MEDIUM FOR DIRECT DIFFERENTIATION OF PLURIPOTENT STEM CELL-DERIVED MESENCHYMAL STEM CELL, METHOD FOR PREPARING MESENCHYMAL STEM CELL BY USING SAME, AND MESENCHYMAL STEM CELL PREPARED THEREBY

Abstract

The present invention relates to a medium for direct differentiation of embryonic stem cell-derived mesenchymal stem cells, a method of preparing mesenchymal stem cells by using same, mesenchymal stem cells prepared thereby, and a cell therapy product comprising the same mesenchymal stem cells. In a medium composition and a method according to an embodiment, mesenchymal stem cells may be prepared at high yield within a short period of time. In addition, the method is simple in preparation procedure because of the absence of an embryoid body formation step and allows homogeneous cells to be prepared, thus advantageously providing a cell therapy product within a reduce period of time, compared to conventional methods.

Claims

1. A medium composition for inducing differentiation of pluripotent stem cells into mesenchymal stem cells comprising a DNA repair agent and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor.

2. The medium composition of claim 1, wherein the DNA repair agent is 3-[(benzylamino)sulfonyl]-4-bromo-N-(4-bromophenyl)benzamide or 4-bromo-N-(4-bromophenyl)-3-[[phenylmethyl)amino]sulfonyl]-benzamide.

3. The method of claim 1, wherein the ROCK inhibitor is one selected from Fasudil, Ripasudil, 4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide, 4-(1-aminoethyl)-N-(1H-pyrrolo(2,3-b)pyridin-4-yl)cyclohexanecarboxamide dihydrochloride, N-(6-fluoro-1H-indazol-5-yl)-1,4,5,6-tetrahydro-2-methyl-6-oxo-4-[4-(trifluoromethyl)phenyl]-3-pyridinecarboxamide, 1-(3-hydroxybenzyl)-3-[4-(pyridin-4-yl)thiazol-2-yl]urea, 2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride, N-[2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl-2,3-dihydro-1,4-benzodioxin-2-carboxamide dihydrochloride], 2-fluoro-N-[[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)phenyl]methyl]benzenemethanamine dihydrochloride, N-[3[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide, (3S)-1-[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinamine dihydrochloride, N-[(1 S)-2-hydroxy-1-phenylethyl]-N′4-[4-(4-pyridinyl)phenyl]-urea, Azaindole-1, and Narciclasine.

4. The medium composition of claim 1, further comprising a TGF-β inhibitor.

5. The medium composition of claim 4, wherein the TGF-β inhibitor is one selected from 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide hydrate, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5-naphthyridine, 4-(5-benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, 44-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate, 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, 2-(3-(6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, 2-[4-(1,3-benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine, 2-(5-chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine, 6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline, 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide hydrate, 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol, and 3-[[5-(6-methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]benzamide.

6. The medium composition of claim 1, wherein the DNA repair agent is contained in a concentration of 5 μM to 20 μM.

7. The medium composition of claim 1, wherein the ROCK inhibitor is contained in a concentration of 5 μM to 20 μM.

8. The medium composition of claim 4, wherein the TGF-β inhibitor is contained in a low concentration of 0.2 μM to 2.0 μM.

9. A method of preparing pluripotent stem cell-derived mesenchymal stem cells, the method comprising: culturing isolated pluripotent stem cells; and inducing differentiation of the cultured pluripotent stem cells into mesenchymal stem cells by culturing the pluripotent stem cells in a medium comprising a DNA repair agent and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor.

10. The method of claim 9, further comprising differentiating the cultured embryonic stem cells into mesodermal cells in the presence of a TGF-β inhibitor.

11. The method of claim 9, further comprising: culturing the differentiation-induced mesenchymal stem cells in a mesenchymal stem cell maturation medium; or subculturing the mesenchymal stem cells cultured in the maturation medium.

12. The method of claim 9, wherein the culturing of isolated pluripotent stem cells is performed in a culture dish coated with a cell adhesion enhancer in the absence of feeder cells.

13. The method of claim 9, wherein the method does not comprise a process of forming an embryoid body.

14. The method of claim 9, wherein the DNA repair agent is 3-[(benzylamino)sulfonyl]-4-bromo-N-(4-bromophenyl)benzamide or 4-bromo-N-(4-bromophenyl)-3-[[phenylmethyl)amino]sulfonyl]-benzamide.

15. The method of claim 9, wherein the ROCK inhibitor is one selected from Fasudil, Ripasudil, 4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide, 4-(1-aminoethyl)-N-(1H-pyrrolo(2,3-b)pyridin-4-yl)cyclohexanecarboxamide dihydrochloride, N-(6-fluoro-1H-indazol-5-yl)-1,4,5,6-tetrahydro-2-methyl-6-oxo-4-[4-(trifluoromethyl)phenyl]-3-pyridinecarboxamide, 1-(3-hydroxybenzyl)-3-[4-(pyridin-4-yl)thiazol-2-yl]urea, 2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride, N-[2-[2-(d imethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl-2,3-dihydro-1,4-benzodioxin-2-carboxamide dihydrochloride], 2-fluoro-N-[[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)phenyl]methyl]benzenemethanamine dihydrochloride, N-[3-[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide, (3S)-1-[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinamine dihydrochloride, N-[(1S)-2-hydroxy-1-phenylethyl]-N′4-[4-(4-pyridinyl)phenyl]-urea, Azaindole-1, and Narciclasine.

16. The method of claim 9, wherein the TGF-β inhibitor is one selected from 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide hydrate, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5-naphthyridine, 4-(5-benzol[1,3]dioxo1-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxo1-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, 4-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate, 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, 2-(3-(6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, 2-[4-(1,3-benzodioxo1-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine, 2-(5-chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine, 6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline, 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide hydrate, 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol, and 3-[[5-(6-methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]benzamide.

17. The method of claim 10, wherein the differentiating the cultured embryonic stem cells into the mesodermal cells in the presence of the TGF-β inhibitor comprises culturing the cells in a medium containing the TGF-β inhibitor for 2 days to 7 days.

18. The method of claim 9, wherein the inducing of differentiation in the medium comprises inducing differentiation in a medium comprising a DNA repair agent, a ROCK inhibitor, and a TGF-β inhibitor for 1 day to 2 days.

19. The method of claim 11, wherein the culturing of the differentiated mesenchymal stem cells in the maturation medium comprises: treating the cells with trypsin; culturing the cells in a Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12 (DMEM/F12) maturation medium; and centrifuging the cells cultured in the maturation medium.

20. The method of claim 11, wherein the subculturing is performed by subculturing the cells in a culture dish coated with a cell adhesion enhancer in the presence of trypsin.

21. The method of claim 20, wherein the subculturing is performed from 1 passage to 10 passages.

22. The method of claim 11, wherein the subculturing comprises removing exfoliated non-mesodermal stem cells and isolating mesodermal stem cells therefrom.

23. The method of claim 9, wherein the pluripotent stem cells are nuclear transfer pluripotent stem cells (NT-hPSC), parthenote-derived human pluripotent stem cells (pn-hPSC), induced pluripotent stem cells (iPSC), or embryonic stem cells (ESC).

24. The method of claim 9, wherein, in the inducing of differentiation into the mesenchymal stem cells, the mesenchymal stem cells are immature mesenchymal stem cells and prepared via the immature mesenchymal stem cells.

25. The method of claim 24, wherein 10% or less of the immature mesenchymal stem cells in the population express a hemangioblast marker VEGFR2 and the differentiation occurs not via hemangioblasts.

26. The method of claim 9, wherein 70% or more of the prepared mesenchymal stem cells in the population are positive for CD29, CD44, CD90, or CD105 marker and 10% or less of the mesenchymal stem cells in the population are negative for TRA-160, CD34, or VEFGR2 marker.

27. The method of claim 9, wherein the mesenchymal stem cells have the ability to differentiate into at least one selected from hematopoietic stem cells, myocytes, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, urinary cells, renal cells, vascular cells, retinal cells, and neuronal cells.

28. Pluripotent stem cell-derived mesenchymal stem cells differentiation-induced in the presence of a DNA repair agent and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor.

29. The mesenchymal stem cells of claim 28, wherein 70% or more of the mesenchymal stem cells in the population are positive for CD29, CD44, CD90, or CD105 marker and 10% or less of the mesenchymal stem cells in the population are negative for TRA-160, CD34, or VEFGR2 marker.

30. A method of preparing a cell therapy product comprising mesenchymal stem cells, the method comprising subculturing the mesenchymal stem cells prepared according to the method of claim 9 for at least 3 passages, wherein a period during which differentiation of pluripotent stem cells is induced and culturing is performed for application to the cell therapy product is from 20 days to 40 days.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0061] FIG. 1 is a schematic diagram of a method for direct differentiation of human embryonic stem cells into mesenchymal stem cells according to an embodiment.

[0062] FIG. 2 shows photographs illustrating cytomorphological features of human embryonic stem cell-derived mesenchymal stem cells according to an embodiment.

[0063] FIG. 3 shows photographs illustrating cytomorphological features of human embryonic stem cell-derived mesenchymal stem cells via an embryoid body-forming system according to a comparative example.

[0064] FIG. 4 is a graph illustrating comparison results between yields of human embryonic stem cell-derived mesenchymal stem cells according to an embodiment (direct differentiation+SB431542+RS1+Y27632) and yields of human embryonic stem cell-derived mesenchymal stem cells prepared using an embryoid body-forming system (differentiation via an embryoid body+SB431542).

[0065] FIG. 5 is a graph illustrating comparison results between differentiation periods of human embryonic stem cell-derived mesenchymal stem cells according to an embodiment (direct differentiation+(SB431542+RS1+Y27632)) required to be applied as a cell therapy product and differentiation periods of human embryonic stem cell-derived mesenchymal stem cells prepared using an embryoid body-forming system (an embryoid body+SB431542)).

[0066] FIG. 6 shows analysis results of cell surface expression markers of human embryonic stem cell-derived mesenchymal stem cells on the 30.sup.th day of culturing (Passage 4) by flow cytometry.

[0067] FIG. 7 shows analysis results of cell surface expression markers of human embryonic stem cell-derived mesenchymal stem cells on the 4.sup.th day of culturing by flow cytometry.

[0068] FIG. 8 is a graph illustrating comparison results between cell surface expression markers of human embryonic stem cell-derived mesenchymal stem cells according to an embodiment on the 30.sup.th day of culturing (Passage 4) and on the 4.sup.th day of culture.

[0069] FIG. 9 is photographs showing differentiation ability of human embryonic stem cell-derived mesenchymal stem cells according to an embodiment.

[0070] FIG. 10 shows photographs illustrating comparison results between cytomorphological features of human embryonic stem cell-derived mesenchymal stem cells according to an embodiment and cytomorphological features of human embryonic stem cell-derived mesenchymal stem cells prepared by treatment with a DNA repair agent or a ROCK inhibitor.

MODE OF DISCLOSURE

[0071] Hereinafter, the present disclosure will be described in more detail with reference to the following examples. However, the following examples are merely presented to exemplify the present disclosure, and the scope of the present disclosure is not limited thereto.

Example 1

Direct Differentiation of Human Embryonic Stem Cell into Mesenchymal Stem Cell

[0072] 1.1. Culture of Human Embryonic Stem Cell

[0073] Human embryonic stem cells were cultured without feeder cells as follows.

[0074] Specifically, a 4-well tissue culture dish having a surface area of 2.0 cm.sup.2 was treated with CTS Cellstart™, which is a cell adhesion enhance consisting of components derived only from humans in a concentration of 160 μl/well at 4° C. for 24 hours. Then, after all of CTS Cellstart™ remaining on the tissue culture dish was removed therefrom at room temperature, a mTeSR™ (StemCells, USA) medium, as an embryonic stem cell conditioned medium, was added to the tissue culture dish in a concentration of 500 μl/well, and then the tissue culture dish was placed in a 5% CO.sub.2 incubator at 37° C.

[0075] Subsequently, human embryonic stem cells (CHA-hES NT 18, CHA university) proliferated on feeder cells were divided into small clumps using a Micro-tip (Axygen, USA) by mechanical sub-passage, and the clumps were seeded on the tissue culture dish placed in the incubator at a density of 15 to 20 clumps/well. Then, the human embryonic stem cells were proliferated without the feeder cells while replacing the medium with a fresh embryonic stem cell conditioned medium (mTeSR™) in a concentration of 500 μl/well every day for 5 days.

[0076] 1.2. Induction of Differentiation of Human Embryonic Stem Cell into Mesodermal Cell

[0077] The human embryonic stem cells proliferated without feeder cells in Example 1.1 were treated with a TGF-β inhibitor to be differentiated into mesodermal cells.

[0078] Specifically, the TGF-β inhibitor (SB431542) was dissolved in dimethylsulfoxide (DMSO, Sigma, USA) in a stock concentration of 1 mM and diluted in an embryonic stem cell differentiation medium (DMEM/F12, 20% (v/v) SR, 1% (v/v) NEAA, 0.1 mM β-mercaptoethanol, and 1% (v/v) penicillin-streptomycin) to a final concentration of 1 μM. Then, the human embryonic stem cells proliferated for 5 days according to Example 1.1 were treated in the embryonic stem cell differentiation medium containing SB431542 for 4 days (Day 0, 1, and 2) to be differentiated into mesodermal cells.

[0079] 1.3. Pretreatment of Mesodermal Cell

[0080] In Example 1.2, the medium was replaced with a fresh medium on the 3.sup.rd day of treatment with the embryonic stem cell differentiation medium, and then the cells were further cultured for 1 day. Specifically, the mesodermal cells were pre-treated by replacing the medium with a differentiation medium containing a 10 μM Rad51 activator (RS1), a 10 μM ROCK inhibitor (Y27632), and a 1 μM TGF-β inhibitor (SB431542).

[0081] 1.4. Induction of Differentiation into Mesenchymal Stem Cell

[0082] The mesodermal cells pre-treated in Example 1. 3 were differentiated into mesenchymal stem cells and subcultured to obtain mesodermal stem cells.

[0083] Specifically, all of the culture medium was removed on the 4.sup.th day of differentiation, and then the cells were washed with PBS mixed with 1% (v/v) penicillin-streptomycin. Then, the resultant was treated with 0.125% trypsin at room temperature to prepare a single cell suspension and neutralized with a mesenchymal stem cell maturation medium (DMEM/F12, 10% (v/v) FBS, 4 ng/ml bFGF, 1% (v/v) NEAA, 0.1 mM β-mercaptoethanol, and 1% (v/v) penicillin-streptomycin), and then centrifuged. Subsequently, the cells obtained by centrifuging were seeded on 1-well/12-well dishes coated with CTS Cellstart™ and continuously subcultured, in the order of 12-well dish (Passage 0)->6-well dish (Passage 1)->T-25 flask (Passage 2)->T-75 flask (Passage 3) whenever a cell confluency reached 80% to 90%, by a method of preparing a single cell suspension via treatment with 0.05% trypsin at room temperature. During this process, non-mesodermal stem cells were dropped and mesodermal stem cells were recovered.

[0084] A method of preparing mesenchymal stem cells according to an embodiment is schematically shown in FIG. 1.

Comparative Example 1

Preparation of Mesenchymal Stem Cell via Embryoid Body-Forming System

[0085] As a control, mesenchymal stem cells were prepared from human embryonic stem cells (hESCs) via an embryoid body-forming system. The mesenchymal stem cells were prepared via the embryoid body-forming system as follows.

[0086] Specifically, human embryonic stem cell lines were co-cultured in a colony form on MEF feeder cells previously prepared in a concentration of 7.5×10.sup.4 cells/well (0.1% gelatin coated dish, 4 well). For formation of an embryoid body (EB), the hESCs were mechanically separated into several groups (2 to 4 clump forms) using a sterilized tip under a dissecting microscope and cultured for 5 days on a 60 mm Petri dish in a Dulbecco's Modified Eagle Medium; Nutrient Mixture F-12 (DMEM/F12) medium supplemented with 20% knockout-serum replacement (KSR, Invitrogen). After EB formation, the cells were cultured one day in a 20% KSR+DMEM/F12 medium (EB medium:medium excluding only bFGF from hESC medium), and then cultured for 2 weeks (13 days) in the EB medium after adding a 1 μM TGF-beta inhibitor (SB431542) thereto, and the medium was replaced once every two days. Then, the cells were transferred to a 6-well plate coated with 0.1% gelatin (30 min, air dry) such that a density of EB was 5 to 7 in one well and further cultured for 16 days in a DMEM (low glucose: 5.5 mM D-glucose (1 g/L)) supplemented with 10% FBS and 1% penicillin-streptomycin (P/S, Invitrogen). After 48 hours of culturing, adhesion of EB and cells extending out of the EB were identified, and the medium was replaced twice a week. After 16 days, the cells extending from the EB were separated using a TrypLE solution (Invitrogen; 500 μl TrypLE per 1 well, 2 min incubation), transferred to a 75T flask coated with 0.1% gelatin, and cultured in a DMEM (low glucose: 5.5 mM D-glucose (1 g/L)) supplemented with 10% FBS and 1% P/S. Next day, mesenchymal stem cells were prepared by subculturing in a DMEM (low glucose; 5.5mM D-glucose (1 g/L) supplemented with 10% FBS, 1% P/S, and subculturing the cells in a MSC proliferation medium including 10% FBS, 1% P/S , 1% nonessential amino acids (NEAA, Invitrogen) and 0.1% 3-mercaptoethanol (Invitrogen) using 0.05% Trypsin-EDTA (1.5 ml per 75T flask, 2 min incubation) whenever a cell confluency is reached.

Comparative Example 2

Preparation of Mesenchymal Stem Cell by Treatment with DNA Repair Agent Alone

[0087] Mesenchymal stem cells were prepared in the same manner as in Example 1, except that both the 10 μM Rad51 activator (RS1) and the 1 μM TGF-β inhibitor (SB431542) were used without using the ROCK inhibitor in Example 1.3.

Comparative Example 3

Preparation of Mesenchymal Stem Cell by Treatment with ROCK Inhibitor Alone

[0088] Mesenchymal stem cells were prepared in the same manner as in Example 1, except that both the 10 μM ROCK inhibitor (Y27632) and the 1 μM TGF-(3 inhibitor (SB431542) were used without using the DNA repair agent in Example 1.3.

[0089] Experimental Example Analysis of Characteristics of Human Embryonic Stem Cell-derived Mesenchymal Stem Cell

[0090] 1. Analysis of Cytomorphological Feature

[0091] For analysis of cytomorphological features of mesenchymal stem cells obtained in Example 1 and Comparative Example 1, a mesenchymal stem cell-specific proliferation pattern (spindle shape) was identified under a phase-contrast microscope (Nikon TE-2000), and the results are shown in FIGS. 2 and 3, respectively.

[0092] FIG. 2 shows photographs illustrating cytomorphological features of human embryonic stem cell-derived mesenchymal stem cells according to an embodiment.

[0093] FIG. 3 shows photographs illustrating cytomorphological features of human embryonic stem cell-derived mesenchymal stem cells via an embryoid body-forming system according to Comparative Example 1.

[0094] As shown in FIG. 2, it was confirmed that the human embryonic stem cell-derived mesenchymal stem cells prepared according to the method according to an embodiment exhibited gradual proliferation in a spindle-like shape similar to that of adult tissue-derived mesenchymal stem cells about on the 10.sup.th day from differentiation and most of the cells had the shape of the mesenchymal stem cells about on the 24.sup.th day from differentiation. On the contrary, as shown in FIG. 3, it was confirmed that the human embryonic stem cell-derived mesenchymal stem cells prepared using the previously reported embryoid body-forming system exhibited proliferation in a spindle shape on the 40.sup.th day.

[0095] These results indicate that cells having mesenchymal stem cell-specific shapes may be obtained at high yield within a shorter period of time according to the method according to an embodiment when compared with the previously reported method.

[0096] 2. Cell Yield Analysis

[0097] The number of proliferating cells were identified to analyze the yield of cells obtained according to the method of Example 1. Specifically, in the process of preparing mesenchymal stem cells according to Example 1 and Comparative Example 1, the cells were stained with a 0.4% (v/v) trypan blue solution in the preparing of the single cell suspension. Then, the number of viable cells was analyzed using a hemocytometer, and the results are shown in FIG. 4.

[0098] In addition, in view of cytomorphological features of Example 1.4, an appropriate time in which the human embryonic stem cell-derived mesenchymal stem cells prepared according to the method of according to an embodiment were applicable as a cell therapy product was 3 passages or more where the mesenchymal stem cell-specific shape, i.e., the spindle shape, was observed. Thus, differentiation time of human embryonic stem cell-derived mesenchymal stem cell lines suitable for application to cell therapy was analyzed in the same manner, and the results are shown in FIG. 5.

[0099] FIG. 4 is a graph illustrating comparison results between yields of human embryonic stem cell-derived mesenchymal stem cells according to an embodiment (direct differentiation+SB431542+RS1+Y27632) and yields of human embryonic stem cell-derived mesenchymal stem cells prepared using an embryoid body-forming system (embryoid body+SB431542).

[0100] FIG. 5 is a graph illustrating comparison results between differentiation periods of human embryonic stem cell-derived mesenchymal stem cells according to an embodiment (direct differentiation+(SB431542+RS1+Y27632)) required to be applied as a cell therapy product and differentiation periods of human embryonic stem cell-derived mesenchymal stem cells prepared using an embryoid body-forming system (embryoid body+SB431542)).

[0101] As shown in FIG. 4, it was confirmed that a large number of cells may be obtained within a short period of time compared to the previously reported method according to the method of preparing human embryonic stem cell-derived mesenchymal stem cells according to an embodiment.

[0102] Also, as shown in FIG. 5, it was confirmed that the differentiation period enough to be applied as a cell therapy product according to the method according to an embodiment may be shortened by about 1.5 times to twice compared to the previously reported embryoid body formation system.

[0103] 3. Analysis of Surface Expression Marker

[0104] For analysis of surface expression markers of the human embryonic stem cell-derived mesenchymal stem cells prepared in Example 1, flow cytometry was performed.

[0105] Specifically, the human embryonic stem cell-derived mesenchymal stem cells (on the 30.sup.th day of culturing (Passage 4)) were washed with PBS supplemented with 1% (v/v) penicillin-streptomycin and then a single cell suspension was prepared using trypsin (0.05%, Trypsin/EDTA, Gibco, USA). Subsequently, the cells were immobilized with a cold 4% formaldehyde solution at room temperature for 30 minutes and washed four times with PBS supplemented with 0.2% (v/v) FBS. Subsequently, APC- or PE-labeled antibody was diluted with PBS supplemented with 0.2% FBS to a final concentration of 5 pg to 15 pg and maintained under dark conditions for 30 minutes at room temperature. Then, the resultant was washed four times with PBS supplemented with 0.2% (v/v) FBS and then expression levels were analyzed by a flow cytometer. As antibodies, antibodies to a pluripotent marker TRA-1-60 (phycoerythrin (PE)-conjugated mouse anti-human TRA-1-60;Cat.560193, BD Pharmingen™), a hematopoietic stem cell marker CD34 (allophycocyanine (APC)-conjugated mouse anti-human CD34; Cat. 555824, BD Pharmingen™), a hemangioblast marker VEGFR2 (APC-conjugated mouse anti-human VEGFR2;Cat. BD Pharmingen™), and mesenchymal stem cell markers CD29 (APC-conjugated mouse anti-human CD29; Cat.559883, BD Pharmingen™) CD44 (APC-conjugated mouse anti-human CD44; Cat.559942, BD Pharmingen™) CD90 (APC-conjugated mouse anti-human CD90; Cat.561971, BD Pharmingen™), and CD105 (APC-conjugated mouse anti-human CD105; Cat.562408, BD Pharmingen™) were used.

[0106] The results of flow cytometry are shown in FIG. 6.

[0107] In addition, to identify whether differentiation according to an embodiment occurs without forming hemangioblasts, expression of surface markers of the cells (Example 1.3) was analyzed on the 4.sup.th day of culturing by flow cytometry, and the results are shown in FIG. 7.

[0108] In addition, analysis results of expression of surface markers of the cells on the 30.sup.th day of culturing (Passage 4) were compared and the results are shown in FIG. 8.

[0109] FIG. 6 shows analysis results of cell surface expression markers of human embryonic stem cell-derived mesenchymal stem cells on the 30.sup.th day of culturing (Passage 4) by flow cytometry.

[0110] FIG. 7 shows analysis results of cell surface expression markers of human embryonic stem cell-derived mesenchymal stem cells on the 4.sup.th day of culturing by flow cytometry.

[0111] FIG. 8 is a graph illustrating comparison results between cell surface expression markers of human embryonic stem cell-derived mesenchymal stem cells according to an embodiment on the 30.sup.th day of culturing (Passage 4) and on the 4.sup.th day of culture.

[0112] As shown in FIG. 6, as a result of identifying expression of the pluripotent marker TRA-1-60, the hematopoietic stem cell marker CD34, and the mesenchymal stem cell markers CD29, CD44, CD90, and CD105 in the human embryonic stem cell-derived mesenchymal stem cells according to an embodiment, it was confirmed that the pluripotent marker TRA-1-60 was not expressed (TRA-1-60: 0.00%), but the mesenchymal stem cell markers were expressed at high rates (CD29: 99.70%; CD44: 99.45%; CD90: 87.15%; and CD105: 96.60%). Therefore, it was confirmed that the human embryonic stem cell-derived mesenchymal stem cells induced by the method according to an embodiment have high mesodermal properties.

[0113] In addition, as shown in FIG. 7, as a result of analyzing surface markers of the cells on the 4.sup.th day of differentiation, it was confirmed that the hematopoietic stem cell marker CD34 was expressed at a high rate (CD34: 69.65%), but the hemangioblast marker VEGFR2 was hardly expressed (VEGFR2: 0.70%).

[0114] In addition, as shown in FIG. 8 indicating a comprehensive result of those of FIGS. 6 and 7, it was confirmed that the mesenchymal stem cell markers were expressed at high rates on the 30.sup.th day (Passage 4) of differentiation, the hematopoietic stem cell marker CD34 was expressed at a high rate and the hemangioblast marker VEGFR2 was expressed at a low rate on the 4.sup.th day of differentiation.

[0115] These results indicate that mesenchymal stem cells were prepared via immature mesenchymal stem cells having characteristics of hematopoietic stem cells without forming hemangioblasts according to the method of preparing human embryonic stem cell-derived mesenchymal stem cells according to an embodiment.

[0116] 4. Analysis of Cell Differentiation Ability

[0117] The differentiation ability of human embryonic stem cell-derived mesenchymal stem cells prepared in Example 1 into adipocytes, osteocytes, and chondrocytes were identified.

[0118] Specifically, differentiation was induced into adipocytes, osteocytes, and chondrocytes using an adipocyte differentiation inducer (StemPro®Adipocyte differentiation kit, Gibco), an osteocyte differentiation inducer (StemPro®Osteocyte differentiation kit, Gibco), and a chondrocyte differentiation inducer (StemPro®Chondrocyte differentiation kit, Gibco), respectively, in accordance with manufacturer's instructions. Cell differentiation was finally identified using staining solutions specific to adipocytes, osteocytes, and chondrocytes (Oil red O, Alizarin red, and Alcian blue, respectively), and the results are shown in FIG. 9.

[0119] FIG. 9 is photographs showing differentiation ability of human embryonic stem cell-derived mesenchymal stem cells according to an embodiment.

[0120] As shown in FIG. 9, in the case of inducing differentiation into adipocytes, lipid droplets stained in red were observed since lipid components in cells were stained by a lipid-specific staining solution Oil red 0. In the case of inducing differentiation into osteocytes, a dark orange color was observed since calcium accumulated in cells was stained by the Alizarin red solution. In the case of inducing differentiation into chondrocytes, a blue color was observed since mucin that is an extracellular matrix of chondrocytes was stained by the chondrocyte-specific staining solution Alcian blue. As a result, it may be confirmed that the mesenchymal stem cells prepared according to an embodiment have multipotency capable of differentiating into adipocytes, osteocytes, and chondrocytes.

[0121] 5. Analysis of Cytomorphological Difference by Period According to Treatment with Single Substance

[0122] For analysis of cytomorphological difference by treatment with the DNA repair agent or the ROCK inhibitor alone on the basis of period, morphologies of cells of Example 1 and Comparative Examples 2 and 3 were analyzed in the same manner as in Experimental Example 1. As a control, cells treated with the TGF-β inhibitor SB431542 alone without being treated with both the DNA repair agent and the ROCK inhibitor were used, and the results are shown in FIG. 10.

[0123] FIG. 10 shows photographs illustrating comparison results between cytomorphological features of human embryonic stem cell-derived mesenchymal stem cells according to an embodiment and cytomorphological features of human embryonic stem cell-derived mesenchymal stem cells prepared by treatment with a DNA repair agent or a ROCK inhibitor.

[0124] As shown in FIG. 10, although the mesenchymal stem cell proliferation pattern of the spindle shape was observed in all groups, the mesenchymal stem cells according to an embodiment (SB431542+RS1+Y27632) exhibited a high yield with the spindle shape first. In addition, uniform proliferation into mesenchymal stem cells was observed after about the 30.sup.th day when treated with SB431542+Y27632 or SB431542+RS1. In the control treated with SB431542 alone, large-sized cells were observed about on the 23.sup.rd day, but most of the cells disappeared on the 30.sup.th day and uniformity deteriorated although cells in the form of mesenchymal stem cells similar to other groups were observed.

[0125] Based on the above-described results, it was confirmed that mesenchymal stem cells may be prepared at high yield within a short period of time according to the method of preparing mesenchymal stem cells according to an embodiment, and the method is simple in preparation procedure because of the absence of an embryoid body formation step and allows homogeneous cells to be prepared, thus advantageously providing a cell therapy product within a reduce period of time, compared to conventional methods.