- / activin / nodal signaling inhibitor is E-616452 (2- [3- (6-methyl-2-pyridinyl) Yl) -1,5-naphthyridine), A-83-01 (3- (6-methyl-2-pyridinyl) -N- phenyl-4- (4-quinolinyl) (3-benzodioxol-5-yl) -5- (2-pyridinyl) -lH-imidazol- Benzamide). &Lt; / RTI &gt;
4. The method of claim 1, wherein the Wnt signal activator is selected from the group consisting of SB216763 (3- (2,4-dichlorophenyl) -4- (1 -methyl-1 H-indol-3-yl) ), SB415286 (3 - [(3-chloro-4-hydroxyphenyl) amino] -4- (2-nitrophenyl) -1H- pyrrole-2,5-dione), Kenpaullone 7,12-dihydro-indolo [3,2-d] - [l] benzazepin-6 (5H) -one), CHIR99021 (9-Bromo-7,12-dihydro- (3-amino-phenyl) -4- (1-methyl-1H) -quinolinone (4-fluorophenyl) -5- (4-pyridyl) -lH- -Imidazole), H-89 (5-isoquinolinesulfonamide), Purmorphamine (2- (1-naphthoxy) -6- (4-morpholinoanilino) -9-cyclohexylpurine) , Or IQ-1 (2- (4-acetyl-phenylazo) -2- [3,3-dimethyl-3,4-dihydro-2H-isoquinoline- (1E) -ylidene] -acetamide) , &Lt; / RTI &gt;
5. 2. The method of claim 1 wherein the ERK signal inhibitor is selected from the group consisting of AS703026 (N - [(2S) -2,3-dihydroxypropyl] -3- [(2-fluoro-4-iodophenyl) Amide), AZD6244 (6- (4-bromo-2-chloroanilino) -7-fluoro-N- (2- hydroxyethoxy) -3- methylbenzimidazole- PDO325901 (N - [(2R) -2,3-dihydroxypropoxy] -3,4-difluoro-2 - [(2- fluoro-4-iodophenyl) amino] ARRY-438162 (5 - [(4-bromo-2-fluorophenyl) amino] -4-fluoro-N- (2- hydroxyethoxy) Carboxamide), RDEA119 ((S) -N- (3,4-difluoro-2 - ((2- fluoro-4-iodophenyl) amino) -6-methoxyphenyl) -1- (2,3-dihydroxypropyl) cyclopropane-1-sulfonamide), GDC0973 ([3,4-difluoro-2- (2- fluoro-4-iodoamylino) phenyl] (R) -3- (2,3-dihydroxypropyl) -6- (2S) -piperidin-2-yl] -azetidin- - Fluorine (3H, 8H) -dione), RO5126766 (3- [2-fluoro-4-iodophenylamino] -8-methylpyrido [ Methyl-7-pyrimidin-2-yloxychromen-2-one) or XL-518 ([(3-fluoro-2- Amino] phenyl] [3-hydroxy-3 - [(2S) -2-piperidinyl] -1- Thienyl] methanone).
6. A method for inducing differentiation of a stem cell into a musculoskeletal progenitor cell, comprising culturing the stem cell in the medium composition of any one of claims 1 to 5.
7. A mucosloskeletal progenitor cell produced by the method of claim 6.
8. 8. The musculoskeletal precursor cell according to claim 7, wherein the cell is a cell capable of differentiating into bone, cartilage, muscle, or tendon.
9. 8. The musculoskeletal precursor cell according to claim 7, wherein the cell is a cell capable of differentiating into an ectoderm or mesoderm.
10. 8. The musculoskeletal precursor cell of claim 7, wherein the cell is positive for CD73, CD105, CD146 or CD166 and negative for CD90.
11. 8. The musculoskeletal precursor cell according to claim 7, wherein said cell is negative for Oct4, Nanog, Sox2 or Gdf3 and positive for Lin28.
12. A pharmaceutical composition for preventing or treating musculoskeletal diseases, comprising the musculoskeletal precursor cells of claim 7.
13. 13. The method of claim 12, wherein the musculoskeletal disease is selected from the group consisting of osteoporosis, osteogenesis, osteogenesis imperfecta, osteopetrosis, osteosclerosis, Paget's disease, bone cancer, arthritis, rickets, , Periodontal disease, segmental bone defect, osteolytic bone disease, primary and secondary hyperparathyroidism, hyperostosis, degenerative arthritis, degenerative knee osteoarthritis, deformed arthropathy, ankle osteoarthritis, deformed arthropathy, deformed arthropathy, Bradycardia nodule, bushard's nodule, deformed moyamoya CM arthropathy, meniscus injury, intervertebral disk degeneration, cruciate ligament brachialis tendon tendon, ligament injuries, arthritis, osteoarthritis of the humerus, , Dry injury disorganized dog, rotator cuff tear, calcific tendinitis, shoulder impulse syndrome, recurrent dislocation, and habitual dislocation. A pharmaceutical composition for preventing or treating musculoskeletal diseases.
14. A cell therapy agent for treating musculoskeletal diseases, comprising the musculoskeletal precursor cell of claim 7.

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