Method for Inducing Differentiated Cell into Mesenchymal Stem Cell, and Combinations of Regulatory Targets Thereof

Abstract

A method for inducing differentiated cells into mesenchymal stem cells (MSCs), and combinations of regulatory targets thereof. The method includes performing a directional induction on the differentiated cells to prepare the mesenchymal stem cells. The directional induction includes treating cells by inhibiting the TGF- signal pathway, inhibiting the activity of PKC, activating the WNT/-catenin signal pathway and activating the cAMP signal pathway. By regulating corresponding signal pathways and/or enzymatic activities by stages, the differentiated cells are induced into the mesenchymal stem cells.

Claims

1. A method for inducing differentiated cells into mesenchymal stem cells, comprising: performing a directional induction on the differentiated cells to prepare the mesenchymal stem cells; and the directional induction comprises treating cells by inhibiting a TGF- signal pathway, inhibiting an activity of PKC, activating a WNT/-catenin signal pathway and activating a cAMP signal pathway.

2. The method for inducing the differentiated cells into the mesenchymal stem cells according to claim 1, wherein the directional induction further comprises activating a RA signaling pathway and/or inhibiting an activity of DNMT and/or inhibiting an activity of HMT and/or inhibiting an activity of histone demethylases and/or inhibiting a JNK signaling pathway and/or inhibiting a ROCK signaling pathway and/or inhibiting an activity of lysine deacetylases.

3. The method for inducing the differentiated cells into the mesenchymal stem cell according to claim 1, further comprising: pretreating the differentiated cells by inhibiting the TGF- signal pathway, activating the WNT/-catenin signal pathway and activating the cAMP signal pathway; or pretreating cells by inhibiting the activity of Lysine deacetylases inhibitors (KDACIs), inhibiting the TGF- signal pathway, activating the WNT/-catenin signal pathway and activating the cAMP signal pathway.

4. The method for inducing the differentiated cells into the mesenchymal stem cells according to claim 1, wherein the TGF- signal pathway is a type I TGF- receptors participated pathway, and the cAMP signal pathway is an EPAC/RAP1 signal pathway.

5. The method for inducing the differentiated cells into the mesenchymal stem cells according to claim 3, comprising: pretreating the differentiated cells for a first time ranging from about 3-10 days to obtain a first treated cells using the method of claim 3; directionally inducing the first treated cells by TGF- signaling pathway inhibitor, PKC signaling pathway inhibitor, WNT/-catenin signaling pathway activator and cAMP signaling pathway activator for a second time ranging from about 2-20 days, or directionally inducing the first treated cells by at least one selected from the group consisting of TGF- signaling pathway inhibitor, PKC inhibitor, WNT/-catenin signaling pathway activator, cAMP signaling pathway activator, RA signaling pathway activator, DNMT inhibitor, HMT inhibitor, histone demethylases inhibitor, JNK signaling pathway inhibitor, ROCK signaling pathway inhibitor and lysine deacetylases inhibitor for a second time ranging from about 2-20 days.

6. The method for inducing the differentiated cells into the mesenchymal stem cells according to claim 1, wherein the differentiated cells are derived from a subject of mammals, including human; and the differentiated cells comprise fibroblasts, epithelial cells, adipocytes or blood cells.

7. A combination of regulatory targets, wherein the combination of regulatory targets is capable of regulating any one of the signaling pathways and/or enzyme activities of claim 1, and the regulatory targets are at least one selected from the group consisting of TGF- receptors, PKC, WNT/-catenin, cAMP, JNK, ROCK, DNMT, HMT, RAR, lysine deacetylases and histone demethylases.

8. A combination of small molecule compounds, wherein the small molecule compounds are capable of acting on any one of regulatory targets of claim 7.

9. Mesenchymal stem cells, prepared by the method of claim 1.

10. A method of performing basic research, clinical treatment, tissue engineering product development and production, comprising: using the mesenchymal stem cells of claim 9 and products of the mesenchymal stem cells as essential materials.

11. A method of researching cell reprogramming, comprising: using the combination of regulatory targets of claim 7 or the combination of small molecule compounds of claim 8 as essential materials in the cell reprogramming.

12. The method for inducing the differentiated cells into the mesenchymal stem cell according to claim 2, further comprising: pretreating the differentiated cells by inhibiting the TGF- signal pathway, activating the WNT/-catenin signal pathway and activating the cAMP signal pathway; or pretreating cells by inhibiting the activity of Lysine deacetylases inhibitors (KDACIs), inhibiting the TGF- signal pathway, activating the WNT/-catenin signal pathway and activating the cAMP signal pathway.

13. The method for inducing the differentiated cells into the mesenchymal stem cells according to claim 2, wherein the TGF- signal pathway is a type I TGF- receptors participated pathway, and the cAMP signal pathway is an EPAC/RAP1 signal pathway.

14. The method for inducing the differentiated cells into the mesenchymal stem cells according to claim 12, comprising: pretreating the differentiated cells for a first time ranging from about 3-10 days to obtain a first treated cells using the method of claim 12; directionally inducing the first treated cells by TGF- signaling pathway inhibitor, PKC signaling pathway inhibitor, WNT/-catenin signaling pathway activator and cAMP signaling pathway activator for a second time ranging from about 2-20 days, or directionally inducing the first treated cells by at least one selected from the group consisting of TGF- signaling pathway inhibitor, PKC inhibitor, WNT/-catenin signaling pathway activator, cAMP signaling pathway activator, RA signaling pathway activator, DNMT inhibitor, HMT inhibitor, histone demethylases inhibitor, JNK signaling pathway inhibitor, ROCK signaling pathway inhibitor and lysine deacetylases inhibitor for a second time ranging from about 2-20 days.

15. The method for inducing the differentiated cells into the mesenchymal stem cells according to claim 2, wherein the differentiated cells are derived from a subject of mammals, including human; and the differentiated cells comprise fibroblasts, epithelial cells, adipocytes or blood cells.

16. The method for inducing the differentiated cells into the mesenchymal stem cells according to claim 3, wherein the differentiated cells are derived from a subject of mammals, including human; and the differentiated cells comprise fibroblasts, epithelial cells, adipocytes or blood cells.

17. The method for inducing the differentiated cells into the mesenchymal stem cells according to claim 4, wherein the differentiated cells are derived from a subject of mammals, including human; and the differentiated cells comprise fibroblasts, epithelial cells, adipocytes or blood cells.

18. The method for inducing the differentiated cells into the mesenchymal stem cells according to claim 5, wherein the differentiated cells are derived from a subject of mammals, including human; and the differentiated cells comprise fibroblasts, epithelial cells, adipocytes or blood cells.

19. The method for inducing the differentiated cells into the mesenchymal stem cells according to claim 12, wherein the differentiated cells are derived from a subject of mammals, including human; and the differentiated cells comprise fibroblasts, epithelial cells, adipocytes or blood cells.

20. A combination of regulatory targets, wherein the combination of regulatory targets is capable of regulating any one of the signaling pathways and/or enzyme activities of claim 2, and the regulatory targets are at least one selected from the group consisting of TGF- receptors, PKC, WNT/-catenin, cAMP, JNK, ROCK, DNMT, HMT, RAR, lysine deacetylases and histone demethylases.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0233] FIG. 1 is a diagram showing cell morphology of reprogramming human skin fibroblasts to induced mesenchymal stem cells by small molecule compounds;

[0234] FIG. 2 is a diagram showing flow cytometric analysis of standard surface marker of natural mesenchymal stem cells to identify induced mesenchymal stem cells;

[0235] FIG. 3 is a diagram showing testing results of tri-lineage differentiation of induced mesenchymal stem cells;

[0236] FIG. 4 is a diagram showing immunosuppressive effects of induced mesenchymal stem cells on T cells;

[0237] FIG. 5 is a diagram showing treatment results of transplantation of induced mesenchymal stem cells into immunodeficient mice to treat femoral defects;

[0238] FIG. 6 is a diagram showing results of cell proliferation and tri-lineage differentiation potential for long-term passage of induced mesenchymal stem cells;

[0239] FIG. 7 is a diagram showing results of tumor formation assay of induced mesenchymal stem cells.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0240] The technical solutions of the present invention are further described in detail below with reference to the drawings and specific embodiments, while the present invention is not limited to the following experimental solutions.

Embodiment 1

1. Isolation of Skin Fibroblasts

[0241] 1.1 A skin biopsy specimen of 1 cm in diameter was derived from donor, and the primary fibroblasts were isolated by tissue adherence method. The isolated fibroblasts were cultured in a basic culture medium containing 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 m/mL streptomycin (Sigma), and High-Glucose DMEM.

[0242] 1.2 Cells were proliferated through passage and fibroblasts at 6.sup.th to 12.sup.th generations were used to transdifferentiate into mesenchymal stem cells. On the day before the initiation of differentiation (Day-1), the cells were seeded at a density of 110.sup.42.510.sup.4/cm.sup.2 in an incubator under a condition of 37 C. and 5% CO.sub.2.

2. Activation of Skin Fibroblasts

[0243] 2.1 At the time of initiation of transdifferentiation (Day 0), the basic medium was completely replaced with a first-stage culture medium for culturing the cells for 4-6 days. The first-stage culture medium contains 10% fetal bovine serum (Hyclone), 100 U/ml penicillin (Sigma), 100 g/ml streptomycin (Sigma), High-Glucose DMEM medium (Gibco), forskolin (2 M-25 M), Repsox (2 M-15 M), CHIR99021 (1 M-10 M), and VPA (0.5 mM-1.5 mM). In this cuture system, the 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at a concentration of 10%-20%; and 100 U/mL penicillin (Sigma) and 100 m/mL streptomycin (Sigma) may not be used. The cells were cultured under the condition of 37 C. and 5% CO.sub.2.

3. Directed Induction of Skin Fibroblasts

[0244] After the treatment of the second step mentioned above was completed, the cell culture medium was completely replaced by a second-stage culture medium. The culture time was ranged from 6 days to 10 days, and the cells were cultured at 37 C. and 5% CO.sub.2 condition. The second-stage culture medium contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), High-Glucose DMEM medium (Gibco), forskolin (2 M-20 M), Repsox (2 M-15 M), CHIR99021 (1 M-10 M), VPA (0.5 mM-1.5 mM), TTNPB (3 M-8 M), AM580 (0.03 M-0.08 M), EPZ004777 (3 M-8 M), Go6983 (1 M-15 M), Y-27632 (3 M-15 M), and L-Ascorbin acid 2-phosphate (0.15 mM-0.25 mM). In this cuture system, the 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at a concentration of 10%-20%; and 100 U/mL penicillin (Sigma) and 100g/mL streptomycin (Sigma) may not be used.

4. Improving the Efficiency of Transdifferentiation to Mesenchymal Stem Cells in the Third Stage

[0245] Subsequently, the cells were cultured in a third-stage culture medium for 3-8 days under the condition of 37 C. and 5% CO.sub.2. The third stage culture medium contains BMP4 (10-20 ng/mL), PDGF-AB (100-250 ng/mL), b-FGF (10-50 ng/mL), 10% fetal bovine serum (Hyclone), 100 U/ml penicillin (Sigma), 100 m/mL streptomycin (Sigma), and High-Glucose DMEM medium (Gibco). In this culture system, 10% fetal bovine serum can also be replaced by 10%-20% serum substitute (invitrogen); and 100 U/mL penicillin (Sigma) and 100m/mL penicillin (Sigma) may not be used.

5. Maintenance Culture and Expansion of Induced Mesenchymal Stem Cells

[0246] Subsequently, the medium was replaced by conventional mesenchymal stem cell culture medium or commercially available mesenchymal stem cell culture medium (Cyagen) for culture and expansion. The conventional mesenchymal stem cell culture medium contains 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), and High-Glucose DMEM medium (Gibco).

Embodiment 2

[0247] 1. Isolation of skin fibroblasts, as described in Embodiment 1.

[0248] 2. At the time of initiation of transdifferentiation (Day 0), the basic culture medium was completely replaced by the following second-stage culture medium. The cells were cultured for 4-12 days under the condition of 37 C. and 5% CO.sub.2. The second-stage culture medium contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 WmLstreptomycin (Sigma), High-Glucose DMEM medium (Gibco), forskolin (2 M-20 M), Repsox (2 M-15 M), CHIR99021 (1 M-10 M), VPA (0.5 mM-1.5 mM), TTNPB (3 M-8 M), AM580 (0.03 M-0.08 M), EPZ004777 (3 M-8 M), Go6983 (1 M-15 M), Y-27632 (3 M-15 M), and L-Ascorbin acid 2-phosphate (0.15 mM-0.25 mM). In this cuture system, the 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at a concentration of 10%-20%; and 100 U/mL penicillin (Sigma) and 100 m/mL streptomycin (Sigma) may not be used.

[0249] 3. Subsequently, the cells were cultured in the following stage culture medium for 3-8 days under 37 C. and 5% CO.sub.2 condition. The stage culture medium contains BMP4 (10-20 ng/mL), PDGF-AB (100-250 ng/mL), b-FGF (10-50 ng/mL), 10% fetal bovine serum (Hyclone), 100 U/ml penicillin (Sigma), 100 m/ml streptomycin (Sigma), and High Glucose DMEM medium (Gibco). In this culture system, 10% fetal bovine serum can also be replaced by serum substitute (invitrogen) at a concentration of 10%-20%; and 100 U/mL penicillin (Sigma) and 100 m/mL streptomycin (Sigma) may not be used.

[0250] 4. Maintenance culture and expansionof induced mesenchymal stem cells

[0251] Subsequently, the medium was replaced with conventional mesenchymal stem cell culture medium or commercially available mesenchymal stem cell culture medium (Cyagen) for culture and expansion. The conventional mesenchymal stem cell culture medium contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100m/mL streptomycin (Sigma), and High-Glucose DMeM medium (Gibco). Embodiment 3

[0252] 1. Isolation of Skin Fibroblasts, as Described in Embodiment 1.

[0253] 2. Activation of Skin Fibroblasts

[0254] 2.1 At the time of initiation of transdifferentiation (Day 0), the basic culture medium was completely replaced by the first-stage culture medium for cuturing the cells for 4-6 days. The first-stage culture medium contains 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 m/mL streptomycin (Sigma), High-Glucose DMeM medium (Gibco), forskolin (2 M-25 M), Repsox (2 M-15 M), CHIR99021 (1 M-10 M), and VPA (0.5 mM-1.5 mM). In this cuture system, the 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at a concentration of 10%-20%; and 100 U/mL penicillin (Sigma) and 100 m/mL streptomycin (Sigma) may not be used. The cells were cultured under 37 C. and 5% CO.sub.2 condition.

[0255] 3. Directed Induction of Skin Fibroblasts

[0256] After the treatment of the second step mentioned above was completed, the cell culture medium was completely replaced by the second-stage culture medium. The culture time was ranging from 6 days to 10 days, and the cells were cultured under 37 C. and 5% CO.sub.2 condition. The second-stage culture medium described contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 m/mL streptomycin (Sigma), High-Glucose DMEM medium (Gibco), forskolin (2 M-20 M), Repsox (2 M-15 M), CHIR99021 (1 M-10 M), VPA (0.5 mM-1.5 mM), TTNPB (3 M-8 M), AM580 (0.03 M-0.08 M), EPZ004777 (3 M-8 M), Go6983 (1 M-15 M), Y-27632 (3 M-15 M), and L-Ascorbin acid 2-phosphate (0.15 mM-0.25mM). In this cuture system, the 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at a concentration of 10%-20%; and100 U/mL penicillin (Sigma) and 100 m/mL streptomycin (Sigma) may not be used.

[0257] 4. Maintenance Culture and Expansion of Induced Mesenchymal Stem Cells

[0258] Subsequently, the medium was replaced by conventional mesenchymal stem cell culture medium or commercially available mesenchymal stem cell culture medium (Cyagen) for culture and expansion. The conventional mesenchymal stem cell culture medium contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), and High-Glucose DMEM medium (Gibco).

Embodiment 4

[0259] 1. Isolation of Skin Fibroblasts, as Described in Embodiment 1.

[0260] 2. Activation of Skin Fibroblasts

[0261] 2.1 At the time of initiation of transdifferentiation (Day 0), the basic culture medium was completely replaced by the first-stage culture medium for 4-6 days. The first stage culture medium contains 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), High-Glucose DMEM medium (Gibco), forskolin (2 M-25 M), Repsox (2 M-15 M), and CHIR99021 (1 M-10 M). In this culture system, the 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at a concentration of 10%-20%; and 100 U/mL penicillin (Sigma) and 100 g/mL streptomycin (Sigma) may not be used. The cells were cultured under 37 C. and 5% CO.sub.2 condition.

[0262] 3. Directed Induction of Skin Fibroblasts

[0263] After the treatment of the second step mentioned above was completed, the cell culture medium was completely replaced by the second-stage culture medium. The culture time was ranging from 6 days to 10 days, and the cells were cultured under 37 C. and 5% CO.sub.2 condition. The second-stage culture medium contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 m/mL streptomycin (Sigma), High-Glucose DMEM medium (Gibco), forskolin (2 M-20 M), Repsox (2 M-15 M), CHIR99021 (1 M-10 M), VPA (0.5 mM-1.5 mM), TTNPB (3 M-8 M), AM580 (0.03M-0.08 M), EPZ004777 (3 M-8 M), Go6983 (1 M-15 M), Y-27632 (3 M-15 M), L-Ascorbin acid 2-phosphate (0.15 mM-0.25mM), and SP600125(1 M-15 M). In this culture system, 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at a concentration of 10%-20%; and 100 U/mL penicillin (Sigma) and 100 g/mL streptomycin (Sigma) may not be used.

[0264] 4. Improving the Efficiency of Transdifferentiation to Mesenchymal Stem Cell in the Third Stage

[0265] Subsequently, the cells were cultured in the third-stage medium for 3-8 days under the condition of 37 C. and 5% CO.sub.2. The third stage culture medium contains BMP4 (10-20 ng/mL), PDGF-AB (100-250 ng/mL), b-FGF (10-50 ng/mL), 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), and High-Glucose DMEM medium (Gibco). In this culture system, 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at a concentration of 10%-20%; and 100 U/mL penicillin (Sigma) and 100 m/mL streptomycin (Sigma) may not be used.

[0266] 5. Maintenance Culture and Expansion of Induced Mesenchymal Stem Cells

[0267] Subsequently, the medium was replaced with conventional mesenchymal stem cell culture medium or commercially available mesenchymal stem cell culture medium (Cyagen) for maintenance and expansion. The conventional mesenchymal stem cell culture medium contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), and High-Glucose DMEM medium (Gibco).

Embodiment 5

[0268] 1. Isolation of Skin Fibroblasts, as Described in Embodiment 1.

[0269] 2. Activation of Skin Fibroblasts

[0270] 2.1 At the time of initiation of transdifferentiation (Day 0), the basic medium was completely replaced by the first stage culture medium for 4-6 days. The first stage culture medium contains 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), High-Glucose DMEM medium (Gibco), forskolin (2 M-25 M), Repsox (2 M-15 M), CHIR99021 (1 M-10 M), andVPA (0.5 mM-1.5 mM). In this culture system, 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at a concentration of 10%-20%; and 100 U/mL penicillin (Sigma) and 100 m/mL streptomycin (Sigma) may not be used. The cells were cultured under 37 C. and 5% CO.sub.2 condition.

[0271] 3. Directed Induction of Skin Fibroblasts

[0272] After the treatment of the second step mentioned above was completed, the cell culture medium was completely replaced by the second-stage culture medium. The culture time was ranging from 6 days to 10 days, and the cells were cultured under 37 C. and 5% CO.sub.2 condition. The second-stage culture medium contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), High-Glucose DMEM medium (Gibco), forskolin (2 M-20 M), Repsox (2 M-15 M), CHIR99021 (1 M-10 M), VPA (0.5 mM-1.5 mM), TTNPB (3 M-8 M), AM580 (0.03 M-0.08 M), EPZ004777 (3 M-8 M), Go6983 (1 M-15 M), Y-27632 (3 M-15 M), L-Ascorbin acid 2-phosphate (0.15 mM-0.25 mM), SP600125 (8 M-12 M), and 5-Aza-2-deoxycytidine (1 M-15 M). In this culture system, 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at concentrations of 10%-20%; and 100 U/mLpenicillin (Sigma) and 100 g/mLstreptomycin (Sigma) also may not be used. 4. Improving the Efficiency of Transdifferentiation to Mesenchymal Stem Cell in the Third Stage

[0273] Subsequently, the cells were cultured in the third-stage medium for 3 to 8 days under the condition of 37 C. and 5% CO.sub.2. The third-stage culture medium contains BMP4 (10-20 ng/mL), PDGF-AB (100-250ng/mL), b-FGF (10-50ng/mL), 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mLstreptomycin (Sigma), and High Glucose DMEM medium (Gibco). In this culture system, 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at a concentration of 10%-20%; and 100 U/mLpenicillin (Sigma) and 100 g/mLstreptomycin (Sigma) also may not be used.

[0274] 5. Maintenance Culture and Expansion of Induced Mesenchymal Stem Cells

[0275] Subsequently, the medium was replaced by conventional mesenchymal stem cell culture medium or commercially available mesenchymal stem cell culture medium (Cyagen) for maintenance and expansion. The conventional mesenchymal stem cell culture medium contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), and High-Glucose DMEM medium (Gibco).

Embodiment 6

[0276] 1. Isolation of Skin Fibroblasts, as Described in Embodiment 1.

[0277] 2. Activation of Skin Fibroblasts

[0278] 2.1 At the time of initiation of transdifferentiation (Day 0), the basic medium was completely replaced by the first-stage culture medium for 4-6 days. The first-stage culture medium contains 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), High-Glucose DMEM medium (Gibco), forskolin (2M-25M), Repsox (2M-15 M), CHIR99021 (1M-10 M), and VPA (0.5 mM-1.5 mM). In this culture system, 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at concentrations of 10%-20%; and 100 U/mL penicillin (Sigma) and 100 g/mL streptomycin (Sigma) may not be used. The cells were cultured under 37 C. and 5% CO.sub.2 condition.

[0279] 3. Directed Induction of Skin Fibroblasts

[0280] After the treatment of the second step mentioned above was completed, the cell culture medium was completely replaced by the second-stage culture medium. The culture time was ranging from 6 days to 10 days, and the cells were cultured under 37 C. and 5% CO.sub.2 condition. The second-stage culture medium contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), High-Glucose DMEM medium (Gibco), forskolin (2 M-20 M), Repsox (2 M-15 M), CHIR99021 (1 M-10 M), VPA (0.5 mM-1.5 mM), TTNPB (3 M-8 M), AM580 (0.03 M-0.08 M), EPZ004777 (3 M-8 M), Go6983 (1 M-15 M), Y-27632 (3 M-15 M), L-Ascorbin acid 2-phosphate (0.15 mM-0.25 mM), SP600125 (8 M-12 M), 5-Aza-2-deoxycytidine (1 M-15 M), Parnate (1 M-10 M). In this culture system, 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at concentrations of 10%-20%; and 100 U/mLpenicillin (Sigma) and 100 m/mLstreptomycin (Sigma) also may not be used.

[0281] 4. Improving the Efficiency of Transdifferentiation to Mesenchymal Stem Cell in the Third Stage

[0282] Subsequently, the cells were cultured in the third-stage medium for 3-8 days under the condition of 37 C. and 5% CO.sub.2. The third stage culture medium contains BMP4 (10-20 ng/mL), PDGF-AB (100-250 ng/mL), b-FGF (10-50 ng/mL), 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mLstreptomycin (Sigma), and High-Glucose DMEM medium (Gibco). In this culture system, 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at concentrations of 10%-20%; and 100 U/ml penicillin (Sigma) and 100 m/ml streptomycin (Sigma) may not be used.

[0283] 5. Maintenance Culture and Expansion of Induced Mesenchymal Stem Cells

[0284] Subsequently, the medium was replaced with conventional mesenchymal stem cell culture medium or commercially available mesenchymal stem cell culture medium (Cyagen) for maintenance and expansion. The conventional mesenchymal stem cell culture medium contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), and High-Glucose DMEM medium (Gibco).

Embodiment 7

[0285] 1. Isolation of Skin Fibroblasts, as Described in Embodiment 1.

[0286] 2. Activation of Skin Fibroblasts

[0287] 2.1 At the time of initiation of transdifferentiation (Day 0), the basic medium was completely replaced by the first-stage culture medium for 4-6 days. The first-stage culture medium contains 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), High-Glucose DMEM medium (Gibco), forskolin (2 M-25 M), Repsox (2 M-15 M), BIO (1 M-10 M), and VPA (0.5 mM-1.5 mM). In this culture system, 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at concentrations of 10%-20%; and 100 U/mL penicillin (Sigma) and 100 g/mL streptomycin (Sigma) may not be used. The cells were cultured under 37 C. and 5% CO.sub.2 condition.

[0288] 3. Directed Induction of Skin Fibroblasts

[0289] After the treatment of the second step mentioned above was completed, the cell culture medium was completely replaced by the second-stage culture medium. The culture time was ranging from 6 days to 10 days, and the cells were cultured under 37 C. and 5% CO.sub.2 condition. The second-stage culture medium contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), High-Glucose DMEM medium (Gibco), forskolin (2 M-20 M), Repsox (2 M-15 M), CHIR99021 (1 M-10 M), VPA (0.5 mM-1.5 mM), TTNPB (3 M-8 M), AM580 (0.03 M-0.08 M), EPZ004777 (3 M-8 M), Go6983 (1 M-15 M), Y-27632 (3 M-15 M), L-Ascorbin acid 2-phosphate (0.15 mM-0.25 mM), SP600125 (8 M-12 M), 5-Aza-2-deoxycytidine (1 M-15 M), and Parnate (1M-10 M). In this culture system, 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at concentrations of 10%-20%; and 100 U/ml penicillin (Sigma) and 100 g/ml streptomycin (Sigma) may not be used.

[0290] 4. Stage 3: Improving the Efficiency of Transdifferentiation to Mesenchymal Stem Cell

[0291] Subsequently, the cells were cultured in the third-stage medium for 3-8 days at 37 C. and 5% CO.sub.2. The third-stage culture medium contains BMP4 (10-20 ng/mL), PDGF-AB (100-250 ng/mL), b-FGF (10-50 ng/mL), 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), and High-Glucose DMEM medium (Gibco). In this culture system, 10% fetal bovine serum can also be replaced by serum substitutes (invitrogen) at concentrations of 10%-20%; and 100 U/ml penicillin (Sigma) and 100 g/ml streptomycin (Sigma) may not be used.

[0292] 5. Maintenance Culture and Expansion of Induced Mesenchymal Stem Cells

[0293] Subsequently, the medium was replaced by conventional mesenchymal stem cell culture medium or commercially available mesenchymal stem cell culture medium (Cyagen) for maintenance and expansion. The conventional mesenchymal stem cell culture medium contains: 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), and High-Glucose DMEM medium (Gibco).

[0294] In the above Embodiments, the human skin fibroblasts are being induced to prepare (are being reprogrammed as) mesenchymal stem cells. The cell morphology of human skin fibroblasts and their induced mesenchymal stem cells (iMSC) is shown in FIG. 1. The detection results of the induced mesenchymal stem cells are shown in Table 1, and FIGS. 2 to 7.

[0295] As shown in FIG. 1, Part A is the morphology of skin fibroblasts; Part B is the morphology of skin fibroblast-induced mesenchymal stem cells obtained by the method in Embodiment 1; Part C is the growth curve of induced mesenchymal stem cells. The iMSCs were derived from two adult individuals, respectively. The cells were cultured in a culture dish and passaged when the confluence was about 90% or passaged every three days. The induced cells had rapid proliferation. Part D is the cell clone formation rate. The higher the clone formation rate, the better the cell viability. Hu Fib represented untreated skin fibroblasts, Hu MSC represented isolated mesenchymal stem cells in vivo, Hu iMSC represented induced mesenchymal stem cells.

TABLE-US-00001 TABLE 1 HuFib HuFib HuFib Hu Hu Antibody 01 02 03 iMSC01 iMSC02 CD29 + + + + + CD73 + + + + + CD105 + + + + + CD90 + + + + + CD24 CD45 CD34

[0296] Table 1 is the flow cytometric analysis of three untreated skin fibroblasts from different adult individuals, namely HuFib01, HuFib02 and HuFib03, and induced mesenchymal stem cells namely Hu iMSC01 and Hu iMSC02. The iMSCs meet the criteria for natural MSC identification which is positive for surface markers of CD29, CD90, CD73 and CD105, and is negative for surface markers of CD24, CD45 and CD34. Untreated skin fibroblasts have the similar characteristics for these markers.

[0297] FIG. 2 is a diagram showing testing results of using another surface marker CD140a to identify mesenchymal stem cells. Compared with untreated fibroblasts Hu Fib, the peaks of Hu iMSC and Hu MSC showed a shift for the protein expression by flow cytometric analysis.

[0298] FIG. 3 is a diagram showing testing results of multipotency of induced mesenchymal stem cells. As shown in FIG. 3, tri-lineage differentiation of Hu iMSC from Embodiment 1 (Part A) and Embodiment 6 (Part B) and Hu Fib (Part C) as a negative control. The generation of osteoblasts, chondrocytes, and adipocytes on day 21 was detected. Alizarin red staining was used for osteogenesis and Alcian blue used for chondrocytes differentiation and oil red O staining used for adipocytes differentiation.

[0299] FIG. 4 is a diagram showing the immunosuppressive effects of induced mesenchymal stem cells on T cells. Compared with Hu Fib, the Hu iMSC and Hu MSC had similar immunosuppressive ability.

[0300] FIG. 5 is a diagram showing treatment results of the transplantation of induced mesenchymal stem cells into immunodeficient mice to treat femoral defects. After 28 days of cell transplantation, microCT was used to detect the defect repair in the transplanted site. The negative control group showed on the left; MSC positive control group (Hu MSC) showed in the middle; and induced mesenchymal stem cell group showed on the right. Compared with the negative control group, the MSC and iMSC group had significant repair effect on femoral defect.

[0301] FIG. 6 is a diagram showing results of the cell proliferation and tri-lineage differentiation potential for long-term passage. As shown in FIG. 6, Part A is the growth curve of Hu iMSC from passage 0 to passage 7. Cells were passaged when the confluence was about 90% or every three days. Part B is the growth curve of skin fibroblasts from passage 0 to passage 10. Part C is the tri-lineage differentiation of Hu iMSC at passage 7 and the generation of osteoblasts, adipocytes and chondrocytes on day 21 (from left to right).

[0302] FIG. 7 is a diagram showing results of the tumor formation assay of induced mesenchymal stem cells. 0.610.sup.5 to 110.sup.5 Hu iMSCs were subcutaneously transplanted into one NOD-SCID mouse and 30 mice were transplanted. No tumor was observed after transplantation for one month.

[0303] It should be understood that, the above-mentioned characteristics of the patent technology would be combined with ones enumerated below (e.g., the Embodiments) to form a new or a preferred technical scheme. Due to limited space, more Embodiments will not be described herein.