METHOD FOR REGULATION OF SELECTIVE DIFFERENTIATION OF MUSCULOSKELETAL STEM CELLS

Abstract

The present disclosure is novel musculoskeletal stem cells (MSSCs) derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), and a medium composition for selectively differentiation into bone, tooth, cartilage, ligament and muscle, and a method for inducing differentiation thereof.

Claims

1. A medium composition inducing of selective differentiation into only muscle or only tendon and ligament from musculoskeletal stem cells (MSSCs) that can be differentiated into bone, tooth, cartilage, tendon, ligament, muscle and adipose; wherein when the medium composition induces selective differentiation into only muscle, the medium composition comprises heparin; wherein when the medium composition induces selective differentiation into only tendon or ligament, the medium composition comprises connective tissue growth factor (CTGF) and ascorbic acid; and wherein the musculoskeletal stem cells have the following characteristics: a) positive for the ectodermal marker nestin (NES); b) positive for the myogenic satellite marker Pax7; c) positive for the mesodermal marker α-SMA; and d) negative for the pluripotency marker LIN28.

2. A pharmaceutical composition for preventing or treating a musculoskeletal disease, comprising the medium composition for inducing differentiation according to claim 1 and musculoskeletal stem cells (MSSCs) as active ingredients.

3. A method for inducing selective differentiation from musculoskeletal stem cells (MSSCs) into only muscle, comprising the step of treating the musculoskeletal stem cells that can differentiate into bone, tooth, cartilage, tendon, ligament, muscle and adipose with the medium composition of claim 1.

4. (canceled)

5. (canceled)

6. A method for inducing selective differentiation from musculoskeletal stem cells (MSSCs) into only tendon or ligament, comprising the step of treating the musculoskeletal stem cells that can differentiate into bone, tooth, cartilage, tendon, ligament, muscle and adipose with the medium composition of claim 1.

7. A medium composition for selectively inducing differentiation into only bone or only tooth from musculoskeletal stem cells (MSSCs) that can be differentiated into bone, tooth, cartilage, tendon, ligament, muscle and adipose; wherein when the medium composition induces selective differentiation into only bone, the medium composition comprises insulin, hyaluronic acid and ascorbic acid; wherein when the medium composition induces selective differentiation into only tooth, the medium composition comprises dental epithelial cells; and wherein the musculoskeletal stem cells have the following characteristics: a) positive for the ectodermal marker nestin (NES); b) positive for the myogenic satellite marker Pax7; c) positive for the mesodermal marker α-SMA; and d) negative for the pluripotency marker LIN28.

8. A pharmaceutical composition for preventing or treating a musculoskeletal disease, comprising musculoskeletal stem cells (MSSCs) pretreated with the medium composition for inducing differentiation according to claim 7 as active ingredients.

9. A method for inducing selective differentiation from musculoskeletal stem cells (MSSCs) into only bone, comprising the step of treating the musculoskeletal stem cells that can differentiate into bone, tooth, cartilage, tendon, ligament, muscle and adipose with the medium composition of claim 7.

10. (canceled)

11. A method for inducing selective differentiation from musculoskeletal stem cells (MSSCs) into only tooth, comprising the step of coating the musculoskeletal stem cells that can differentiate into bone, tooth, cartilage, tendon, ligament, muscle and adipose with dental epithelial cells, wherein the musculoskeletal stem cells have the following characteristics: a) positive for the ectodermal marker nestin (NES); b) positive for the myogenic satellite marker Pax7; c) positive for the mesodermal marker α-SMA; and d) negative for the pluripotency marker LIN28.

12. The medium composition according to claim 1, wherein the musculoskeletal stem cells further have the following characteristics: e) negative for the mesenchymal stem cell marker CD90; f) negative for the mesenchymal stem cell marker CD271; g) positive for the pluripotency marker DPPA4; h) negative for the mesodermal markers T and nodal; i) positive for the neuroectodermal marker Pax6; j) positive for the intestinal stem cell marker LGR5; k) negative for the chondrocyte marker SOX9; or l) negative for the myoblast marker MyoD.

13. A method for treating damaged tendon or ligament, comprising a step of administering musculoskeletal stem cells (MSSCs) that can differentiate into bone, tooth, cartilage, tendon, ligament, muscle and adipose treated with the medium composition of claim 1 to a damaged tendon or ligament area of a subject.

14. A method for treating arthritis, comprising a step of administering musculoskeletal stem cells (MSSCs) that can differentiate into bone, tooth, cartilage, tendon, ligament, muscle and adipose into the articular cavity of a subject, wherein the musculoskeletal stem cells have the following characteristics: a) positive for the ectodermal marker nestin (NES); b) positive for the myogenic satellite marker Pax7; c) positive for the mesodermal marker α-SMA; and d) negative for the pluripotency marker LIN28.

15. The method according to claim 3, wherein the musculoskeletal stem cells further have the following characteristics: e) negative for the mesenchymal stem cell marker CD90; f) negative for the mesenchymal stem cell marker CD271; g) positive for the pluripotency marker DPPA4; h) negative for the mesodermal markers T and nodal; i) positive for the neuroectodermal marker Pax6; j) positive for the intestinal stem cell marker LGR5; k) negative for the chondrocyte marker SOX9; or l) negative for the myoblast marker MyoD.

16. The medium composition according to claim 7, wherein the musculoskeletal stem cells further have the following characteristics: e) negative for the mesenchymal stem cell marker CD90; f) negative for the mesenchymal stem cell marker CD271; g) positive for the pluripotency marker DPPA4; h) negative for the mesodermal markers T and nodal; i) positive for the neuroectodermal marker Pax6; j) positive for the intestinal stem cell marker LGR5; k) negative for the chondrocyte marker SOX9; or l) negative for the myoblast marker MyoD.

17. The method according to claim 6, wherein the musculoskeletal stem cells further have the following characteristics: e) negative for the mesenchymal stem cell marker CD90; f) negative for the mesenchymal stem cell marker CD271; g) positive for the pluripotency marker DPPA4; h) negative for the mesodermal markers T and nodal; i) positive for the neuroectodermal marker Pax6; j) positive for the intestinal stem cell marker LGR5; k) negative for the chondrocyte marker SOX9; or l) negative for the myoblast marker MyoD.

18. The method according to claim 9, wherein the musculoskeletal stem cells further have the following characteristics: e) negative for the mesenchymal stem cell marker CD90; f) negative for the mesenchymal stem cell marker CD271; g) positive for the pluripotency marker DPPA4; h) negative for the mesodermal markers T and nodal; i) positive for the neuroectodermal marker Pax6; j) positive for the intestinal stem cell marker LGR5; k) negative for the chondrocyte marker SOX9; or l) negative for the myoblast marker MyoD.

19. The method according to claim 11, wherein the musculoskeletal stem cells further have the following characteristics: e) negative for the mesenchymal stem cell marker CD90; f) negative for the mesenchymal stem cell marker CD271; g) positive for the pluripotency marker DPPA4; h) negative for the mesodermal markers T and nodal; i) positive for the neuroectodermal marker Pax6; j) positive for the intestinal stem cell marker LGR5; k) negative for the chondrocyte marker SOX9; or l) negative for the myoblast marker MyoD.

20. The method according to claim 13, wherein the musculoskeletal stem cells further have the following characteristics: e) negative for the mesenchymal stem cell marker CD90; f) negative for the mesenchymal stem cell marker CD271; g) positive for the pluripotency marker DPPA4; h) negative for the mesodermal markers T and nodal; i) positive for the neuroectodermal marker Pax6; j) positive for the intestinal stem cell marker LGR5; k) negative for the chondrocyte marker SOX9; or l) negative for the myoblast marker MyoD.

21. The method according to claim 14, wherein the musculoskeletal stem cells further have the following characteristics: e) negative for the mesenchymal stem cell marker CD90; f) negative for the mesenchymal stem cell marker CD271; g) positive for the pluripotency marker DPPA4; h) negative for the mesodermal markers T and nodal; i) positive for the neuroectodermal marker Pax6; j) positive for the intestinal stem cell marker LGR5; k) negative for the chondrocyte marker SOX9; or l) negative for the myoblast marker MyoD.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0195] FIGS. 1A to 1E show the characteristics of hMSSCs derived from hESCs. FIG. 1A shows the change of cell morphology of hESCs subcultured using a musculoskeletal stem cell induction medium, from passage 7 to passage 19. FIG. 1B shows a result of observing the expression of the pluripotency markers OCT4, NANOG, SOX2 and LIN28 in hMSSCs by immunocytochemistry. FIG. 1C shows a result of investigating the expression of pluripotency, ectodermal, mesodermal and endodermal markers in hESCs, hMSCs and hMSSCs at passages 7 and 17 twice through RNA sequencing. FIG. 1D shows a result of measuring the expression of cell surface antigens by flow cytometry for characterization of hMSSCs. FIG. 1E shows a result of measuring the expression of tissue-specific markers of different lineages by immunocytochemistry for characterization of hMSSCs. In the figures, DAPI indicates stained nuclei. In the figures, blue triangles indicate β-galactosidase-positive cells.

[0196] FIGS. 2A to 2E show a result of comparing the in-vitro differentiation capacity of hMSSCs with other types of cells. FIG. 2A shows a result of comparing the in-vitro bone, cartilage and adipose differentiation capacity of hMSCs and hMSSCs. FIG. 2B shows a result of confirming the potential of hMSSCs for differentiation into skeletal muscle by immunocytochemistry for the skeletal muscle cell-specific marker MYH9. C2C12 cells were used as a skeletal muscle cell positive control group. FIGS. 2C and 2D show a result of confirming the absence of the potential of hMSSCs for differentiation into endothelial cells by immunocytochemistry for the endothelial cell-specific markers CD31 and VE-cadherin. FIG. 2E show a result of confirming the absence of the potential of hMSSCs for differentiation into nerve cells by immunocytochemistry for the nerve cell-specific marker MAP2. Neural stem cells derived from H9 hESCs were used as a positive control group.

[0197] FIGS. 3A to 3C show a result of measuring the differentiation potential of hMSSCs in vivo. FIG. 3A-a shows a result of confirming the formation of typical muscle, adipose and tendon by H&E staining when hMSSCs were transplanted into the kidney. FIG. 3A-b shows a result of confirming the differentiation into muscle, adipose and tendon cells when hMSSCs were transplanted into the kidney with immunohistochemistry for the muscle-specific marker pMLC, the adipose-specific marker PPAR-gamma (PPAr), or the tendon- or ligament-specific marker SCX. hLA shows a result of staining of a human cell-specific marker for confirming the presence of human-derived cells. FIG. 3B-a shows a result of confirming the formation of bone in the kidney where hMSSCs were transplanted by micro-CT scan. FIGS. 3B-b and 3B-c show a result of confirming the formation of bone by H&E and pentachrome immunohistochemistry. FIG. 3B-d shows a result of confirming the expression of the human cell marker hLA (human leukocyte antigen), the bone markers Osx (osterix), Runx2, DMP1 and OCN (osteocalin), and the blood vessel marker vWF in osteogenic tissue by immunohistochemistry. FIG. 3C shows a result of confirming cartilage formation when hMSSCs were transplanted into the subcutaneous tissue by H&E and toluidine blue immunohistochemistry for chondrocytes. In addition, the expression of the cartilage marker Coln (collagen type II) was confirmed by immunohistochemistry.

[0198] FIGS. 4A and 4B show a result of confirming the effect of hMSSCs on recovery from fracture. FIG. 4A shows that bone was formed by the cells of mouse, not by hMSCs transplanted into the fracture area. FIG. 4A-a shows a result of micro-CT at 2, 4 and 6 weeks after the transplantation of hMSCs into the fracture area. FIG. 4A-b shows a result of H&E immunohistochemistry of the thighbone including the fracture area into which hMSCs were transplanted. FIG. 4A-c is an enlarged image of the red square in FIG. 4A-b. FIG. 4A-d shows a result of confirming that the transplanted hMSCs were not differentiated into bone cells by immunohistochemistry for the bone cell marker Runx2 and the human cell marker hLA. FIG. 4B shows that bone was formed by differentiation of hMSSCs when the hMSCs when the hMSSCs were transplanted, unlike the hMSCs. FIG. 4B-a shows a result of micro-CT at 2, 4 and 6 weeks after the transplantation of hMSSCs into the fracture area. FIG. 4B-b shows a result of H&E immunohistochemistry of the thighbone including the fracture area into which hMSSCs were transplanted. FIG. 4B-c is an enlarged image of the red square in FIG. 4B-b. FIG. 4B-d shows a result of confirming that the transplanted hMSSCs were differentiated into bone cells by immunohistochemistry for the bone cell marker Runx2 and the human cell marker hLA.

[0199] FIGS. 5A to 5D show a result of investigating whether hiPSCs are also differentiated into hMSSCs like hESCs. FIG. 5A shows a result of investigating the expression level of the pluripotency markers Oct4, Nanog, Sox2 and Lin28 in hMSSCs derived from hiPSCs by immunocytochemistry. FIG. 5B shows a result of investigating the expression of specific cell surface antigens in hMSSCs derived from hiPSCs by flow cytometry. FIG. 5C shows a result of confirming the in-vitro bone, cartilage and adipose differentiation capacity of hMSSCs derived from hiPSCs. FIG. 5D shows a result of differentiating hMSSCs derived from hiPSCs into skeletal muscle by culturing using a skeletal muscle differentiation medium and conducting immunocytochemistry for the skeletal muscle marker MYH9.

[0200] FIG. 6 shows a result of comparing the expression level of CD44 in hMSSCs induced with a CM medium and an hMSSC induction medium by flow cytometry.

[0201] FIG. 7 shows a result of investigating the effect of an hMSSC medium ingredients deficiency on differentiation into cartilage or bone by staining with alcian blue which confirms cartilage differentiation and ALP and alizarin red S which confirm osteogenic differentiation.

[0202] FIG. 8 shows the selective differentiation of hMSSCs derived from hESCs into muscle. hMSSCs (1×10.sup.6 cells) differentiated from hESCs were transplanted into the subcutaneous tissue of BALB/c nude mouse after being mixed with fibrin glue. After 7 weeks, the transplanted area was removed and then stained with hematoxylin & eosin.

[0203] FIG. 9 shows the selective differentiation of hMSSCs derived from hESCs into tendon or ligament. hMSSCs were pretreated with 50 ng/mL connective tissue growth factor and 25 μg/mL ascorbic acid for 2 days and the pretreated cells (1×10.sup.6 cells) were transplanted into the subcutaneous tissue of BALB/c nude mouse after being mixed with 100 μL of fibrin glue. After 5 weeks, the transplanted area was removed and then stained with hematoxylin & eosin. It was also stained with an antibody against hLA which binds to a human cell nucleus marker to confirm whether the differentiated tissue is derived from the hMSSCs, and immunofluorescence staining with an antibody against the tendon or ligament marker SCX marker to confirm whether the differentiated tissue is tendon or ligament.

[0204] FIG. 10 shows the selective differentiation of hMSSCs derived from hESCs into bone. hMSSCs derived from hESCs were prepared into a cell aggregate by a hanging drop method using N2B27 medium supplemented with 10 μg/mL hyaluronic acid, 5 μg/mL insulin and 25 μg/mL ascorbic acid and transplanted into the kidney of BALB/c nude mouse after culturing for 3 days. 5 weeks later, the transplanted area was removed and then subjected to hematoxylin & eosin staining, micro-CT and immunostaining. FIG. 10A shows the image of the kidney extracted 5 weeks after the transplantation of the hMSSC cell aggregate. FIG. 10B shows the micro-CT image of the kidney of FIG. 10A. FIG. 10C shows the typical bone formation by H&E staining in the hard tissue area on micro-CT image 4 weeks after transplantation of the hMSSC cell aggregates into the kidney. FIG. 10D shows a result of investigating the expression of the human cell marker hLA (human leukocyte antigen) and the cartilage marker ColII (collagen type II) in cells in osteogenic tissue by immunohistochemistry. FIG. 10E shows a result of investigating the expression of the human cell marker hLA (human leukocyte antigen) and the bone marker Osx (osterix) in cells of osteogenic tissue by immunohistochemistry.

[0205] FIG. 11 shows the selective differentiation of hMSSCs derived from hESCs into tooth. FIG. 11A shows the image of the kidney extracted 6 weeks after the transplantation of an hMSSC cell aggregate into the kidney of mouse by coating with mouse dental epithelial cell. K indicates kidney, B indicates bone, and T indicates tooth. FIG. 11B shows the micro-CT image of the kidney of FIG. 11A. FIG. 11C shows a hematoxylin & eosin staining image of the tooth of FIG. 11A. FIG. 11D shows a result of investigating the expression of the human cell marker hLA for the tooth of FIG. 11A by immunohistochemistry. To-PRO-3 shows a result of staining nuclei.

[0206] FIG. 12 shows a result of investigating the possibility of treatment of damaged ligament with hMSSCs derived from hESCs in vivo. It was confirmed by H&E staining and immunofluorescence staining for the human cell-specific marker hLA and the tendon- or ligament-specific marker SCX that broken ligament was connected as the hMSSCs were differentiated.

[0207] FIG. 13 shows a result of investigating the possibility of treatment of damaged cartilage with hMSSCs derived from hESCs in vivo. It was confirmed that the severity of arthritis was decreased to half or lower in the groups to which hMSSCs were injected (PBS and Sol groups).

BEST MODE

[0208] Hereinafter, the present disclosure will be described in more detail through examples. However, these examples are only for describing the present disclosure more specifically, and it will be obvious to those having ordinary knowledge in the art to which the present disclosure belongs that the scope of the present disclosure is not limited by the examples.

EXAMPLES

Example 1. Experimental Animals

[0209] 7- to 10-week old Balb/c-nude background mice (body weight 20-24 g) were purchased from Orient Bio (Seongnam, Korea). All animal experiments were performed according to the guidelines of the Jeonbuk National University Animal Care and Use Committee. The animals were accommodated under controlled-temperature (21-24° C.) and 12:12-hour light-dark cycle environments and were given free access to water and feed.

Example 2.1. Induction of Differentiation from hESCs into hMSSCs

[0210] H9 hESCs (human embryonic stem cells) were purchased from WiCell (Madison, Mich., USA). The hESCs were cultured on CF1 mouse embryonic fibroblast (MEF) feeder cells whose cell division was blocked by mitomycin C treatment. A hESC culture medium was prepared with DMEM/F12 (Invitrogen, USA) supplemented with 20% knockout serum replacement (hereinafter, referred to as KSR, Invitrogen, USA), 1 mM glutamine (Invitrogen, USA), 1% nonessential amino acids (Invitrogen, USA), 0.1 mM β-mercaptoethanol (Invitrogen, USA), 0.1% penicillin/streptomycin (Invitrogen, USA) and 15 ng/mL bFGF (R&D Systems, USA).

[0211] A medium for inducing differentiation from hESCs into hMSSCs (human musculoskeletal stem cells) (hereinafter, referred to as “MSSC medium”) was prepared with the following composition:

[0212] 1) 250 ng/mL human noggin (Koma Biotech, Korea),

[0213] 2) 20 ng/mL human LIF (Koma Biotech, Korea),

[0214] 3) 15 ng/mL basic fibroblast growth factor (FGF; R&D Systems, USA) (FGF2 signaling activator),

[0215] 4) 3 μM CHIR99021 (Cayman, USA) (Wnt signaling activator),

[0216] 5) 1 μM PD0325901 (Cayman, USA) (ERK (extracellular signal-regulated kinase) signaling inhibitor),

[0217] 6) 10 μM SB431542 (Tocris, United Kingdom) (TGF-β/activin/nodal (TGF-β/activin/nodal) signaling inhibitor),

[0218] 7) Others: 10% knockout serum replacement (Invitrogen, USA), 1% N2 supplement (GIBCO, USA), 2% B27 supplement (GIBCO, USA), 1% nonessential amino acids (GIBCO, USA), 43% DMEM/F12 (GIBCO, USA), 43% Neurobasal (GIBCO, USA), 1 mM glutamine, 0.1 mM β-mercaptoethanol, 0.1% penicillin-streptomycin and 5 mg/mL bovine serum albumin (GIBCO, USA).

[0219] The hESCs were treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor (Y-27632, 10 μM, Calbiochem, Germany) and a PKC (protein kinase C) inhibitor (Go6983, 2.5 μM, Sigma, USA) for 24 hours in order to enhance survivability. Then, the hESCs were trypsinized by treating with TrypLE (Life Technologies, USA) and were induced to differentiate into hMSSCs by culturing with the MSSC medium on a culture dish coated with vitronectin+gelatin (1 ng/mL, Sigma, USA) until passage 7. The differentiated MSSCs were identified to be stably identical from passage 5, and the cells cultured for 10 passages were deposited on October 10, 2018 in the Korean Cell Line Bank and were given the accession number KCLRF-BP-00460.

Example 2.2. Induction of Differentiation from iPSCs into hMSSCs

[0220] hiPSCs (human induced pluripotent stem cells) were obtained by introducing the OCT4, KLF4, SOX2 and cMYC genes into BJ fibroblasts (ATCC®CRL2522™) using Sendai virus according to the method developed by Hasegawa et al. (Fusaki et al., 2009, PNAS 85, 348-362). The hiPSCs were cultured on CF1 mouse embryonic fibroblast (MEF) feeder cells whose cell division was blocked by mitomycin C treatment. A hiPSC culture medium was prepared with DMEM/F12 (Invitrogen, USA) supplemented with 20% knockout serum replacement (hereinafter, referred to as KSR, Invitrogen, USA), 1 mM glutamine (Invitrogen, USA), 1% nonessential amino acids (Invitrogen, USA), 0.1 mM β-mercaptoethanol (Invitrogen, USA), 0.1% penicillin/streptomycin (Invitrogen, USA) and 15 ng/mL bFGF (R&D Systems, USA).

[0221] A medium for inducing differentiation of hiPSCs into hMSSCs (human musculoskeletal stem cells) (hereinafter, referred to as “MSSC medium”) was prepared with the following composition:

[0222] 1) 250 ng/mL human noggin (Koma Biotech, Korea),

[0223] 2) 20 ng/mL human LIF (Koma Biotech, Korea),

[0224] 3) 15 ng/mL basic fibroblast growth factor (FGF) (R&D Systems, USA) (FGF2 signaling activator),

[0225] 4) 3 μM CHIR99021 (Cayman, USA) (Wnt signaling activator),

[0226] 5) 1 μM PD0325901 (Cayman, USA) (ERK (extracellular signal-regulated kinase) signaling inhibitor),

[0227] 6) 10 μM SB431542 (Tocris, United Kingdom) (TGF-β/activin/nodal (TGF-β/activin/nodal) signaling inhibitor),

[0228] 7) Others: 10% knockout serum replacement (Invitrogen, USA), 1% N2 supplement (GIBCO, USA), 2% B27 supplement (GIBCO, USA), 1% nonessential amino acids (GIBCO, USA), 43% DMEM/F12 (GIBCO, USA), 43% Neurobasal (GIBCO, USA), 1 mM glutamine, 0.1 mM β-mercaptoethanol, 0.1% penicillin-streptomycin and 5 mg/mL bovine serum albumin (GIBCO, USA).

[0229] The hiPSCs were treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor (Y-27632, 10 μM, Calbiochem, Germany) and a PKC (protein kinase C) inhibitor (Go6983, 2.5 μM, Sigma, USA) for 24 hours in order to enhance survivability. Then, the hiPSCs were trypsinized by treating with TrypLE (Life Technologies, USA) and were induced to differentiate into hMSSCs by culturing with the MSSC medium on a culture dish coated with vitronectin+gelatin (1 ng/mL, Sigma, USA) until passage 7. The differentiated MSSCs were identified to be stably identical from passage 5.

Example 3. Immunohistochemistry

[0230] Samples obtained by injecting the hMSSCs differentiated in Example 2 into the subcutaneous tissue and kidney of Balb/c-nude were fixed overnight at 4° C. in 2% paraformaldehyde (PFA; Wako, Japan). For a sample to investigate differentiation into bone, decalcification was conducted at 4° C. for 2 weeks in PBS (pH 7.2) using 0.4 M EDTA. Then, the samples were dehydrated using ethanol and xylene sequentially, embedded in paraffin or OCT in sucrose, and cut to 5 μm thickness. The specimen was stained with H&E (Cosmobio, Japan).

Example 4. Fluorescence Immunoassay

[0231] “Immunofluorescence staining” was performed as follows.

[0232] The samples obtained by injecting the hMSSCs into the subcutaneous tissue and kidney of Balb/c-nude were fixed overnight at 4° C. in 2% paraformaldehyde (PFA; Wako, Japan). All the samples were decalcified with Morse's solution. The samples were embedded in paraffin (Leica Biosystems, Germany) and then cut to 5 μm thickness. After blocking the specimen for 15 minutes in 3% hydrogen peroxide, the samples were incubated at 4° C. overnight with primary antibodies. The primary antibodies treated on the specimen were as follows: mouse monoclonal antibody against HLA class I (Abcam, United Kingdom), goat polyclonal antibody against collagen type II (Santacruz, USA), and antibody against osterix (Abcam, USA). Alexa 555 (Invitrogen, USA) and Alexa 488 (Invitrogen, USA) IgGs were used as secondary antibodies. The immunostained specimen was counterstained with TO-PRO3 (Invitrogen, USA) to visualize nuclei. The fluorescence-labeled cut surface was imaged with the Leica DM 5000 microscope (Leica Microsystems, Germany) or a confocal microscope (LSM510; Carl Zeiss, Germany) and analyzed with the Zen software.

Example 5. Flow Cytometry

[0233] After separating the hMSSCs of Examples 2.1 and 2.2 into a single-cell suspension by treating with trypsin/EDTA and blocking nonspecific binding with 2% BSA in PBS, the cells were reacted with monoclonal antibodies against Sca, CD2, CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD31, CD34, CD44, CD45, CD51, CD56, CD73, CD90, CD105, CD146, CD166, CD235a and CD271 (BD Biosciences, USA) in a buffer solution [1×PBS, 1% BSA and 0.01% sodium azide] and then washed. The cells were reacted with Alexa Fluor 488 secondary mouse IgGs (Invitrogen, USA), washed and then analyzed using a flow cytometer (FACStar Plus flow cytometer, BD Biosciences, USA). Normal mouse IgGs (BD Biosciences, USA) were used as a negative control group.

Example 6.1. Differentiation of Human Mesenchvmal Stem Cells (hMSCs) and hMSSCs into Osteoblasts In Vitro

[0234] In order to differentiate the hMSSCs of Examples 2.1 and 2.2 into osteoblasts, the cells were cultured in an osteogenic differentiation medium (StemPro® osteogenic differentiation kit, Life Technologies, USA) under the condition of 37° C. and 5% CO.sub.2 for 14 days. Alkaline phosphatase (Roche, Switzerland) staining and alizarin red S (Sigma, USA) staining were conducted to observe osteogenesis. The differentiation of hMSCs (Lonza, Switzerland) into osteoblasts was also compared in the same manner.

Example 6.2. Differentiation of Human Mesenchvmal Stem Cells (hMSCs) and hMSSCs Into Adipocytes In Vitro

[0235] In order to differentiate the hMSSCs of Examples 2.1 and 2.2 into adipocytes, the cells were cultured in an adipogenic differentiation medium (StemPro® adipogenic differentiation kit, Life Technologies, USA) under the condition of 37° C. and 5% CO.sub.2 for 14 days. Oil red O (Sigma, USA) staining was conducted to observe adipogenesis. The differentiation of hMSCs (Lonza, Switzerland) into adipocytes was also compared in the same manner.

Example 6.3. Differentiation of Human Mesenchymal Stem Cells (hMSCs) and hMSSCs into Chondrocytes In Vitro

[0236] In order to differentiate the hMSSCs of Examples 2.1 and 2.2 into chondrocytes, the cells were resuspended in a chondrogenic differentiation medium (StemPro® chondrogenic differentiation kit, Life Technologies, USA) and then centrifuged. For formation of micromass, the formed pellets were resuspended in a differentiation medium to 1×10.sup.5 cells/μL and then 5 μL of the cell solution was dropped on the center of a 96-well plate. After incubating the micromass for 2 hours under a high-humidity condition and adding a warmed chondrogenic differentiation medium, incubation was performed in an incubator under the condition of 5% CO.sub.2 and 37° C. The culture medium was re-feded with 3- to 4-day intervals. After 14 days, the chondrogenic pellets were stained with alcian blue. The differentiation of hMSCs (Lonza, Switzerland) into chondrocytes was also compared in the same manner.

[0237] Example 7.1. Differentiation Capacity of hMSSCs into Endothelial Cells In Vitro

[0238] It was investigated whether the hMSSCs of Example 2.1 differentiate into endothelial cells (ECs). The hMSSCs were differentiated by culturing with an EC differentiation medium (endothelial growth medium (EGM)-2, Lonza, Walkersville, Md., USA) supplemented with 50 ng/mL VEGF (vascular endothelial growth factor: ProSpec, Rehovot, Israel) and 10 ng/mL bFGF (basic fibroblast growth factor; ProSpec, Rehovot, Israel) for 6 days. The differentiation was confirmed by immunocytochemistry.

Example 7.2. Differentiation Capacity of hMSSCs into Skeletal Muscle Cells In Vitro

[0239] It was investigated whether the hMSSCs of Examples 2.1 and 2.2 differentiate into skeletal muscle cells. The hMSSCs were differentiated by culturing with a skeletal muscle differentiation medium (DMEM supplemented with 2% B27) for 2 weeks on a Matrigel-coated coverslip. The differentiation was confirmed by immunocytochemistry.

Example 8. Induction of Differentiation of hMSSCs into Nerve Cells In Vitro

[0240] For differentiation into nerve cells, the hMSSCs of Example 2.1 were plated on a polyornithine- and laminin-coated culture dish. After 2 days, the culture medium was exchanged with a neural differentiation medium (Neurobasal medium supplemented with 2% B27, 2 mM GlutaMAX and antibiotics). From day 7, 0.5 mM dibutyl cAMP (Sigma, USA) was added every day for 3 days. As a control group, human neural stem cells derived from H9 hESCs (GIBCO, USA) were differentiated into nerve cells in the same manner. The differentiation was confirmed by immunocytochemistry.

Example 9.1. Differentiation Capacity of hMSSCs in Mouse Kidney

[0241] In order to measure the differentiation capacity of the hMSSCs of Example 2.1 in the kidney of mouse, the hMSSCs were cultured with an MSCGM-CD medium (Lonza, Switzerland) for 2-5 passages and collected as single cells. The hMSSCs (2×10.sup.5 cells) were cultured in an agarose gel well with DMEM+20% FBS for 2 days to form a cell aggregate, which were transplanted into the kidney capsule of Balb/c nude mouse. Immunohistochemistry and immunofluorescence staining were performed 4 weeks after the transplantation.

Example 9.2. Differentiation Capacity of hMSSCs in Mouse Subcutaneous Tissue

[0242] In order to measure the differentiation capacity of the hMSSCs of Example 2.1 in the subcutaneous tissue of mouse, the hMSSCs were cultured with an MSCGM-CD medium (Lonza, Switzerland) for 2-5 passages and collected as single cells. The hMSSCs (2×10.sup.5 cells) were loaded in fibrin glue (Greenplast®, Green Cross, Korea) to which 1 μg/mL hyaluronic acid (Sigma, USA) was added and then transplanted into the subcutaneous tissue of Balb/c nude mouse. Immunohistochemistry and immunofluorescence staining were performed 4 weeks after the transplantation.

Example 10.1. Bone Formation Test using hMSCs

[0243] For analysis of osteogenesis of hMSCs in a thighbone fracture model, hMSCs (Lonza, Switzerland) were cultured with an MSCGM-CD medium (Lonza, Switzerland) for 7 passages, collected as single cells and then absorbed into a collagen membrane (SK Bioland, Korea) cut to a size of 1 mm×1 mm. After perforating one tibia of a 6-week-old Balb/c-nude mouse by about 1 mm using a drill (Bosch Professional, Germany), the hMSCs absorbed in the collagen membrane were inserted into the fracture area of the mouse. The mouse was anesthetized every 2 weeks and micro-CT (Skyscan 1076, Antwerp, Belgium) images were obtained for the fracture area. Immunohistochemistry and immunofluorescence staining were performed 6 weeks later.

Example 10.2. Bone Formation Test using hMSSCs

[0244] For analysis of osteogenesis of hMSSCs in a thighbone fracture model, the hMSSCs of Example 2.1 were cultured with an MSCGM-CD medium (Lonza, Switzerland) for 2-5 passages, collected as single cells and then absorbed into a collagen membrane (SK Bioland, Korea) cut to a size of 1 mm×1 mm. After perforating one tibia of a 6-week-old Balb/c-nude mouse by about 1 mm using a drill (Bosch Professional, Germany), the hMSSCs absorbed in the collagen membrane were inserted into the fracture area of the mouse. The mouse was anesthetized every 2 weeks and micro-CT (Skyscan 1076, Antwerp, Belgium) images were obtained for the fracture area. Immunohistochemistry and immunofluorescence staining were performed 6 weeks later.

Example 11. Micro-CT

[0245] The bone formed in the kidney in which the hMSSCs were transplanted in Example 10.1 was scanned by micro-CT (Skyscan 1076, Antwerp, Belgium) to obtain 3D CT (computed tomography) images. Then, the data were digitalized with a frame grabber and the resulting images were transmitted to a computer using the Comprehensive TeX Archive Network (CTAN) topographic reconstruction software.

Example 12. Measurement of SCX, Runx2 and MYH9 mRNA Expression Levels

[0246] RNAs were extracted from the transplant of the hMSSCs of Example 2.1 in the kidney using 500 μL of Trizol (Life Technologies, USA) according to the manufacturer's protocol. After treating the transplant derived from hMSSCs in the kidney with DNAse (RQ1 DNase, Promega, USA), 500 ng of RNAs were reversely transcribed to cDNAs using oligo-d(T) and random hexamers according to the Superscript III RT (Life Technologies, USA) first-strand cDNA synthesis protocol. qRT-PCR was conducted on the StepOne Plus PCR cycler (Applied Biosystems) using Sybr green (Applied Biosystems, Foster City, Calif.). mRNA expression data were analyzed using the ΔΔCT method and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for gene detection. The primers necessary for the qRT-PCR were purchased from Qiagen (USA). As a control group, RNAs were extracted from hMSSCs and qRT-PCR was conducted in the same manner.

Example 13. Confirmation of Differentiation of hMSSCs Derived from hESCs

Senescence Marker

[0247] Differentiation from hESCs into hMSSCs was induced as described in Example 2 and the morphological change of the induced hMSSCs was observed. The result is shown in FIG. 1A. As seen from FIG. 1A, it was confirmed that the undifferentiated single H9 hESCs were differentiated into cells with fibroblast morphology within 7 passages. They grew with similar morphologies for 10 passages or longer, from passage 7 until passage 17, and showed positive response to staining with the senescence marker β-galactosidase since passage 19, suggesting that aging was progressed.

Confirmation of Pluripotency Marker by Immunofluorescence Method

[0248] The expression of pluripotency markers in the hMSSCs after 7 passages or longer since the induction from the hESCs was observed by the immunofluorescence method, and the result is shown in FIG. 1B. For comparison, the expression of the pluripotency markers in H9 hESCs was investigated by the immunofluorescence method.

[0249] As seen from FIG. 1B, the H9 hESCs were positive for all of OCT4, NANOG, SOX2 and LIN28, suggesting that they have pluripotency. In contrast, the hMSSCs derived from the H9 hESCs were negative for OCT4, NANOG, SOX2 and LIN28.

Confirmation of Pluripotency, Ectodermal, Mesodermal and Endodermal Markers through RNA Sequencing

[0250] The expression of pluripotency, ectodermal, mesodermal and endodermal markers in hESCs, hMSCs and hMSSCs at passages 7 and 17 was investigated through RNA sequencing, and the result is shown in FIG. 1C. The expression of the mRNAs of the pluripotency markers TDGF, NANOG, POU5F1, SOX2, DPPA4, LEFTY1, GDF3, etc. was confirmed in the H9 hESCs (hESC-1, hESC-2). In contrast, for the hMSSCs derived from the H9 hESCs, the expression of the pluripotency marker DPPA4 was observed but the expression of the pluripotency markers TDGF, NANOG, POU5F1, LEFTY1 and GDF3 was not observed. The expression level of DPPA4 was comparable to that in the H9 hESCs.

[0251] The expression of DPPA4 was not observed in the human mesenchymal stem cells. In addition, the hMSSCs were positive for the ectodermal marker NES, were positive for most mesodermal markers except for DES and the early mesodermal markers T and nodal, and were negative for most endodermal markers. In particular, NES was not expressed in the mesenchymal stem cells.

Confirmation of Mesenchymal Stem Cell Markers through Expression of Cell Surface Antigens

[0252] The expression of surface antigens of hMSSCs was measured as shown in FIG. 1D. When the expression of mesenchymal stem cell-specific cell surface antigens was investigated, the expression of the mesenchymal stem cell markers CD44, CD51, CD73, CD105, CD146 and CD166 was observed in the hMSSCs, but the expression of the mesenchymal stem cell markers CD90 and CD271 was not observed in the hMSSCs. In addition, the expression of the blood-associated cell surface markers CD2, CD3, CD7, CD8, CD11b, CD14, CD19, CD20, CD31, CD34 and CD56 was not observed, but the expression of the pre-B cell marker CD10 was observed.

Confirmation of Other Cell-Specific Markers

[0253] The expression of tissue-specific markers of different lineages was analyzed to investigate the characteristics of hMSSCs as shown in FIG. 1E. The mesodermal marker alpha smooth muscle actin (a-SMA), the neuroectodermal marker Pax6, the myogenic satellite marker Pax7, the intestinal stem cell marker LGR5, etc. were expressed, whereas the chondrocyte marker SOX9, the myoblast marker MyoD, etc. were not expressed. This suggests that the hMSSCs are progenitor cells prior to differentiation into chondrocytes and muscle cells.

Example 14. Differentiation Capacity of hMSSCs In Vitro

[0254] In-vitro osteogenesis, chondrogenesis and adipogenesis were tested for hMSCs and the hMSSCs of Example 2.1 (Example 6), and the result is shown in FIG. 2A. From FIG. 2A, it was confirmed that the hMSCs were differentiated into bone, cartilage and adipose in vitro. Meanwhile, the hMSSCs were differentiated into bone and cartilage but were hardly differentiated into adipose under the same condition for the hMSCs in vitro. In other words, it was confirmed that there was a functional difference from mesenchymal stem cells.

Differentiation Capacity into Skeletal Muscle

[0255] It was investigated whether the hMSSCs of Example 13 have the potential to differentiate into skeletal muscle.

[0256] The hMSSCs were cultured for 2 weeks in a skeletal muscle differentiation medium (DMEM containing 2% B27) on a Matrigel-coated coverslip, and then immunofluorescence assay was performed for the skeletal muscle marker MYH9. The result is shown in FIG. 2B. C2C12 cells were used as a positive control group. As shown in FIG. 2B, it was confirmed that the skeletal muscle-specific marker MYH9 was expressed when the hMSSCs were cultured in the skeletal muscle differentiation medium, suggesting that the hMSSCs have the potential to differentiate into skeletal muscle.

Differentiation Capacity into Endothelial Cells

[0257] It was investigated whether the hMSSCs of Example 13 have the potential to differentiate into endothelial cells.

[0258] The hMSSCs were cultured for 6 days in an EC differentiation medium (endothelial growth medium (EGM)-2; Lonza, Walkersville, MD) supplemented with 50 ng/mL VEGF (vascular endothelial growth factor: ProSpec, Rehovot, Israel) and 10 ng/mL bFGF (basic fibroblast growth factor; ProSpec), and then immunofluorescence assay was performed for the endothelial cell markers CD31 and VE-cadherin. The result is shown in FIGS. 2C and 2D. HUVECs were used as a positive control group for endothelial cell differentiation. As shown in FIG. 2C and FIG. 2D, the expression of CD31 and VE-cadherin was not observed in the hMSSCs, suggesting that the hMSSCs lack the potential to differentiate into endothelial cells. In contrast, the expression of the markers was observed in the control group HUVECs.

Differentiation Capacity into Nerve Cells

[0259] hMSSCs were incubate for 7 days in a neural differentiation medium (Neurobasal medium containing 2% B27, 2 mM GlutaMAX and antibiotics) and then cultured for 3 days while adding 0.5 mM dibutyl cAMP (Sigma) every day. Then, immunofluorescence assay was performed for the nerve cell differentiation marker MAP2. The result is shown in FIG. 2E. NSCs (neuronal stem cells) were used as a positive control group for nerve cell differentiation. As shown in FIG. 2E, the cell morphology of the NSCs was changed to that of nerve cells and the expression of the nerve cell-specific marker MAP2 was observed, suggesting that the cells were differentiated into nerve cells. In contrast, the hMSSCs showed no change in cell morphology and the expression of MAP2 was not observed, suggesting that they lack the potential to differentiate into nerve cells.

[0260] Although the hMSSCs were positive for the ectodermal marker NES as confirmed in Example 13, they did not differentiate into nerve cells. It was confirmed that the hMSSCs can differentiate into the mesoderm, more specifically into bone, cartilage and muscle.

Example 15. Confirmation of Differentiation of hMSSCs into Bone, Cartilage, Muscle, Adipose and Tendon In Vivo

[0261] In order to measure the differentiation potential of the hMSSCs induced in the same manner as in Example 2 in vivo, the hMSSCs were transplanted into the kidney (Example 9.1) and subcutaneous tissue (Example 9.2) of an immunodeficient mouse. After transplanting the hMSSCs into the kidney of the mouse and staining tissues with H&E 3-4 weeks later, immunofluorescence staining was performed for bone-, muscle-, adipose-, tendon- and ligament-specific markers and the cell nuclei were counterstained with TO-PRO3. The result is shown in FIG. 3A and FIG. 3B.

[0262] FIG. 3A shows the images obtained 4 weeks after culturing the hMSSCs in an MSCGM-CD medium (Lonza, Switzerland) for 2-5 passages and transplanting them into the kidney. The H&E staining result shows that muscle, adipose, tendon and ligament were formed in the kidney (FIG. 3A-a). As a result of confirming the differentiated muscle tissue derived from hMSSCs, the differentiation into skeletal muscle was observed but the differentiation into smooth muscle was not observed. In contrast, when human MSCs were transplanted under the same condition, muscle, adipose, tendon, etc. were not formed at all (data not shown). In addition to, for immunohistochemistry analysis confirmed that each differentiated tissue was positive for the muscle marker phospho-myosin light chain (pMLC), the adipose marker PPAR-gamma (PPAr), the tendon or ligament marker scleraxis (SCX), etc., and was also positive for the human cell marker hLA (human leukocyte antigen). In this reason, it can be seen that the transplanted hMSSCs were differentiated into muscle, adipose and tendon (or ligament) cells (This is contrary to the in-vitro test result showing no differentiation into adipose.) (FIG. 3A-b).

[0263] FIG. 3B-a a micro-CT scan image showed that hard tissue, i.e., bone, was formed at the hMSSCs transplanted site into the kidney.

[0264] FIGS. 3B-b and c show a result of confirming bone formation by H&E staining and pentachrome immunohistochemistry. It can be seen that the transplanted hMSSCs were differentiated into bone in the kidney capsule.

[0265] FIG. 3B-d shows the result of immunohistochemical assay for the transplanted site. It was confirmed that the cells in the tissue were positive for the human cell marker hLA (human leukocyte antigen), the bone markers Osx (osterix), Runx2, DMP1, OCN (osteocalin), etc. and the blood vessel marker vWF, confirming that bone was formed. Therefore, it can be seen that the transplanted hMSSCs were differentiated into bone.

[0266] FIG. 3C shows that the hMSSCs transplanted into the mouse subcutaneous tissue after being loaded in fibrin glue to which hyaluronic acid was added were differentiated into chondrocytes. The formation of cartilage was confirmed through H&E staining and toluidine blue staining.

[0267] Taken together, it was confirmed that the hMSSCs of the present disclosure can differentiate into cartilage, muscle, tendon (or ligament) and bone at the transplanted site and have superior differentiation capacity.

Example 16. Confirmation of Fracture Recovery Effect of hMSSCs

[0268] In order to investigate the fracture recovery effect of hMSSCs induced in the same manner as in Example 2, bone formation test was performed as in Example 10. The result is shown in FIG. 4A and FIG. 4B.

[0269] It was confirmed that bone was formed at the fracture site about 6 weeks after hMSC transplantation using the femur fracture model. However, the results showed that the bone formation site was positive for the bone marker Runx2, whereas it was negative for the human cell marker hLA. This suggests that the bone formation was not caused by hMSCs but the cells of the mouse itself formed the bone (FIG. 4A). In contrast, hMSSCs were transplanted under the same condition, bone was formed at the fracture site about 6 weeks later, and it was confirmed that the osteogenic site was positive for Runx2 and positive for the human cell marker hLA. Thus, It was confirmed that bone was formed by differentiation of hMSSCs (FIG. 4B).

Example 17. Induction of Differentiation from hiPSCs into hMSSCs and Characterization of Induced hiPSCs

[0270] hiPSC (human induced pluripotent stem cells) were prepared by reprogramming embryonic IMR90 fibroblasts by sendai virus-mediated overexpression of OCT4, KLF4, SOX2 and MYC according to the protocol developed by Hasegawa et al. (Fusaki et al., 2009).

[0271] hMSSCs (hereinafter, iPS-hMSSCs) were obtained by inducing hMSSCs from hiPSCs as in Example 2. The expression level of the pluripotency markers Oct4, Nanog, Sox2 and Lin28 in the iPS-hMSSCs was investigated by immunofluorescence assay and RT-PCR. The result is shown in FIG. 5A.

[0272] As seen from FIG. 5A, it was confirmed that iPS cells were positive for all of OCT4, NANOG, SOX2 and LIN28, suggesting that they have pluripotency. In contrast, the iPS-hMSSCs were negative for all the pluripotency markers OCT4, NANOG, SOX2 and LIN28.

[0273] FIG. 5B shows a result of measuring the expression of surface antigens for the iPS-hMSSCs. When the expression of mesenchymal stem cell-specific cell surface antigens was investigated, it was confirmed that, among the mesenchymal stem cell markers, CD44, CD51, CD73, CD105, CD146 and CD166 were expressed in the iPS-hMSSCs but CD90 and CD271 were not expressed in the iPS-hMSSCs. In addition, the pre-B cell marker CD10 was expressed whereas the blood-associated cell surface markers CD2, CD3, CD7, CD8, CD14, CD20 and CD56 were not expressed.

[0274] Also, the osteogenesis, chondrogenesis and adipogenesis of the iPS-hMSSCs were investigated in the same manner as in Example 14, and the result is shown in FIG. 5C. As seen from FIG. 5C, it was confirmed that the hMSSCs derived from the hiPSCs were differentiated into bone and cartilage in vitro but were hardly differentiated into adipose.

[0275] In addition, the iPS-hMSSCs were cultured for 2 weeks in a skeletal muscle differentiation medium (DMEM containing 2% B27) on a Matrigel-coated coverslip and then immunofluorescence assay was performed for the skeletal muscle marker MYH9. The result is shown in FIG. 5D. C2C12 cells were used as a positive control group. As seen from FIG. 5D, it was confirmed that the hMSSCs derived from the hiPSCs have the potential to differentiate into skeletal muscle.

[0276] Taken together, it was confirmed that the hMSSCs derived from the hiPSCs have the same characteristics as the hMSSCs derived from the hECSs, suggesting that hMSSCs can be obtained using hiPSCs instead of hECSs.

Example 18. Differentiation Capacity of hMSSCs Derived from hiPSCs In Vivo

Transplantation into Kidney

[0277] After transplanting the hMSSCs of Example 17 into mouse kidney, tissue was stained with H&E 3-4 weeks later. It was confirmed that typical muscle, adipose and tendon (or ligament) were formed in the kidney. As a result of immunohistochemical assay, the transplanted site was positive for the muscle marker phospho-myosin light chain (pMLC), the adipose marker PPAR-gamma (PPAr), the tendon or ligament marker scleraxis (SCX), etc. and was also positive for the human cell marker hLA (human leukocyte antigen). Also, it was positive for the bone markers Osx (osterix), Runx2, DMP1, OCN (osteocalin), etc. As a result, it was confirmed that the hMSSCs induced from the iPSCs can differentiate into muscle, adipose, tendon (or ligament) and bone.

Transplantation into Subcutaneous Tissue

[0278] When the hMSSCs of Example 17 were transplanted into mouse subcutaneous tissue after being loaded in fibrin glue to which hyaluronic acid was added, it was confirmed through H&E staining and toluidine blue staining that the hMSSCs can differentiate into cartilage.

Example 19. Comparison of Differentiation Capacity of Noggin-Containing MSSC Medium and Conditioned Medium-Containing CM Medium

[0279] The differentiation capacity of a medium (hereinafter, referred to as “CM medium”) obtained by replacing human noggin (Life Technologies), which is the ingredient 1) of the seven ingredients of the MSSC medium of Example 2, with conditioned medium (culture supernatant obtained after culturing CF1 cells for 24 hours using complete medium wherein DMEM/F12 is replaced with knockout DMEM (knockout DMEM supplemented with 20% knockout serum replacement (Invitrogen, USA), 1 mM glutamine, 1% nonessential amino acids (Invitrogen, USA), 0.1 mM β-mercaptoethanol, 0.1% penicillin-streptomycin and 5 mg/mL bovine serum albumin) (the remaining ingredients 2)-7) are the same) was compared with that of the MSSC medium.

[0280] Noggin is generally used to maintain the characteristics of hESCs during culturing (Chaturvedi G, Simone P D, Ain R, Soares M J, Wolfe M W. Noggin maintains pluripotency of human embryonic stem cells grown on Matrigel. Cell Prolif. 2009 August; 42(4): 425-33). Contrarily to the previously known mechanism, it significantly increased the tendency toward the mesoderm. As can be seen from Table 1, the tendency of osteogenic differentiation was increased 10 times or greater when noggin was contained, as compared to when the CM medium was used.

TABLE-US-00001 TABLE 1 Differentiation tendency of MSSC medium vs. CM medium (number of differentiations out of 20 observations) Induction medium Bone Muscle Tendon Adipose CM medium  1/20 20/20  2/20  2/20 Noggin-containing medium 15/20 20/20 10/20 12/20

[0281] In addition, the expression level of CD44 was compared for the two media. After inducing differentiation using the CM-containing medium (CM medium) and the noggin-containing medium (MSSC medium), the expression level of CD44 was measured in the same manner as in Example 6. As a result, it was confirmed that the expression level of CD44 was increased remarkably when the noggin-containing MSSC medium was used as compared to when the CM medium was used (FIG. 6).

[0282] During osteogenic differentiation, the formation of endochondral bone occurs necessarily after chondrogenesis. CD44 is known to play an essential role in chondrogenesis (Wu S C, Chen C H, Chang J K, Fu Y C, Wang C K, Eswaramoorthy R, Lin Y S, Wang Y H, Lin S Y, Wang G J, Ho M L: Hyaluronan initiates chondrogenesis mainly via cd44 in human adipose-derived stem cells. J Appl Physiol (1985) 2013; 114: 1610-1618). As a result, it was confirmed that use of the MSSC medium rather than the CM medium is suitable for osteogenic differentiation.

[0283] When hMSSCs were transplanted into the kidney, the cells differentiated by the hMMSC medium showed 1-2 weeks faster differentiation as compared to the cells differentiated by the CM medium. The difference in differentiation speed when the CM medium was used and when the hMMSC medium was used is shown in Table 2.

TABLE-US-00002 TABLE 2 Differentiation speed of MSSC medium vs. CM medium (increase in mRNA level as compared to before transplantation of hMSSCs) mRNA Week 1 Week 2 Week 3 Week 4 MYH9 CM 1.3 ± 0.1 2.1 ± 0.1 5.1 ± 0.3 12.5 ± 3.1 MYH9 MSSC 2.2 ± 0.3 4.4 ± 0.4 20.1 ± 3.1  23.1 ± 3.4 Runx2 CM 1.2 ± 0.3 1.8 ± 0.3 3.6 ± 0.3  6.5 ± 3.1 Runx2 MSSC 2.1 ± 0.2 4.3 ± 0.3 7.1 ± 0.3 13.3 ± 3.1 SCX CM 1.3 ± 0.2 2.3 ± 1.2 5.2 ± 1.3 10.7 ± 2.2 SCX MSSC 2.1 ± 0.2 4.7 ± 1.5 12.1 ± 0.3  16.5 ± 2.9

Example 20. Comparison of Synergistic Effect for Combinations of Ingredients of MSSC Medium

[0284] The differentiation capacity of the MSSC medium of Example 2 was compared for the cases where one of the ingredients 1)-6) was missing and the case where all the ingredients were contained. As a result, it was confirmed that differentiation into cartilage (alcian blue) or bone (ALP and alizarin red S) was not achieved well when one of the ingredients 1)-6) was absent (FIG. 7, Table 3).

TABLE-US-00003 TABLE 3 Comparison of differentiation capacity of MSSC medium vs. medium with one ingredient missing FGF-2 Ingredients TGF-β/activin/ ERK signaling of MSSC nodal signaling signaling Wnt signaling Noggin activator medium 6 factors activator (−) hLIF (−) inhibitor (−) activator (−) (−) (−) 1) Noggin ◯ ◯ ◯ ◯ ◯ X ◯ 2) LIF ◯ ◯ X ◯ ◯ ◯ ◯ 3) FGF-2 ◯ ◯ ◯ ◯ ◯ ◯ X signaling activator 4) Wnt ◯ ◯ ◯ ◯ X ◯ ◯ signaling activator 5) ERK ◯ ◯ ◯ X ◯ ◯ ◯ signaling inhibitor 6) ◯ X ◯ ◯ ◯ ◯ ◯ TGF-β/activin/ nodal signaling inhibitor Remarks Muscle Muscle and Cartilage Cartilage and Bone MSSCs MSSCs (including cartilage differentiation bone differentiation not not adipose), differentiation not achieved differentiation inhibited induced induced cartilage and not achieved inhibited osteogenic differentiation achieved as desired

Example 21. Induction of Selective Differentiation of hMSSCs into Muscle, Ligament, Cartilage and Bone In Vivo

[0285] In order to measure the possibility of selective differentiation of hMSSCs, hMSSCs were transplanted into the kidney (kidney capsule) or subcutaneous tissue of Balb/c nude mouse by pretreating with various growth factors such as connective tissue growth factor, transforming growth factor, insulin, fibroblast growth factor, etc. and then preparing into a cell aggregate (4×10.sup.5 cells) or mixing with a cell carrier. 3-6 weeks after the transplantation, tissue was analyzed after removing the transplanted cells.

Example 22. Confirmation of Induced Differentiation of hMSSCs into Muscle

[0286] For investigation of the selective differentiation capacity of the hMSSCs induced in the same manner as in Example 2 into muscle in vivo, hMSSCs were mixed with fibrin glue to which heparin was added and then transplanted into the subcutaneous tissue of an immunodeficient mouse. 7 weeks after the transplantation of hMSSCs, tissue was stained with H&E and the STEM101 antibody (Takara, Japan) binding to the human cell nucleus marker in order to identify whether the differentiated tissue was derived from the hMSSCs. In addition, the differentiated tissue was immunofluorescence staining with an antibody against the muscle marker MyoD (Thermo Fisher Scientific Inc., USA) in order to identify muscle cells. The cell nuclei were counterstained with DAPI. The result is shown in FIG. 8. As shown in FIG. 8, it was confirmed that the hMSSCs were differentiated into muscle only.

Example 23. Confirmation of Induced Differentiation of hMSSCs into Tendon or Ligament

[0287] For investigation of the selective differentiation capacity of the hMSSCs induced in the same manner as in Example 2 into tendon or ligament in vivo, hMSSCs were pretreated with 50 ng/mL connective tissue growth factor and 25 μg/mL ascorbic acid for 2 days in an MSCGM-CD medium (Lonza, Switzerland) and the pretreated cells (1×10.sup.6 cells) were transplanted into the subcutaneous tissue of an immunodeficient mouse after mixing with 100 μL of Matrigel (BD Sciences, USA). 5 weeks after the transplantation of hMSSCs, tissue was stained with H&E and an antibody against hLA binding to the human cell nucleus marker (Abcam, United Kingdom) in order to identify whether the differentiated tissue was derived from the hMSSCs. In addition, the differentiated tissue was immunofluorescence staining with an antibody against the tendon or ligament marker SCX (Antibodies-Online, USA) in order to identify whether the differentiated tissue was tendon or ligament. The result is shown in FIG. 9. As shown in FIG. 9, it was confirmed that the hMSSCs were differentiated into tendon or ligament only.

Example 24. Confirmation of Induced Differentiation of hMSSCs into Bone

[0288] For investigation of the selective differentiation capacity of the hMSSCs induced in the same manner as in Example 2 into bone in vivo, hMSSCs were pretreated with 5 μg/mL insulin, 25 μg/mL ascorbic acid and 1 μg/mL hyaluronic acid for 2 days in a medium supplemented with 10% knockout serum replacement (Invitrogen, USA), 1% N2 supplement (GIBCO, USA), 2% B27 supplement (GIBCO, USA), 43% DMEM/F12 (GIBCO, USA) and 43% Neurobasal (GIBCO, USA), prepared into a cell aggregate and then transplanted into the subcutaneous tissue of an immunodeficient mouse. 5 weeks after the transplantation of hMSSCs, tissue was stained with H&E. In addition, the differentiated tissue was immunofluorescence staining for the cartilage- and bone-specific markers collage type II (colII) and osterix (osx) and cell nuclei were counterstained with DAPI. The result is shown in FIG. 10. As shown in FIG. 10, it was confirmed that the hMSSCs were differentiated into bone only through endochondral ossification.

Example 25. Confirmation of Induced Differentiation of hMSSCs into Tooth

[0289] For investigation of the selective differentiation capacity of the hMSSCs induced in the same manner as in Example 2 into tooth in vivo, hMSSCs were prepared into a cell aggregate, coated with mouse dental epithelial cells and then transplanted into the subcutaneous tissue of an immunodeficient mouse. 6 weeks after the transplantation of hMSSCs, tissue was stained with H&E. In addition, the differentiated tissue was immunofluorescence staining for the human cell-specific marker hLA and cell nuclei were counterstained with To-PRO-3. The result is shown in FIG. 11. As shown in FIG. 11, it was confirmed that the hMSSCs were differentiated into tooth only. In FIG. 11A, K indicates kidney tissue, T indicates tooth tissue, and B indicates bone tissue. FIG. 11B shows the micro-CT image of the kidney of FIG. 11A. FIG. 11C shows the image of the H&E-stained tissue. FIG. 11D shows the fluorescence image obtained by staining with the antibody against human leukocyte antigen (hLA). To-PRO-3 shows the result of counterstaining cell nuclei.

Example 25. Therapeutic Effect of hMSSCs on Ligament Damage

[0290] In order to evaluate the possibility of treating damaged ligament with hMSSCs induced in the same manner as in Example 2 in vivo, the posterior thoracolumbar ligament of an immunodeficient mouse was damaged by cutting along a perpendicular direction and then a cell aggregate prepared by mixing hMSSCs (2×10.sup.6 cells) with 10 μL of fibrin glue was transplanted into the damaged site. 6 weeks after the transplantation of the hMSSCs, tissue was stained with H&E and immunofluorescence staining was performed for the human cell-specific marker hLA and the tendon- or ligament-specific marker SCX. The result is shown in FIG. 12. Cell nuclei were counterstained with To-PRO-3. As seen from FIG. 12, it was confirmed that the broken ligament was connected as the hMSSCs were differentiated.

Example 26. Therapeutic Effect of hMSSCs on Arthritis

[0291] In order to evaluate the possibility of treating damaged cartilage with hMSSCs induced in the same manner as in Example 2 in vivo, destabilization of medial meniscus (DMM) surgery was performed on the left joint of 10-week-old C57BL/6 mice and hMSSCs (3×10.sup.5) were injected into the articular cavity 4-6 weeks later. Experimental groups were divided into a sham control group (Sham), a group to which DMM was performed and the cells were injected after thawing, spinning down and resuspending in PBS (hMSSC-PBS), and a group to which the cells contained in a freezing solution were injected without any treatment (hMSSC-Sol). At week 8, samples were acquired, fixed, decalcified in 10% EDTA solution for 4 weeks, sectioned (5 μm) and then stained with safranin O. The severity of arthritis was evaluated by the method proposed by the Osteoarthritis Research Society International (OARSI). As shown in FIG. 13, the arthritis index (OA score) was increased in the DMM group as compared to the Sham group, suggesting that osteoarthritis was induced well. In the hMSSC-injected groups, the severity of arthritis was decreased to half or lower, and there was no difference between the PBS and Sol groups. At week 6 after the injection of hMSSCs, the severity of arthritis was decreased as compared to at week 4, but there was no statistical difference. It was confirmed that the hMSSCs are therapeutically effective even when they are stored by freeze-drying and administered immediately after thawing (FIG. 13).

[0292] Although the specific exemplary embodiments of the present disclosure have been described in detail, it will be obvious to those having ordinary knowledge in the art that they are merely specific examples and the scope of the present disclosure is not limited by them. It is to be noted that the substantial scope of the present disclosure is defined by the appended claims and their equivalents.

ACCESSION NUMBER

[0293] Depository agency: Korean Cell Line Bank

[0294] Accession number: KCLRFBP00460

[0295] Date of deposition: 2018 Oct. 10