KR20110105665A - Method inducing differentiation from skin-derived precursor cells to neural and mesenchymal lineage cells and differentiation-inducing composition used thereof - Google Patents

Abstract

The present invention relates to a method for differentiating mouse or human skin-derived precursor cells (SKPs) into neural and mesodermal cells, and to a composition for inducing differentiation, wherein the SKPs are individually individual conditions. Mesenchymal Stem Cells (MSCs), which are formed from special neurons, such as peripheral nerve cells and Schwann cells, or mesenchymal stem cells (MSCs) formed from SKPs, are specially cultured in each individual condition to treat bone cells, chondrocytes, adipocytes and muscle cells. A method for differentiating into mesodermal cells such as cells and a composition for inducing differentiation used therein. By using the composition for inducing differentiation and the method of inducing differentiation of the present invention, SKPs or MSCs obtained from tissues such as subcutaneous adipose tissue or skin of adult subjects can be easily differentiated into neural cells and mesodermal cells, thereby injuring the nervous system. It may be useful for the treatment of and replacement of cells of mesodermal tissues and structures.

Description

Method inducing differentiation from skin-derived precursor cells to neural and mesenchymal lineage cells and differentiation--inducing composition used

The present invention relates to a method for differentiating skin-derived precursor cells (SKPs) into neural and mesodermal cells and a composition for inducing differentiation.

Stem cells or precursors (stem cells / precursors) are in vitro (in vitro) models that study the potential resources of specialized cells used in regenerative medicine (Non-Patent Document 1). The most prominent use of such multipotent adult human precursors is for therapeutic cell transplantation and replacement. Although it was previously believed that adult stem cells have limited differentiation potential, it has recently been discovered that several types of cells can be produced from adult stem cells derived from various tissues such as bone marrow, fat and skin (Non-Patent Documents). 2 to 4). Due to the surprisingly diverse differentiation repertoire of different adult stem cell types, studies are increasingly focused on the possibility of adult stem cells as a realistic substitute for embryonic stem cells (Non Patent Literatures 5 and 6).

Recently, the separation of skin-derived precursor cells (SKPs) has been successful not only in the dermis of embryos, newborns and adult rats, but also in the skin of adult humans, which are very easy to obtain tissues (Non-Patent Document 2). SKPs have similar characteristics to embryonic neural crest stem cells (NCSCs) (Non Patent Literatures 7 and 8), mesenchymally-derived smooth muscle cells, adipocytes (non-patents). Patent Documents 2 and 12) and osteoblasts and chondrocytes (Non-Patent Document 6), as well as peripheral neural cells (Schwann cells) (Non-Patent Documents 9 and 10) ) And catecholaminergic neurons (Non-Patent Document 11). SKPs isolated from skins of rodents and mammals form spheres in serum-free medium and contain types of cells that are not found in the skin at all, such as neurons. To differentiate into neural and mesodermal cells. SKPs are representative embryonic neural crest-related precursor cells produced in the skin during embryoogenesis and are maintained in a small number in adults (Non Patent Literature 7). Mammary tissues of the facial area and upper body of mammals are known to originate from cranial neural crest cells. Several studies using the chick-quail chimera system, developing transgenic mice, and human embryonic stem cell-derived neural crest cells The role of neural crest cells in mesoderm tissue has been found in (Non-Patent Documents 13 to 15). Multipotent adult SKPs have potential potential for the treatment of injured nervous systems (Non Patent Literature 9), and may also be used for cell replacement of mesodermal tissues and structures. SKPs can be isolated and propagated from adult human skin (Non-Patent Document 12), potentially becoming a representative autologous cellular resource.

Despite numerous efforts, it remains to be seen whether SKPs can differentiate into mesodermal cell types useful for treatment. There are ethical issues associated with the use of fetal tissue and some cumbersome and painful process issues related to bone marrow extraction, so resources of mesenchymal stem cells (MSCs) can be distributed to the adult subcutaneous adipose tissue or skin. Ideally, you will build a method of obtaining from tissue that is readily available. Although dermal MSCs can differentiate into morphologically identifiable cell types, the potential for differentiation that such dermal MSCs can differentiate into mesodermal tissues such as bone, cartilage, fat and muscle in vitro. Very few studies have shown multipotency.

Accordingly, the present inventors confirmed that mouse skin and human foreskin-derived SKPs can be induced differentiation into mesodermal and nervous system cells based on similarities between known SKPs and NCSCs, and culture conditions most suitable for such differentiation. By establishing the present invention, the present invention has been completed.

<Previous Documents>

< Non Patent Literature >

Joseph NM, Morrison SJ. Toward an understanding of the physiological function of Mammalian stem cells. Dev Cell 2005; 9: 173-183.

2. Toma JG, Akhavan M, Fernandes KJ et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 2001; 3: 778-784.

3. Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418: 41-49.

4. Dicker A, Le Blanc K, Astrom G et al. Functional studies of mesenchymal stem cells derived from adult human adipose tissue. Exp Cell Res 2005; 308: 283-290.

5. Joshi CV, Enver T. Plasticity revisited. Curr Opin Cell Biol 2002; 14: 749-755.

6. Lavoie JF, Biernaskie JA, Chen Y et al. Skin-derived precursors differentiate into skeletogenic cell types and contribute to bone repair. Stem Cells Dev 2009; 18: 893-906.

7. Fernandes KJ, McKenzie IA, Mill P et al. A dermal niche for multipotent adult skin-derived precursor cells. Nat Cell Biol 2004; 6: 1082-1093.

8. Wong CE, Paratore C, Dours-Zimmermann MT et al. Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin. J Cell Biol 2006; 175: 1005-1015.

9. McKenzie IA, Biernaskie J, Toma JG et al. Skin-derived precursors generate myelinating Schwann cells for the injured and dysmyelinated nervous system. J Neurosci 2006; 26: 6651-6660.

10. Biernaskie JA, McKenzie IA, Toma JG et al. Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny. Nat Protoc 2006; 1: 2803-2812.

11. Fernandes KJ, Kobayashi NR, Gallagher CJ et al. Analysis of the neurogenic potential of multipotent skin-derived precursors. Exp Neurol 2006; 201: 32-48.

12. Toma JG, McKenzie IA, Bagli D et al. Isolation and characterization of multipotent skin-derived precursors from human skin. Stem Cells 2005; 23: 727-737.

13. Le Douarin NM, Creuzet S, Couly G et al. Neural crest cell plasticity and its limits. Development 2004; 131: 4637-4650.

14. Takashima Y, Era T, Nakao K et al. Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 2007; 129: 1377-1388.

15. Lee G, Kim H, Elkabetz Y et al. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat Biotechnol 2007; 25: 1468-1475.

An object of the present invention is to provide a method for differentiating mouse or human skin-derived progenitor cells (SKPs) into neural and mesodermal cells and a composition for inducing differentiation.

In order to achieve the above object, the present invention is a skin-derived precursor cell (SKP) to the skin-derived neurotrophic factor (BDNF), NT3 (neurotrophin-3) and nerve growth factor (nerve growth factor (NGF)) It provides a method for inducing differentiation of peripheral nerve cells from skin-derived progenitor cells (SKP), comprising culturing in a serum-free medium containing.

In addition, the present invention includes peripheral nerve cells from skin-derived progenitor cells (SKP), including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and nerve growth factor (NGF). Provided is a composition for inducing differentiation.

In addition, the present invention is a skin-derived precursor cell (SKP) is a serum-free containing dibutyryl cyclic AMP (dbcAMP), bFGF and neuregulin (neuregulin) Provided is a method for inducing Schwann cell differentiation from skin-derived progenitor cells (SKP), comprising culturing in medium.

The present invention also provides a composition for inducing the differentiation of Schwann cells from skin-derived progenitor cells (SKP), including dibutyryl cyclic AMP (dbcAMP), bFGF and neuregulin (neuregulin) to provide.

In addition, the present invention mesenchymal stem cells (MSCs) beta-glycerol phosphate (β-glycerol phosphate), dexamethasone (dexamethasone), ascorbic acid (ascorbic acid) and fetal bovine serum (FBS) It provides a method for inducing osteoblast differentiation from mesenchymal stem cells (MSCs) comprising culturing in the medium containing the.

In addition, the present invention from beta-glycerol phosphate (β-glycerol phosphate), dexamethasone (dexamethasone), ascorbic acid (ascorbic acid) and fetal bovine serum (fetal bovine serum, FBS), from mesenchymal stem cells (MSCs) Provided is a composition for inducing differentiation of osteogenic cells.

In addition, the present invention is a medium containing mesenchymal stem cells (MSCs) to TNF-β (transforming growth factor-β), ascorbic acid (ascorbic acid) and fetal bovine serum (FBS) It provides a method of inducing chondrogenic differentiation from mesenchymal stem cells (MSCs) comprising culturing.

The present invention also provides chondrogenic cells from mesenchymal stem cells (MSCs), including transforming growth factor-β (TNF-β), ascorbic acid and fetal bovine serum (FBS). Provided is a composition for inducing differentiation.

In addition, the present invention is mesenchymal stem cells (MSCs) 1-methyl-3-isobutylxanthine (1-methyl-3-isobutylxanthine), dexamethasone (dexamethasone), insulin (insulin), indomethacin ( It provides a method of inducing adipocyte differentiation from mesenchymal stem cells (MSCs), comprising culturing in a medium containing indomethacin) and fetal bovine serum (FBS).

In addition, the present invention is 1-methyl-3-isobutylxanthine (1-methyl-3-isobutylxanthine), dexamethasone (dexamethasone), insulin (insulin), indomethacin (indomethacin) and fetal bovine serum (FBS) It provides a composition for inducing differentiation of adipogenic cells from mesenchymal stem cells (MSCs), including.

In addition, the present invention comprises the step of culturing the mesenchymal stem cells (MSCs) in a medium containing fetal bovine serum (FBS), mesenchymal stem cells (MSCs) from the muscle cells (MSCs) myogenic cells).

In addition, the present invention provides a composition for inducing differentiation of myogenic cells from mesenchymal stem cells (MSCs), including fetal bovine serum (FBS).

Using the composition for inducing differentiation and the method of inducing differentiation of the present invention, SKPs or MSCs obtained from tissues easily obtained such as subcutaneous adipose tissue or skin of an adult subject can be easily differentiated into neural cells and mesodermal cells. It can be usefully used for the treatment and cell replacement of mesodermal tissues and structures.

1 is a diagram showing the formation and staged differentiation of SKPs from the skin of an adult mouse,
(A) is a schematic design of the experiment (top) and representative images (bottom) of spherical SKPs on days 7, 14 and 21;
(B) is an image of the cell appearance after exposing SKP-derived mesodermal precursors to a medium containing serum;
(C) is a graph showing the results of FACS analysis of the surface labeling profile of SKP-derived mesodermal precursors.
2 is a diagram of the formation and staged differentiation of SKPs from adult human foreskin,
(A) is a schematic design of the experiment (top) and representative images (bottom) of spherical SKPs on days 7, 14 and 21;
(B) is an image of the cell appearance after exposing SKP-derived mesodermal precursors to a medium containing serum;
(C) is a graph showing the results of FACS analysis of the surface labeling profile of SKP-derived mesodermal precursors.
3 is a diagram showing the results of differentiation of mouse-derived SKPs into peripheral neurons and Schwann cells,
(A) is a diagram showing representative images of differentiated SKPs staining peripheral neuronal specific labels by immunocytochemistry;
(B) is a graph showing the results of analyzing peripheral neuron specific marker genes by real-time PCR;
(C) shows a representative image of differentiated SKPs stained by Schwann cell-specific label by immunocytochemistry,
(D) is a graph showing the results of analyzing Schwann cell specific marker genes by real-time PCR;
(E) is a graph showing the results of analyzing the peripheral neuronal specific marker genes by reverse transcriptase PCR;
(F) is a graph showing the results of the analysis of Schwann cell-specific marker genes by reverse transcriptase PCR.
4 is a diagram showing the results of differentiation of human-derived SKPs into peripheral neurons and Schwann cells,
(A) is a diagram showing representative images of differentiated SKPs staining peripheral neuronal specific labels by immunocytochemistry;
(B) is a graph showing the results of analyzing peripheral neuron specific marker genes by real-time PCR;
(C) shows a representative image of differentiated SKPs stained by Schwann cell-specific label by immunocytochemistry,
(D) is a graph showing the results of analyzing Schwann cell specific marker genes by real-time PCR;
(E) is a graph showing the results of analyzing the peripheral neuronal specific marker genes by reverse transcriptase PCR;
(F) is a graph showing the results of the analysis of Schwann cell-specific marker genes by reverse transcriptase PCR.
5 is a diagram showing the results of differentiation of mouse SKPs-derived MSCs into bone cells.
(A) is a picture showing histologic staining of differentiated MSCs with alizarin red;
(B) is a graph showing the results of analyzing bone cell specific marker genes by real-time PCR;
(C) is a graph showing the results of analysis of reverse cell transcriptase PCR of bone cell specific marker genes.
6 is a diagram showing the results of differentiation of human SKPs-derived MSCs into osteoblasts.
(A) is a picture showing histologic staining of differentiated MSCs with alizarin red;
(B) is a graph showing the results of analyzing bone cell specific marker genes by real-time PCR;
(C) is a graph showing the results of analysis of reverse cell transcriptase PCR of bone cell specific marker genes.
7 is a diagram showing the results of differentiation of mouse SKPs-derived MSCs into chondrocytes.
(A) is a picture showing histologic staining of differentiated MSCs with alcian blue;
(B) is a graph showing the results of analyzing chondrocyte specific marker genes by real-time PCR;
(C) is a graph showing the results of analyzing the chondrocyte specific marker gene by reverse transcriptase PCR.
8 is a diagram showing the results of differentiation of human SKPs-derived MSCs into chondrocytes.
(A) is a picture showing histologic staining of differentiated MSCs with alcian blue;
(B) is a graph showing the results of analyzing chondrocyte specific marker genes by real-time PCR;
(C) is a graph showing the results of analyzing the chondrocyte specific marker gene by reverse transcriptase PCR.
9 is a diagram showing the results of differentiation of mouse SKPs-derived MSCs into adipocytes.
(A) is a picture showing histologic staining of differentiated MSCs with oil red O;
(B) is a graph showing the results of analyzing the adipocyte-specific marker genes by real-time PCR;
(C) is a graph showing the results of the analysis of the adipocyte-specific marker gene by reverse transcriptase PCR.
10 is a diagram showing the results of differentiation of human SKPs-derived MSCs into adipocytes.
(A) is a picture showing tissue staining of differentiated MSCs with oil red O;
(B) is a graph showing the results of analyzing the adipocyte-specific marker genes by real-time PCR;
(C) is a graph showing the results of the analysis of the adipocyte-specific marker gene by reverse transcriptase PCR.
11 is a diagram showing the results of differentiation from mouse SKPs-derived MSCs into muscle cells.
(A) is a diagram showing representative images of differentiated MSCs stained with muscle cell specific labels by immunocytochemistry;
(B) is a graph showing the results of analyzing muscle cell specific marker genes by real-time PCR;
(C) is a graph showing the results of analyzing the myocyte-specific marker genes by reverse transcriptase PCR.
12 is a diagram showing the results of differentiation of human SKPs-derived MSCs into muscle cells.
(A) is a diagram showing representative images of differentiated MSCs stained with muscle cell specific labels by immunocytochemistry;
(B) is a graph showing the results of analyzing muscle cell specific marker genes by real-time PCR;
(C) is a graph showing the results of analyzing the myocyte-specific marker genes by reverse transcriptase PCR.

Hereinafter, the present invention will be described in detail.

The present invention is a skin-derived precursor cells (SKPs), serum-derived neurotrophic factor (BDNF), NT3 (neurotrophin-3) and nerve growth factor (nerve growth factor (NGF)) containing serum (serum) It provides a method for inducing differentiation of peripheral nerve cells from skin-derived progenitor cells (SKPs), comprising culturing in a free medium.

The present invention also provides peripheral nerve cells from skin-derived progenitor cells (SKPs), including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and nerve growth factor (NGF). Provided is a composition for inducing differentiation.

The concentration of the BDNF is preferably 40 to 60 ng / ml, more preferably 45 to 55 ng / ml, most preferably 50 ng / ml, but is not limited thereto. In addition, the concentration of NT3 is preferably 1 to 20 ng / ml, more preferably 5 to 15 ng / ml, most preferably 10 ng / ml, but is not limited thereto. The concentration of the nerve growth factor is preferably 40 to 60 ng / ml, more preferably 45 to 55 ng / ml, most preferably 50 ng / ml, but is not limited thereto.

The culture is preferably performed in a culture dish coated with polyornithine and laminin, and the culture dish is 15 μg / ml polyornithine and 1 μg / ml laminin More preferably, but is not limited thereto. In addition, the serum-free medium is preferably serum-free DMEM / F12, but is not limited thereto. In addition, the skin-derived progenitor cells (SKPs) are preferably derived from the back skin of a mouse or human foreskin (foreskin), but is not limited thereto.

In order to confirm the effect of the method of inducing differentiation of peripheral nerve cells of the present invention, in a specific embodiment of the present invention, 15 μg / ml polyornithine and 1 skin-derived SKPs such as mouse and skin 50 ng / ml of brain-derived neurotrophic factor (BDNF), 10 ng / ml of NT3 (neurotrophin-3) and 50 mg / ml of nerve growth factor in culture plates coated with μg / ml laminin 5 × 10 ³ to 20 × 10 ³ cells were cultured with serum-free DMEM / F12 medium containing growth factor (NGF). The medium was replaced every two days and maintained for two weeks without passage to induce differentiation into peripheral nerve cells.

Cells differentiated as described above were analyzed by immunocytochemistry using antibodies against TuJ1 and peripherin. As a result, TuJ1 and Peripherin, which are peripheral neuron-specific markers in the cells differentiated as described above, It was confirmed that it was detected (see A of FIG. 3 and A of FIG. 4).

In addition, total RNA was isolated from the cells differentiated as described above, and the expression of Peripherin and TuJ1 genes was analyzed by Reverse Transcriptase PCR and Real-Time PCR. While genes were not expressed or less expressed in SKPs, it was confirmed that Periperin and TuJ1 genes were remarkably expressed in the cells differentiated as described above (see B, E of FIG. 3 and B, E of FIG. 4).

From the above results, it can be seen that SKPs derived from skin and human foreskin cells such as mice are differentiated into peripheral neural cells by the above method.

In addition, the present invention is a skin-derived precursor cells (SKPs) is a serum-free containing dibutyryl cyclic AMP (dbcAMP), bFGF and neuregulin (skin-derived precursor cells, SKPs) Provided is a method for inducing Schwann cell differentiation from skin-derived progenitor cells (SKPs), comprising culturing in a medium.

In addition, the present invention provides a composition for inducing the differentiation of Schwann cells from skin-derived progenitor cells (SKPs), including dibutyryl cyclic AMP (dbcAMP), bFGF and neuregulin (neuregulin) to provide.

The concentration of dbcAMP is preferably 1 to 20 mM, more preferably 5 to 15 mM, most preferably 10 mM, but is not limited thereto. In addition, the concentration of bFGF is preferably 1 to 20 ng / ml, more preferably 5 to 15 ng / ml, most preferably 10 ng / ml, but is not limited thereto. The concentration of neuregulin is preferably 10 to 30 ng / ml, more preferably 15 to 25 ng / ml, most preferably 20 ng / ml, but is not limited thereto.

The culture is preferably performed in a culture dish coated with polyornithine and laminin, and the culture dish is 15 μg / ml polyornithine and 1 μg / ml laminin More preferably, but is not limited thereto. In addition, the serum-free medium is preferably serum-free DMEM / F12, but is not limited thereto. In addition, the skin-derived progenitor cells (SKPs) are preferably derived from the back skin of a mouse or human foreskin (foreskin), but is not limited thereto.

In order to confirm the effect of the Schwann cell differentiation induction method of the present invention, in a specific embodiment of the present invention, 15 μg / ml polyornithine and 1 μg / Radish containing 10 mM dibutyryl cyclic AMP (dbcAMP), 10 ng / ml bFGF and 20 ng / ml neuregulin in a culture dish coated with ml laminin Cells of 5 × 10 ³ to 20 × 10 ³ were cultured with serum-free DMEM / F12 medium. The medium was changed every two days and maintained for 2 weeks without passage to induce differentiation into Schwann cells.

As a result of analyzing the cells differentiated as described above by immunocytochemistry using antibodies against S100β and GFAP, it was confirmed that Svanβ-specific markers S100β and GFAP were detected in the cells differentiated as described above (Fig. C of 3 and C of FIG. 4).

In addition, total RNA was isolated from the cells differentiated as described above, and the expression of S100β and GFAP genes was analyzed by reverse transcriptase PCR (Reverse Transcriptase PCR) and real-time PCR (Real-Time PCR). While not expressed or less expressed in SKPs, it was confirmed that the S100β and GFAP genes are significantly expressed in the cells differentiated as described above (see D, F of FIG. 3 and D, F of FIG. 4).

From the above results, it can be seen that SKPs derived from skin and human foreskin cells such as mice are differentiated into Schwann cells by the above method.

In addition, the present invention mesenchymal stem cells (MSCs) beta-glycerol phosphate (β-glycerol phosphate), dexamethasone (dexamethasone), ascorbic acid (ascorbic acid) and fetal bovine serum (FBS) It provides a method for inducing osteoblast differentiation from mesenchymal stem cells (MSCs) comprising culturing in the medium containing the.

In addition, the present invention from beta-glycerol phosphate (β-glycerol phosphate), dexamethasone (dexamethasone), ascorbic acid (ascorbic acid) and fetal bovine serum (fetal bovine serum, FBS), from mesenchymal stem cells (MSCs) Provided is a composition for inducing differentiation of osteogenic cells.

The concentration of the beta-glycerol phosphate is preferably 1 to 20 mM, more preferably 5 to 15 mM, most preferably 10 mM, but is not limited thereto. The concentration of dexamethasone is preferably 1 to 20 μM, more preferably 5 to 15 μM, and most preferably 10 μM, but not always limited thereto. The concentration of ascorbic acid is preferably 100 to 300 μM, more preferably 150 to 250 μM, and most preferably 200 μM, but not always limited thereto. The fetal calf serum is preferably included 5 to 15%, more preferably 7 to 12%, most preferably 10%, but is not limited thereto.

The culture is preferably performed in a culture dish coated with polyornithine, laminin and fibronectin, and the culture dish is 15 μg / ml polyornithine, 1 μg / ml Laminin of (laminin) and is coated with 10 ng / ml of fibronectin (fibronectin), but is not limited thereto. In addition, the medium is preferably alpha-minimal essential medium (α-MEM), but is not limited thereto.

The mesenchymal stem cells (MSCs) are preferably derived from various adult tissues such as bone marrow, adipose tissue, cord blood, peripheral blood, neonatal tissues, placenta and skin-derived progenitor cells (SKPs). More preferably, the mesenchymal stem cells (MSCs) are derived from skin-derived progenitor cells (SKPs), and the mesenchymal stem cells (MSCs) are the following methods 1) to 3):

1) removing skin-derived progenitor cells (SKPs) and diluting with DMEM / F12 medium containing 20 ng / ml EGF, 20 ng / ml bFGF and 1X B27;

2) The diluted skin-derived progenitor cells (SKPs) of step 1) were coated with 15 μg / ml polyornithine, 1 μg / ml laminin and 10 ng / ml fibronectin Culturing with DMEM / F12 medium containing 20 ng / ml EGF, 20 ng / ml bFGF and 1 × (B) of B27; And

3) replacing the medium of step 2) with an alpha-minimal essential medium (α-MEM) containing 10% FBS (fetal bovine serum) without a growth factor. It is most preferably produced through, but is not limited thereto.

In addition, the skin-derived progenitor cells (SKPs) are preferably derived from the back skin of a mouse or human foreskin (foreskin), but is not limited thereto.

In order to confirm the effect of the method for inducing osteogenic differentiation of the present invention, in specific embodiments of the present invention, 10 mM beta-glycerol phosphate (β-glycerol) was formed from MSCs formed from skin and human foreskin-derived SKPs such as mice. phosphate, 10 μM dexamethasone, 200 μM ascorbic acid and 10% fetal bovine serum (FBS) with α-minimal essential medium (α) -MEM) for 2 weeks. The medium was changed every 2 days and induced differentiation into osteogenic cells while maintaining for 2 weeks without passage.

As a result of visualizing mineral deposits through histological staining using alizarin red S solution, the cells differentiated as described above were observed. It was confirmed that the inorganic material is distributed in intracellular nodules and some cells growing in monolayers (see FIG. 5A and FIG. 6A).

In addition, total RNA was isolated from the cells differentiated as described above, and osteoblast specific marker genes (for mouse-derived samples: ALP, Osteopontin, BSP and Osteocalcin, and for human-derived samples: ALP, Cbfa-1, and BSP). And Osteocalcin) expression was analyzed by Reverse Transcriptase PCR and Real-Time PCR. As a result, the specific marker gene was not expressed or less expressed in MSCs. In human-derived cells, ALP, Cbfa-1, Osteopontin, BSP and Osteocalcin genes were remarkably expressed (see B, C of FIG. 5 and B, C of FIG. 6).

From the above results, it can be seen that SKPs-derived MSCs are differentiated into osteocytes by the above method.

In addition, the present invention is a medium containing mesenchymal stem cells (MSCs) to TNF-β (transforming growth factor-β), ascorbic acid (ascorbic acid) and fetal bovine serum (FBS) It provides a method of inducing chondrogenic differentiation from mesenchymal stem cells (MSCs) comprising culturing.

The present invention also provides chondrogenic cells from mesenchymal stem cells (MSCs), including transforming growth factor-β (TNF-β), ascorbic acid and fetal bovine serum (FBS). Provided is a composition for inducing differentiation.

The concentration of TNF-β is preferably 1 to 20 ng / ml, more preferably 5 to 15 ng / ml, most preferably 10 ng / ml, but is not limited thereto. The concentration of ascorbic acid is preferably 100 to 300 mM, more preferably 150 to 250 mM, most preferably 200 mM, but is not limited thereto. The fetal calf serum is preferably included 5 to 15%, more preferably 7 to 12%, most preferably 10%, but is not limited thereto.

The culture is preferably performed in a culture dish coated with polyornithine, laminin and fibronectin, and the culture dish is 15 μg / ml polyornithine, 1 μg / ml Laminin of (laminin) and is coated with 10 ng / ml of fibronectin (fibronectin), but is not limited thereto. In addition, the medium is preferably alpha-minimal essential medium (α-MEM), but is not limited thereto.

The mesenchymal stem cells (MSCs) are preferably derived from various adult tissues such as bone marrow, adipose tissue, cord blood, peripheral blood, neonatal tissues, placenta and skin-derived progenitor cells (SKPs). More preferably, the mesenchymal stem cells (MSCs) are derived from skin-derived progenitor cells (SKPs), and the mesenchymal stem cells (MSCs) are the following methods 1) to 3):

1) removing skin-derived progenitor cells (SKPs) and diluting with DMEM / F12 medium containing 20 ng / ml EGF, 20 ng / ml bFGF and 1X B27;

2) The diluted skin-derived progenitor cells (SKPs) of step 1) were coated with 15 μg / ml polyornithine, 1 μg / ml laminin and 10 ng / ml fibronectin Culturing with DMEM / F12 medium containing 20 ng / ml EGF, 20 ng / ml bFGF and 1 × (B) of B27; And

3) replacing the medium of step 2) with an alpha-minimal essential medium (α-MEM) containing 10% FBS (fetal bovine serum) without a growth factor. It is most preferably produced through, but is not limited thereto.

In addition, the skin-derived progenitor cells (SKPs) are preferably derived from the back skin of a mouse or human foreskin (foreskin), but is not limited thereto.

In order to confirm the effect of the method of inducing chondrogenic differentiation of the present invention, in specific embodiments of the present invention, MSNs formed from skin and human foreskin-derived SKPs such as mice are transformed by 10 ng / ml TNF-β (TNF-β). factor-β), re-suspended in alpha-minimal essential medium (α-MEM) containing 200 mM ascorbic acid and 10% fetal bovine serum (FBS) Afterwards, aliquots containing 1.0 × 10 ⁶ cells in 5-ml defined medium for 3 minutes at 390x in 15-ml polypropylene conical tubes were used to produce pellets. Centrifugation was carried out. The medium was changed every two days and the differentiation into chondrogenic cells was induced by culturing the pellets for 4 weeks at 37 ° C.

Cells differentiated as described above were analyzed by histological staining using alcian blue 8GX solution, which is a chondrocyte specific stain, and the cells differentiated as described above were stained with alcian blue. In addition, it was confirmed that a multi-layer rich matrix (matrix) was generated, and the presence of a proteoglycan-rich extracellular matrix (extracellular matrix) (A of FIG. 7 and A of FIG. 8). Reference).

In addition, total RNA was isolated from the cells differentiated as described above, and chondrocyte specific marker genes (for mouse-derived samples: ATF3, col2α1, Ihh and Sox5, and for human-derived samples: Aggrecan, ATF3, col2α1, Ihh). , Sox5, Sox6, and Sox9) expression was analyzed by Reverse Transcriptase PCR and Real-Time PCR, whereby the specific marker genes are not expressed or less expressed in MSCs. It was confirmed that Aggrecan, ATF3, col2α1, Ihh, Sox5, Sox6 and Sox9 genes were significantly expressed in differentiated mouse or human-derived cells (see B, C of FIG. 7 and B, C of FIG. 8).

From the above results, it can be seen that SKPs-derived MSCs are differentiated into chondrocytes by the above method.

In addition, the present invention is mesenchymal stem cells (MSCs) 1-methyl-3-isobutylxanthine (1-methyl-3-isobutylxanthine), dexamethasone (dexamethasone), insulin (insulin), indomethacin ( It provides a method of inducing adipocyte differentiation from mesenchymal stem cells (MSCs), comprising culturing in a medium containing indomethacin) and fetal bovine serum (FBS).

In addition, the present invention is 1-methyl-3-isobutylxanthine (1-methyl-3-isobutylxanthine), dexamethasone (dexamethasone), insulin (insulin), indomethacin (indomethacin) and fetal bovine serum (FBS) It provides a composition for inducing differentiation of adipogenic cells from mesenchymal stem cells (MSCs), including.

The concentration of 1-methyl-3-isobutylxanthine is preferably 0.1 to 1.0 mM, more preferably 0.2 to 0.7 mM, most preferably 0.5 mM, but not always limited thereto. The concentration of dexamethasone is preferably 0.1 to 2 mM, more preferably 0.5 to 1.5 mM, most preferably 1 mM, but is not limited thereto. The concentration of indomethacin is preferably 30 to 70 mM, more preferably 40 to 60 mM, most preferably 50 mM, but is not limited thereto. The fetal calf serum is preferably included 5 to 15%, more preferably 7 to 12%, most preferably 10%, but is not limited thereto.

The culture is preferably performed in a culture dish coated with polyornithine, laminin and fibronectin, and the culture dish is 15 μg / ml polyornithine, 1 μg / ml Laminin of (laminin) and is coated with 10 ng / ml of fibronectin (fibronectin), but is not limited thereto. In addition, the medium is preferably alpha-minimal essential medium (α-MEM), but is not limited thereto.

The mesenchymal stem cells (MSCs) are preferably derived from various adult tissues such as bone marrow, adipose tissue, cord blood, peripheral blood, neonatal tissues, placenta and skin-derived progenitor cells (SKPs). More preferably, the mesenchymal stem cells (MSCs) are derived from skin-derived progenitor cells (SKPs), and the mesenchymal stem cells (MSCs) are the following methods 1) to 3):

1) removing skin-derived progenitor cells (SKPs) and diluting with DMEM / F12 medium containing 20 ng / ml EGF, 20 ng / ml bFGF and 1X B27;

2) The diluted skin-derived progenitor cells (SKPs) of step 1) were coated with 15 μg / ml polyornithine, 1 μg / ml laminin and 10 ng / ml fibronectin Culturing with DMEM / F12 medium containing 20 ng / ml EGF, 20 ng / ml bFGF and 1 × (B) of B27; And

3) replacing the medium of step 2) with an alpha-minimal essential medium (α-MEM) containing 10% FBS (fetal bovine serum) without a growth factor. It is most preferably produced through, but is not limited thereto.

In addition, the skin-derived progenitor cells (SKPs) are preferably derived from the back skin of a mouse or human foreskin (foreskin), but is not limited thereto.

In order to confirm the effect of the method of inducing differentiation of adipogenic cells of the present invention, in the specific example of the present invention, MSCs formed from SKPs derived from skin and human foreskin such as mouse are 0.5 mM 1-methyl-3-isobufil. Xanthine (1-methyl-3-isobutylxanthine), 1 mM dexamethasone, 10 μg / ml insulin, 50 mM indomethacin and 10% fetal bovine serum, Incubated for 2 weeks with alpha-minimal essential medium (α-MEM) containing FBS). The medium was changed every two days and maintained for 4 weeks without passage to induce differentiation into adipogenic cells.

Cells differentiated as described above were analyzed by histological staining using an oil red O solution, which is a fat cell specific staining solution. lipid-rich vacuoles were accumulated (see FIG. 9A and FIG. 10A).

In addition, by separating the total RNA from the cells differentiated as described above, the expression of adipocyte-specific marker genes (C / EBPa, LPL and PPARγ) was reverse transcription enzyme PCR (Reverse Transcriptase PCR) and real-time PCR (Real-Time PCR) As a result, it was confirmed that the specific marker genes were not expressed or less expressed in MSCs, whereas C / EBPa, LPL and PPARγ genes were remarkably expressed in the differentiated mouse or human-derived cells (Fig. 9). B, C and B, C of FIG. 10).

From the above results, it can be seen that SKPs-derived MSCs are differentiated into adipocytes by the above method.

In addition, the present invention comprises the step of culturing mesenchymal stem cells (MSCs) in a culture plate containing medium containing fetal bovine serum (FBS), from mesenchymal stem cells (MSCs) It provides a method for inducing myogenic cells differentiation.

In addition, the present invention provides a composition for inducing differentiation of myogenic cells from mesenchymal stem cells (MSCs), including fetal bovine serum (FBS).

The fetal calf serum is preferably included 5 to 15%, more preferably 7 to 12%, most preferably 10%, but is not limited thereto.

The culture is preferably performed in a culture dish coated with polyornithine, laminin and fibronectin, and the culture dish is 15 μg / ml polyornithine, 1 μg / ml Laminin of (laminin) and is coated with 10 ng / ml of fibronectin (fibronectin), but is not limited thereto. In addition, the medium is preferably alpha-minimal essential medium (α-MEM), but is not limited thereto.

The mesenchymal stem cells (MSCs) are preferably derived from various adult tissues such as bone marrow, adipose tissue, cord blood, peripheral blood, neonatal tissues, placenta and skin-derived progenitor cells (SKPs). More preferably, the mesenchymal stem cells (MSCs) are derived from skin-derived progenitor cells (SKPs), and the mesenchymal stem cells (MSCs) are the following methods 1) to 3):

1) removing skin-derived progenitor cells (SKPs) and diluting with DMEM / F12 medium containing 20 ng / ml EGF, 20 ng / ml bFGF and 1X B27;

2) The diluted skin-derived progenitor cells (SKPs) of step 1) were coated with 15 μg / ml polyornithine, 1 μg / ml laminin and 10 ng / ml fibronectin Culturing with DMEM / F12 medium containing 20 ng / ml EGF, 20 ng / ml bFGF and 1 × (B) of B27; And

3) replacing the medium of step 2) with an alpha-minimal essential medium (α-MEM) containing 10% FBS (fetal bovine serum) without a growth factor. It is most preferably produced through, but is not limited thereto.

In addition, the skin-derived progenitor cells (SKPs) are preferably derived from the back skin of a mouse or human foreskin (foreskin), but is not limited thereto.

In order to confirm the effect of the method of inducing differentiation of adipogenic cells of the present invention, in specific embodiments of the present invention, 10% fetal bovine serum (MSCs) formed from skin and human foreskin-derived SKPs, such as a mouse, FBS) was incubated for 6 weeks with alpha-minimal essential medium (α-MEM), and the culture was carried out with subculture when the cell density reached 90 &lt; &gt; Differentiation into.

Cells differentiated as described above were analyzed by immunocytochemistry using antibodies against smooth muscle actin alpha (SMAα) and desmin, resulting in muscle cell specific markers in the differentiated cells. It was confirmed that SMA and desmine were detected (see FIG. 11A and FIG. 12A).

In addition, total RNA was isolated from the cells differentiated as described above, and reverse transcription of muscle cell specific marker genes (SMAα, SMAγ, Caldesmon and Smootheiln for mouse-derived samples: MYH11 and Smootheiln for human-derived samples). Analysis of enzymes (Reverse Transcriptase PCR) and Real-Time PCR (Real-Time PCR) revealed that the specific marker genes are not expressed in MSCs or are less expressed, whereas SMAα in mouse or human-derived cells differentiated as described above. , SMAγ, Caldesmon, MYH11 and Smootheiln gene was confirmed to be remarkably expressed (see B, C of Figure 11 and B, C of Figure 12).

From the above results, it can be seen that SKPs-derived MSCs are differentiated into myocytes by the above method.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are intended to specifically illustrate the present invention, the content of the present invention is not limited by the following examples.

< Example 1 > Skin-derived progenitor cells ( SKPs Manufacturing

Skin cells in the back skin of C57 / BL6 mice (Orient, Seongnam, Korea) aged 6 to 7 according to the method approved by the Experimental Animal Committee of Seoul National University. skin cells were isolated and operated according to the guidelines of the Institutional Review Board (IRB) on the Human Subjects Research and Ethics Committee at the Seoul National University Dental Hospital. Skin cells were isolated from the foreskin of a 1 to 3 year old patient. Skin samples consisting of epidermis and dermis are incised and cut into small sections, followed by calcium- and magnesium-exclusion Hanks balanced salt solution with 0.1% Trypsin-EDTA at 37 ° C. -and magnesium-free Hanks' balanced salt solution, CMF-HBSS; Invitrogen, Carlsbad, CA, USA) for 60 minutes. Partially submerged skin sections were filtered out mechanically using a 40-μm cell strainer (40-μm cell strainer; BD Biosciences, Bedford, Mass., USA). After washing and re-filtering the filtered cells, mouse skin cells were harvested in 1-fold (1 ×) B-27 (Invitrogen), 20 ng / ml basic fibroblast growth factor (PefTechTech, Rocky Hill) in a 35-mm petri dish. , NJ, USA) and 20 ng / ml of EGF (epidermal growth factor; PeproTech, Rocky Hill, NJ, USA) incubated for 1 week in DMEM / F12 (vol / vol, 1: 1) medium, human Foreskin cells were cultured in 1-fold (1X) B-27 (Invitrogen), 20 ng / ml basic fibroblast growth factor (PeProTech, Rocky Hill, NJ, USA), 20 ng / ml EGF in 35-mm culture dishes. (epidermal growth factor; PeproTech, Rocky Hill, NJ, USA) and DMEM / F12 (vol / vol, 1: 1) containing 10 ng / ml of human leukemia inhibitory factor; Millipore, Dedford, Mass., USA Cultured for 1 week. Growth factors were added every 2 days without medium change. For repeated serial sphere formation assays, the process of isolating spherical cells with accutase (accutase, Innovative Cell Technologies, San Diego, Calif., USA), followed by subculture Once weekly for three weeks.

As a result, at each passage, the globular cells were separated into single cells, and then proliferated into new globular cells (A in FIG. 1 and A in FIG. 2), and skin cells such as mice and the like. It can be seen that SKPs are formed in human foreskin cells.

< Example 2 > Peripheral nerve cells peripheral nerve cells Eruption to

<2-1> SKPs Peripheral Into nerve cells  Differentiation induction

Skin and human foreskin-derived SKPs formed through the procedure of <Example 1> were isolated with accutase (assutase, Innovative Cell Technologies, San Diego, CA, USA), and then 15 μg / ml polyoni 50 ng / ml of brain-derived neurotrophic factor (BDNF), 10 ng / ml of neurotrophin-3 (NT3) and 50 mg / ml in a culture dish coated with polyornithine and 1 μg / ml laminin 5 × 10 ³ to 20 × 10 ³ cells were cultured with a serum-free DMEM / F12 medium containing a nerve growth factor (NGF). The medium was changed every two days and maintained for two weeks without passage.

<2-2> Immunocytochemistry ( immunocytochemistry ) Eruption

The cells differentiated as in <2-1> were fixed for 30 minutes with 2% paraformaldehyde at room temperature, and permeabilized with 0.1% Triton X-100. . After blocking for 60 minutes with PBS containing 1% bovine serum albumin (BSA), cells were treated with TuJ1 (1: 200; R & D System, minneapolis, MN, USA) and peripherin, 1:50; incubated with primary antibody against Santa Cruz Biotechnology, Santa Cruz, Ca, USA for 1 hour at room temperature, followed by secondary antibody (FITC-bound goat anti-mouse IgG and Cy3-binding) Goat anti-rabbit IgG) and nuclei counterstained with DAPI. The images were observed and recorded using a fluorescence microscope (Olympus FV300; Olympus, Tokyo, Japan) equipped with a CDD camera.

As a result, it was confirmed that the peripheral neuron specific markers TuJ1 and periprine (A of FIG. 3 and A of FIG. 4).

<2-3> Reverse transcriptase PCR ( Reverse Transcriptase PCR ) Analysis to identify differentiation

According to the manufacturer's instructions, TRI-reagent (TRI-reagent; Molecular Research Center, Cincinnati, OH, USA) was used to isolate total RNA from cells differentiated as in <2-1>. RNA was denatured at 70 ° C. for 10 minutes and placed on ice for 5 minutes, and cDNA was prepared using reverse transcriptase (Invitrogen) and random hexamers. In addition, specific marker genes (periprine and TuJ1) were amplified by reverse transcriptase PCR using cDNA corresponding to 133 ng of total RNA and primers having a final concentration of 300 nM. In the reverse transcriptase PCR reaction, the primers for the periprine gene include a forward primer (5'-CCACTCTGTCCCGCCTAGAA-3 ') as shown in SEQ ID NO: 1 and a reverse primer (5'-TGCAGGTCTCGAAGTTCCTCTT-3') as shown in SEQ ID NO: 2. The primers for the TuJ1 gene were used as forward primers (5'-TCCGCCTGCCTTTTCGTCTCTA-3 ') as depicted in SEQ ID NO: 3 and reverse primers (5'-AGTTGCCGCTGGGGTCTATGC-3') as depicted in SEQ ID NO: 4. The PCR reaction was incubated at 95 ° C. for 2 minutes, followed by 30 cycles of 20 seconds at 95 ° C., 10 seconds at 60 ° C., and 4 seconds at 70 ° C. After the PCR reaction as described above, the reaction was analyzed by 1.5% agarose gel electrophoresis, and visualized by EtBr (ethidium bromide) staining method. In the reverse transcriptase-RCR experiment, a GAPDH gene using a forward primer (5'-ATGACTCCACTCACGGCAAAT-3 ') as set out in SEQ ID NO: 49 and a reverse primer (5'-GGGTCTCGCTCCTGGAAGAT-3') as set out in SEQ ID NO: 50 was used. It was used as a control.

As a result, the two specific marker genes were not expressed in SKPs, but it was confirmed that the expression of the periperin and TuJ1 genes was confirmed in the cells differentiated as in <2-1> (Fig. 3E and Fig. 4). E).

<2-4> real time PCR ( Real - Time PCR ) Analysis to identify differentiation

7500 using SYBR ^(R) Green PCR Master Mix (Takara, Otsu, Japan) containing cDNA corresponding to 33 ng of total RNA and a final concentration of 300 nM using the cDNA prepared in <2-3>. Specific marker genes (periprine and TuJ1) were amplified via a 7500 Real-time PCR System (Applied Biosystems, Foster City, Calif., USA). In the real-time PCR reaction, the primer for the periprine gene is used as a forward primer (5'-CCACTCTGTCCCGCCTAGAA-3 ') as shown in SEQ ID NO: 1 and a reverse primer (5'-TGCAGGTCTCGAAGTTCCTCTT-3') as shown in SEQ ID NO: 2. As a primer for the TuJ1 gene, a forward primer (5'-TCCGCCTGCCTTTTCGTCTCTA-3 ') as shown in SEQ ID NO: 3 and a reverse primer (5'-AGTTGCCGCTGGGGTCTCGTG-3') as shown in SEQ ID NO: 4 were used. The PCR reaction was incubated at 95 ° C. for 4 minutes, followed by 40 cycles of 15 seconds at 95 ° C., 20 seconds at 60 ° C., and 34 seconds at 72 ° C. For analysis of the data, cycle threshold values were determined through automated threshold analysis, and the calculated cycle threshold values were exported to Microsoft Excel. In addition, as described by the manufacturer (Applied Biosystems), the relative expression of each target mRNA was calculated using a relatively cyclic threshold method.

As a result, in the case of mouse-derived samples, it was confirmed that the expression level of periper was increased by about 4000-fold and the expression level of TuJ1 was increased by about 25-fold in the cells differentiated as in <2-1> compared to SKPs. (FIG. 3B). In addition, in the case of human-derived samples, the expression level of peripherin was increased by about 18-fold and the expression level of TuJ1 was increased by about 9-fold in cells differentiated as in <2-1> compared to MSCs ( 4B).

Accordingly, it can be seen that SKPs derived from skin such as mouse and human foreskin cells are differentiated into peripheral neural cells by the method of <2-1>.

< Example 3 Schwann cells Schwann cells Differentiation into

<3-1> SKPs Of differentiation into Schwann cells

Skin and human foreskin-derived SKPs formed through the procedure of <Example 1> were isolated with accutase (assutase, Innovative Cell Technologies, San Diego, CA, USA), and then 15 μg / ml polyoni 10 mM dibutyryl cyclic AMP (dbcAMP), 10 ng / ml bFGF and 20 ng / ml neurons in a culture dish coated with polyornithine and 1 μg / ml laminin Cells of 5 × 10 ³ to 20 × 10 ³ were cultured with serum-free DMEM / F12 medium containing neuregulin. The medium was changed every two days and maintained for two weeks without passage.

<3-2> Immunocytochemistry ( immunocytochemistry ) Eruption

The cells differentiated as in <3-1> were fixed for 30 minutes with 2% paraformaldehyde at room temperature, and permeabilized with 0.1% Triton X-100. . After blocking for 60 minutes with PBS containing 1% bovine serum albumin (BSA), cells were blocked with S100β (1: 100; Sigma-Aldrich) and GFAP (flial fibrillary acidic protein, 1: 500). Incubated with primary antibodies against Chemicon, Temecula, Ca, USA for 1 hour at room temperature, followed by secondary antibodies (FITC-bound goat anti-mouse IgG and Cy3-binding goat anti-rabbit IgG) The nuclei were counterstained with DAPI. The images were observed and recorded using a fluorescence microscope (Olympus FV300; Olympus, Tokyo, Japan) equipped with a CDD camera.

As a result, it was confirmed that Schwann cell specific labels S100β and GFAP were detected (B of FIG. 3 and B of FIG. 4).

<3-3> Reverse transcriptase PCR ( Reverse Transcriptase PCR ) Analysis to identify differentiation

According to the manufacturer's instructions, total RNA was isolated from cells differentiated as in <3-1> using TRI-reagent (TRI-reagent; Molecular Research Center, Cincinnati, OH, USA). RNA was denatured at 70 ° C. for 10 minutes and placed on ice for 5 minutes, and cDNA was prepared using reverse transcriptase (Invitrogen) and random hexamers. In addition, specific marker genes (GFAP and S100β) were amplified by reverse transcriptase PCR using cDNA corresponding to 133 ng of total RNA and primers having a final concentration of 300 nM. In the reverse transcriptase PCR reaction, the primer for the GFAP gene is used as a forward primer (5'-CATGCCACGCTTCTCCTTGTCTC-3 ') as shown in SEQ ID NO: 5 and a reverse primer (5'-GCTCGCTCGCCCGTGTCT-3') as shown in SEQ ID NO: 6). As primers for the S100β gene, a forward primer (5'-CCTTTCAGGATGATCGCTTTG-3 ') as shown in SEQ ID NO: 7 and a reverse primer (5'-ATGGACAGCAGAGGGAGCAA-3') as shown in SEQ ID NO: 8 were used. The PCR reaction was incubated at 95 ° C. for 2 minutes, followed by 30 cycles of 20 seconds at 95 ° C., 10 seconds at 60 ° C., and 4 seconds at 70 ° C. After the PCR reaction as described above, the reaction was analyzed by 1.5% agarose gel electrophoresis, and visualized by EtBr (ethidium bromide) staining method. In the reverse transcriptase-RCR experiment, a GAPDH gene using a forward primer (5'-ATGACTCCACTCACGGCAAAT-3 ') as set out in SEQ ID NO: 49 and a reverse primer (5'-GGGTCTCGCTCCTGGAAGAT-3') as set out in SEQ ID NO: 50 was used. It was used as a control.

As a result, it was confirmed that the two specific marker genes were not expressed or less expressed in SKPs, whereas the expression of GFAP and S100β genes was confirmed in the cells differentiated as in <3-1> (FIG. 3F and 4F).

<3-4> real time PCR ( Real - Time PCR ) Analysis to identify differentiation

7500 using SYBR ^(R) Green PCR Master Mix (Takara, Otsu, Japan) containing cDNA corresponding to 33 ng of total RNA and a final concentration of 300 nM using the cDNA prepared in <3-3>. Specific marker genes (GFAP and S100β) were amplified via a 7500 Real-time PCR System (Applied Biosystems, Foster City, Calif., USA). In the real-time PCR reaction, a primer for the GFAP gene was used as a forward primer (5'-CATGCCACGCTTCTCCTTGTCTC-3 ') as shown in SEQ ID NO: 5 and a reverse primer (5'-GCTCGCTCGCCCGTGTCT-3') as shown in SEQ ID NO: 6 As a primer for the S100β gene, a forward primer (5'-CCTTTCAGGATGATCGCTTTG-3 ') as shown in SEQ ID NO: 7 and a reverse primer (5'-ATGGACAGCAGAGGGAGCAA-3') as shown in SEQ ID NO: 8 were used. The PCR reaction was incubated at 95 ° C. for 4 minutes, followed by 40 cycles of 15 seconds at 95 ° C., 20 seconds at 60 ° C., and 34 seconds at 72 ° C. For analysis of the data, cycle threshold values were determined through automated threshold analysis, and the calculated cycle threshold values were exported to Microsoft Excel. In addition, as described by the manufacturer (Applied Biosystems), the relative expression of each target mRNA was calculated using a relatively cyclic threshold method.

As a result, in the case of mouse-derived samples, the expression level of S100β was increased by about 110-fold and the expression level of GFAP was increased by about 30-fold in cells differentiated as in <3-1> compared to SKPs ( 3D). In addition, in the case of human-derived samples, the expression level of S100β was increased by about 300-fold and the expression level of GFAP was increased by about 1000-fold in cells differentiated as in <3-1> compared to SKPs (Fig. 4, D).

Accordingly, it can be seen that SKPs derived from skin such as mouse and human foreskin cells are differentiated into Schwann cells by the method of <3-1>.

< Example 4 > SKPs from MSCs Eruption

<4-1> SKPs from MSCs Production

In order to prepare MSCs, SKPs derived from skin and human foreskin such as mice formed through the procedure of <Example 1> were separated with accutase (assutase, Innovative Cell Technologies, San Diego, CA, USA), and then 20 15 μg / ml polyornithine, 1 μg / ml laminin, resuspended in 1 ml of DMEM / F12 medium containing ng / ml EGF, 20 ng / ml bFGF and 1x (1X) B27 (laminin) and plate cultured in 10 ng / ml fibronectin coated plates, after 4 hours containing 20 ng / ml EGF, 20 ng / ml bFGF and 1x (1X) of B27 9 ml of DMEM / F12 medium was added. After 24 hours, the medium was replaced with α-minimal essential medium (α-MEM) containing 10% FBS (fetal bovine serum) without growth factor, Each time cell density was 80%, it was passaged.

As a result, it was confirmed that the cells were attached to the culture plate and proliferated in a well-spread morphology (B of FIG. 1 and B of FIG. 2), and MSCs were formed in SKPs derived from skin and human foreskin cells such as mice. have.

<4-2> Flow cell  analysis( Flow cytometry )

After differentiating the cells differentiated as described in <4-1> using trypsin, the aliquots containing 1.0 × 10 ⁶ cells were collected in 5-ml round-bottom test tubes. Produced. After washing with PBS containing 0.2% of FBS, spin-down, it was blocked for 30 minutes at 4 ℃ with PBS containing 1% BSA and 0.2% FBS. The cells were labeled with MSCs specific marker molecules CD29 (1:10), CD44 (1:10), CD51 (1:10), CD73 (1:40), CD106 (1: 7; BD Pharmingen, San Diego, CA , USA), CD49F (1:10; Chemicon), CD 133 (1:50), CD 146 (1: 1000; Abcam), and Stro-1 (1: 7; Santa Cruz Biotechnology) Incubate for 1 hour on ice together, then wash with PBS containing 0.2% FBS, again incubate for 1 hour on ice with FITC-labeled secondary antibody, and finally The cells were analyzed using a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).

As a result, it was confirmed that most of the cells differentiated as in <4-1> were positive cells for the MSCs specific labeling molecules (C of FIG. 1 and C of FIG. 2), and <4-1>. It can be seen that SKPs derived from skin and human foreskin cells, such as mice, are differentiated into MSCs.

< Example 5 > SKPs -origin MSCs From bone cells ( osteocyte Eruption to

<5-1> SKPs -origin MSCs From bone cells ( osteocyte Differentiation into

SKPs-derived MSCs formed through the process of <4-1> were 10 mM beta-glycerol phosphate (β-glycerol phosphate) and 10 μM dexamethasone at low density (4.0 × 10 ³ cells / 24-well). for 2 weeks with alpha-minimal essential medium (α-MEM) containing dexamethasone, 200 μM ascorbic acid and 10% fetal bovine serum (FBS) Incubated. The medium was changed every two days and maintained for two weeks without passage.

The cells differentiated through the above process formed aggregates or nodules and accumulated calcium significantly.

<5-2> tissue staining

In order to visualize the mineral deposits of the cells differentiated as in <5-1>, the cells were fixed with ice-cold 95% ethanol at -20 ° C, and alizarin red S solution. S solution; secondary distilled water with 40 mM alizarin red S, pH 4.2) for 1 hour. Cells stained as above were washed 5 times with secondary distilled water, washed again with phosphate-buffered saline (PBS) for 15 minutes, and then observed under a microscope.

As a result, abundant mineral content was observed in the cells differentiated as in <5-1>, and it was confirmed that the inorganic material was distributed in the cells in the nodule and in some cells growing in the monolayer (Fig. 5A and 6). A).

<5-3> Reverse transcriptase PCR ( Reverse Transcriptase PCR ) Analysis to identify differentiation

According to the manufacturer's instructions, total RNA was isolated from cells differentiated as in <5-1> using a TRI-reagent (TRI-reagent; Molecular Research Center, Cincinnati, OH, USA). RNA was denatured at 70 ° C. for 10 minutes and placed on ice for 5 minutes, and cDNA was prepared using reverse transcriptase (Invitrogen) and random hexamers. In addition, specific marker genes (for mouse-derived samples: ALP, Osteopontin, BSP and Osteocalcin, and human-derived samples) were identified by reverse transcriptase PCR using cDNA corresponding to 133 ng of RNA and primers having a final concentration of 300 nM. ALP, Cbfa-1, BSP and Osteocalcin) were amplified. In the reverse transcriptase PCR reaction, the primer for the ALP gene was used as a forward primer (5'-GTGCCCTGACTGAGGCTGTC-3 ') as shown in SEQ ID NO: 9 and a reverse primer (5'-GGATCATCGTGTCCTGCTCAC-3') as shown in SEQ ID NO: 10. The primer for the Cbfa-1 gene was a forward primer (5'-GCCTTCAAGGTGGTAGCCC-3 ') as shown in SEQ ID NO: 11 and a reverse primer (5'-CGTTACCCGCCATGACAGTA-3') as shown in SEQ ID NO: 12, and BSP The primer for the gene was used as the forward primer (5'-CACCCCAAGCACAGACTTTTG-3 ') as shown in SEQ ID NO: 13 and the reverse primer (5'-TCCTCGTCGCTTTCCTTCAC-3') as shown in SEQ ID NO: 14, and the primer for the Osteocalcin gene was used. The forward primer (5'-CCGGGAGCAGTGTGAGCTTA-3 ') as set out in SEQ ID NO: 15 and the reverse primer (5'-TAGATGCGTTTGTAGGCGGTC-3') as set out in SEQ ID NO: 16 were used, and the Osteopontin gene was used. For primers for the forward primer (5'-GATGCCACAGATGAGGACCTC-3 ') as shown in SEQ ID NO: 17 and the reverse primer (5'-CTGGGCAACAGGGATGACAT-3') as shown in SEQ ID NO: 18 was used. The PCR reaction was incubated at 95 ° C. for 2 minutes, followed by 30 cycles of 20 seconds at 95 ° C., 10 seconds at 60 ° C., and 4 seconds at 70 ° C. After the PCR reaction as described above, the reaction was analyzed by 1.5% agarose gel electrophoresis, and visualized by EtBr (ethidium bromide) staining method. In the reverse transcriptase-RCR experiment, a GAPDH gene using a forward primer (5'-ATGACTCCACTCACGGCAAAT-3 ') as set out in SEQ ID NO: 49 and a reverse primer (5'-GGGTCTCGCTCCTGGAAGAT-3') as set out in SEQ ID NO: 50 was used. It was used as a control.

As a result, the specific marker genes are not expressed or less expressed in MSCs, whereas ALP, Cbfa-1, Osteopontin, BSP and Osteocalcin genes are remarkably expressed in mouse or human-derived cells differentiated as in <5-1>. It was confirmed that it is expressed (C of FIG. 5 and C of FIG. 6).

<5-4> real time PCR ( Real - Time PCR ) Analysis to identify differentiation

7500 using SYBR ^(R) Green PCR Master Mix (Takara, Otsu, Japan) containing cDNA corresponding to 33 ng of total RNA and a final concentration of 300 nM using the cDNA prepared in <5-3>. Specific marker genes (for mouse-derived samples: ALP, Osteopontin, BSP and Osteocalcin, for human-derived samples: ALP, via 7500 Real-time PCR System; Applied Biosystems, Foster City, CA, USA) Cbfa-1, BSP and Osteocalcin) were amplified. In the real-time PCR reaction, a primer for the ALP gene was used as a forward primer (5'-GTGCCCTGACTGAGGCTGTC-3 ') as shown in SEQ ID NO: 9 and a reverse primer (5'-GGATCATCGTGTCCTGCTCAC-3') as shown in SEQ ID NO: 10. For primers for the Cbfa-1 gene, a forward primer (5'-GCCTTCAAGGTGGTAGCCC-3 ') as shown in SEQ ID NO: 11 and a reverse primer (5'-CGTTACCCGCCATGACAGTA-3') as shown in SEQ ID NO: 12 were used. The primer for was used as the forward primer (5'-CACCCCAAGCACAGACTTTTG-3 ') as shown in SEQ ID NO: 13 and the reverse primer (5'-TCCTCGTCGCTTTCCTTCAC-3') as shown in SEQ ID NO: 14, and the primer for the Osteocalcin gene was sequenced. A forward primer (5'-CCGGGAGCAGTGTGAGCTTA-3 ') as depicted in No. 15 and a reverse primer (5'-TAGATGCGTTTGTAGGCGGTC-3') as depicted in SEQ ID NO: 16 were used for the Osteopontin gene. One primer used a forward primer (5'-GATGCCACAGATGAGGACCTC-3 ') as shown in SEQ ID NO: 17 and a reverse primer (5'-CTGGGCAACAGGGATGACAT-3') as shown in SEQ ID NO: 18. The PCR reaction was incubated at 95 ° C. for 4 minutes, followed by 40 cycles of 15 seconds at 95 ° C., 20 seconds at 60 ° C., and 34 seconds at 72 ° C. For analysis of the data, cycle threshold values were determined through automated threshold analysis, and the calculated cycle threshold values were exported to Microsoft Excel. In addition, as described by the manufacturer (Applied Biosystems), the relative expression of each target mRNA was calculated using a relatively cyclic threshold method.

As a result, in the case of mouse-derived samples, the expression level of ALP was increased by about 40-fold, the expression level of Osteopontin was increased by about 24-fold, and the expression of BSP in cells differentiated as in <5-1> compared to MSCs. The amount was increased about 18 times, the amount of Osteocalcin expression was confirmed to increase about 50 times (B of Figure 5). In addition, in the case of human-derived samples, the expression level of ALP was increased by about 80-fold and the expression level of Cbfa-1 was increased by about 4-fold in cells differentiated as in <5-1> compared to MSCs. The amount of expression increased about 45 times, and the amount of osteocalcin expression was confirmed to increase about 2 times (B of FIG. 6).

Therefore, it can be seen that SKPs-derived MSCs are differentiated into osteocytes by the method of <5-1>.

< Example 6 > SKPs -origin MSCs From chondrocytes ( chondrocyte Eruption to

<6-1> SKPs -origin MSCs From chondrocytes ( chondrocyte Differentiation into

SKPs-derived MSCs formed through the process of <4-1> were 10 ng / ml of transforming growth factor-β (TNF-β), 200 mM of ascorbic acid and 10% fetal bovine serum ( After resuspending with alpha-minimal essential medium (α-MEM) containing fetal bovine serum (FBS), 1.0 × in 5-ml defined medium for pellet production. Aliquots containing 10 ⁶ cells were centrifuged for 3 min at 390x in 15-ml polypropylene conical tubes. The pellets were incubated at 37 ° C. for 4 weeks with medium replacement every two days.

<6-2> tissue staining

The cells differentiated as in <6-1> were washed with PBS, fixed with 10% formalin for 5 minutes, and then infiltrated with Tissue-Tek ^(R) OCT Compound (SAKURA Finetek torrance, CA, USA). , cryostat (Leica CM3050 S; Leica Microsystems Nussloch GmbH, Heidelberger, Germany) was cut to 5-um. Sections were hydrated and stored for 3 minutes with 3% acetic acid, then 3% cold with 8GX solution (1% alcian blue 8GX), a chondrocyte specific stain Acetic acid, pH 2.5) for 30 minutes and then washed with running water for 2 minutes. The tissue staining sample prepared as above was observed under a microscope.

As a result, the cells differentiated as in <6-1> were stained with Alcian blue, and confirmed to occur in a form containing a rich matrix of multiple layers, and proteoglycan-rich extracellular cells. It was confirmed that an extracellular matrix was present (A of FIG. 7 and A of FIG. 8).

<6-3> Reverse transcriptase PCR ( Reverse Transcriptase PCR ) Analysis to identify differentiation

According to the manufacturer's instructions, total RNA was isolated from cells differentiated as described above using TRI-reagent (TRI-reagent; Molecular Research Center, Cincinnati, OH, USA). RNA was denatured at 70 ° C. for 10 minutes and placed on ice for 5 minutes, and cDNA was prepared using reverse transcriptase (Invitrogen) and random hexamers. In addition, specific marker genes (for mouse-derived samples: ATF3, col2α1, Ihh and Sox5, and human-derived samples) were identified by reverse transcriptase PCR using cDNA corresponding to 133 ng of RNA and primers having a final concentration of 300 nM. Aggrecan, ATF3, col2α, Ihh, Sox5, Sox6 and Sox9) were amplified. In the reverse transcriptase PCR reaction, the primer for the Aggrecan gene is used as a forward primer (5'-TGAGTGAAACCACCTCTGCATT-3 ') as shown in SEQ ID NO: 25 and a reverse primer (5'-GACGCCTCGCCTTCTTGA-3') as shown in SEQ ID NO: 26. As a primer for the ATF3 gene, a forward primer (5'-CCTCCTGGGTCACTGGTATTTG-3 ') represented by SEQ ID NO: 27 and a reverse primer (5'-TCCGATGGCAGAGGTGTTTAT-3') described in SEQ ID NO: 28 were used. For primers, the forward primer (5'-CCGGCCCTCAAGGATTTCAA-3 ') described in SEQ ID NO: 29 and the reverse primer (5'-TCCCAGCTTCACCGTCGTCA-3') described in SEQ ID NO: 30 were used. A forward primer described as 31 (5'-CTGCCCAGGCTAAGCTCCTT-3 ') and a reverse primer described as SEQ ID NO: 32 (5'-GATTTCCCCCTCCATGCAA-3') were used for the Sox5 gene. One primer used a forward primer (5'-AACAGTCCACCACCCAAAAGC-3 ') as shown in SEQ ID NO: 33 and a reverse primer (5'-AGGTGGGTGATGCAGGTGAT-3') as shown in SEQ ID NO: 34, and the primer for the Sox6 gene was SEQ ID NO: A forward primer described as SEQ ID NO: 35 (5'-CACCAGGAAGAGACTTACGAATTAGA-3 ') and a reverse primer described as SEQ ID NO: 36 (5'-CGCGCCTCCTGAATGG-3') were used, and the primers for the Sox9 gene were forward described as SEQ ID NO: 37 A primer (5'-TTACCCACTTGTGGCCAATCA-3 ') and a reverse primer (5'-GCTTGCTCTGAAGAGGGTTTAAAA-3') described in SEQ ID NO: 37 were used. The PCR reaction was incubated at 95 ° C. for 2 minutes, followed by 30 cycles of 20 seconds at 95 ° C., 10 seconds at 60 ° C., and 4 seconds at 70 ° C. After the PCR reaction as described above, the reaction was analyzed by 1.5% agarose gel electrophoresis, and visualized by EtBr (ethidium bromide) staining method. In the reverse transcriptase-RCR experiment, a GAPDH gene using a forward primer (5'-ATGACTCCACTCACGGCAAAT-3 ') as set out in SEQ ID NO: 49 and a reverse primer (5'-GGGTCTCGCTCCTGGAAGAT-3') as set out in SEQ ID NO: 50 was used. It was used as a control.

As a result, the specific marker genes were not expressed or less expressed in MSCs, whereas Aggrecan, ATF3, col2α1, Ihh, Sox5, Sox6 and Sox9 genes were remarkably expressed in the cells differentiated as described in <6-1>. It was confirmed (C of FIG. 7 and C of FIG. 8).

<6-4> real time PCR ( Real - Time PCR ) Analysis to identify differentiation

7500 using SYBR ^(R) Green PCR Master Mix (Takara, Otsu, Japan) containing cDNA corresponding to 33 ng of total RNA and a final concentration of 300 nM using the cDNA prepared in <6-3>. Specific marker genes (for mouse-derived samples: ATF3, col2α1, Ihh and Sox5, for human-derived samples: Aggrecan, via the 7500 Real-time PCR System; Applied Biosystems, Foster City, CA, USA) ATF3, col2α, Ihh, Sox5, Sox6 and Sox9) were amplified. In the real-time PCR reaction, a primer for the Aggrecan gene was used as a forward primer (5'-TGAGTGAAACCACCTCTGCATT-3 ') as shown in SEQ ID NO: 25 and a reverse primer (5'-GACGCCTCGCCTTCTTGA-3') as shown in SEQ ID NO: 26. For primers for the ATF3 gene, a forward primer (5'-CCTCCTGGGTCACTGGTATTTG-3 ') as shown in SEQ ID NO: 27 and a reverse primer (5'-TCCGATGGCAGAGGTGTTTAT-3') as shown in SEQ ID NO: 28 were used. Primer was used as the forward primer (5'-CCGGCCCTCAAGGATTTCAA-3 ') as shown in SEQ ID NO: 29 and the reverse primer (5'-TCCCAGCTTCACCGTCGTCA-3') as shown in SEQ ID NO: 30, primers for the Ihh gene is SEQ ID NO: 31 Forward primers (5'-CTGCCCAGGCTAAGCTCCTT-3 ') and reverse primers (5'-GATTTCCCCCTCCATGCAA-3') as set forth in SEQ ID NO: 32 were used. Reamers used a forward primer (5'-AACAGTCCACCACCCAAAAGC-3 ') as depicted in SEQ ID NO: 33 and a reverse primer (5'-AGGTGGGTGATGCAGGTGAT-3') as depicted in SEQ ID NO: 34, and the primer for the Sox6 gene was SEQ ID NO: 35 A forward primer (5'-CACCAGGAAGAGACTTACGAATTAGA-3 ') and a reverse primer (5'-CGCGCCTCCTGAATGG-3') described in SEQ ID NO: 36 were used, and the primers for the Sox9 gene were forward primers described in SEQ ID NO: 37. (5'-TTACCCACTTGTGGCCAATCA-3 ') and reverse primers (5'-GCTTGCTCTGAAGAGGGTTTAAAA-3') as set forth in SEQ ID NO: 37 were used. The PCR reaction was incubated at 95 ° C. for 4 minutes, followed by 40 cycles of 15 seconds at 95 ° C., 20 seconds at 60 ° C., and 34 seconds at 72 ° C. For analysis of the data, cycle threshold values were determined through automated threshold analysis, and the calculated cycle threshold values were exported to Microsoft Excel. In addition, as described by the manufacturer (Applied Biosystems), the relative expression of each target mRNA was calculated using a relatively cyclic threshold method.

As a result, in mouse-derived samples, the expression level of ATF3 was increased by about 12-fold, the expression level of col2α1 was increased by about 150-fold, and the expression of Ihh in cells differentiated as in <6-1> compared to MSCs. The amount was increased about 100 times, the expression level of Sox5 was confirmed to increase about 4 times (B of Figure 7). In addition, in the case of human-derived samples, the expression level of Aggrecan increased about 1.5-fold, the expression level of ATF3 increased about 60-fold, and the expression amount of col2α1 in the cells differentiated as in <6-1> compared to MSCs. Was increased by about 1800-fold and Ihh expression increased by about 4000-fold. In addition, the expression level of Sox5 and Sox6 was increased about 25 times, the expression level of Sox9 was confirmed to increase about 30 times (B of Figure 8).

Therefore, it can be seen that SKPs-derived MSCs are differentiated into chondrocytes by the method of <6-1>.

< Example 7 > SKPs -origin MSCs From fat cells ( adipocyte Eruption to

<7-1> SKPs -origin MSCs From fat cells ( adipocyte Differentiation into

SKPs-derived MSCs formed through the process of <4-1> were 0.5 mM 1-methyl-3-isobufilzantine (1-methyl-3- at low density (2.5 × 10 ⁴ cells / 24-well)). isobutylxanthine, 1 mM dexamethasone, 10 μg / ml insulin, 50 mM indomethacin and 10% fetal bovine serum (FBS) Incubated with essential medium (α-minimal essential medium, α-MEM) for 2 weeks. The medium was changed every 2 days and maintained for 4 weeks without passage.

<7-2> tissue staining

The cells differentiated as in <7-1> were washed with PBS, fixed with 10% formalin for 5 minutes, washed with 60% isopropanol, and completely dried. It was stained with an oil red O solution (60% isopropanol containing 0.42% oil red O) for 10 minutes, washed four times with secondary distilled water, and then observed under a microscope.

As a result, it was confirmed that lipid-rich vacuoles accumulate in the cells differentiated as in <7-1> (A of FIG. 9 and A of FIG. 10).

<7-3> Reverse transcriptase PCR ( Reverse Transcriptase PCR ) Analysis to identify differentiation

According to the manufacturer's instructions, total RNA was isolated from cells differentiated as described above using TRI-reagent (TRI-reagent; Molecular Research Center, Cincinnati, OH, USA). RNA was denatured at 70 ° C. for 10 minutes and placed on ice for 5 minutes, and cDNA was prepared using reverse transcriptase (Invitrogen) and random hexamers. The specific marker genes (C / EBPa, LPL and PPARγ) were amplified by reverse transcriptase PCR using cDNA corresponding to 133 ng of total RNA and primers having a final concentration of 300 nM. In the reverse transcriptase PCR reaction, the primer for the C / EBPa gene is a forward primer of SEQ ID NO: 19 (5'-TCAGACCAGAAAGCTGAGTTGTG-3 ') and a reverse primer of SEQ ID NO: 20 (5'-TGGTCCCCGTGTCCTCCTA-3'). The primer for the LPL gene was a forward primer (5'-CATCGAGAGGATCCGAGTGAA-3 ') as shown in SEQ ID NO: 21 and a reverse primer (5'-TGCTGAGTCCTTTCCCTTCTG-3') as shown in SEQ ID NO: 22, and PPARγ As primers for the gene, a forward primer (5'-AACTGCCGGATCCACAAAAA-3 ') as shown in SEQ ID NO: 23 and a reverse primer (5'-GCCCAAACCTGATGGCATT-3') as shown in SEQ ID NO: 24 were used. The PCR reaction was incubated at 95 ° C. for 2 minutes, followed by 30 cycles of 20 seconds at 95 ° C., 10 seconds at 60 ° C., and 4 seconds at 70 ° C. After the PCR reaction as described above, the reaction was analyzed by 1.5% agarose gel electrophoresis, and visualized by EtBr (ethidium bromide) staining method. In the reverse transcriptase-RCR experiment, a GAPDH gene using a forward primer (5'-ATGACTCCACTCACGGCAAAT-3 ') as set out in SEQ ID NO: 49 and a reverse primer (5'-GGGTCTCGCTCCTGGAAGAT-3') as set out in SEQ ID NO: 50 was used. It was used as a control.

As a result, it was confirmed that the specific marker genes were not expressed or less expressed in MSCs, whereas C / EBPa, LPL and PPARγ genes were remarkably expressed in the cells differentiated as in <7-1> (Fig. 9). C and C) of FIG. 10.

<7-4> real time PCR ( Real - Time PCR ) Analysis to identify differentiation

7500 using SYBR ^(R) Green PCR Master Mix (Takara, Otsu, Japan) containing cDNA corresponding to 33 ng of total RNA and a final concentration of 300 nM using the cDNA prepared in <7-3>. Specific marker genes (C / EBPa, LPL and PPARγ) were amplified through a 7500 Real-time PCR System (Applied Biosystems, Foster City, Calif., USA). In the real-time PCR reaction, the primers for the C / EBPa gene include a forward primer (5'-TCAGACCAGAAAGCTGAGTTGTG-3 ') as shown in SEQ ID NO: 19 and a reverse primer (5'-TGGTCCCCGTGTCCTCCTA-3') as shown in SEQ ID NO: 20. The primer for the LPL gene was used as a forward primer (5'-CATCGAGAGGATCCGAGTGAA-3 ') as shown in SEQ ID NO: 21 and a reverse primer (5'-TGCTGAGTCCTTTCCCTTCTG-3') as shown in SEQ ID NO: 22, and the PPARγ gene. Primers for were used forward primers (5'-AACTGCCGGATCCACAAAAA-3 ') as depicted in SEQ ID NO: 23 and reverse primers (5'-GCCCAAACCTGATGGCATT-3') as depicted in SEQ ID NO: 24. The PCR reaction was incubated at 95 ° C. for 4 minutes, followed by 40 cycles of 15 seconds at 95 ° C., 20 seconds at 60 ° C., and 34 seconds at 72 ° C. For analysis of the data, cycle threshold values were determined through automated threshold analysis, and the calculated cycle threshold values were exported to Microsoft Excel. In addition, as described by the manufacturer (Applied Biosystems), the relative expression of each target mRNA was calculated using a relatively cyclic threshold method.

As a result, in the case of mouse-derived samples, C / EBPa expression was increased by about 300-fold, LPL expression was increased by 40-fold, and PPARγ in cells differentiated as in <7-1> compared to MSCs. In addition, in the case of human-derived samples, C / EBPa expression was increased about 1.5-fold in cells differentiated as in <7-1> compared to MSCs, and LPL was increased by about 30-fold. The expression level of was increased by about 300 times, the expression level of PPARγ was confirmed to increase by about 15 times (B of Figure 9 and B of Figure 10).

Therefore, it can be seen that SKPs-derived MSCs are differentiated into adipocytes by the method of <7-1>.

< Example 8 > Into myogenic cells  Eruption

<8-1> SKPs -origin MSCs from Into myocytes  Differentiation induction

SKPs-derived MSCs formed through the process of <4-1> and alpha-minimal essential medium (α-MEM) containing 10% fetal bovine serum (FBS) and Incubated together for 6 weeks. The culture was passaged each time the cell density reached 90%.

<8-2> Immunocytochemistry ( immunocytochemistry ) Eruption

The cells differentiated as in <8-1> were fixed with 2% paraformaldehyde for 30 minutes at room temperature, and permeabilized with 0.1% Triton X-100. . After blocking for 60 minutes with PBS containing 1% bovine serum albumin (BSA), the cells were removed using SMAα (smooth muscle actin alpha, 1: 100; Sigma-Aldrich) and desmin (desmin, 1: 100; incubated with primary antibody against DAKO, Glostrup, Denmark for 1 hour at room temperature, followed by secondary antibody (FITC-bound goat anti-mouse IgG and Cy3-bound goat anti-rabbit IgG) ) And nuclei counterstained with DAPI. The images were observed and recorded using a fluorescence microscope (Olympus FV300; Olympus, Tokyo, Japan) equipped with a CDD camera.

As a result, it was confirmed that muscle cell specific markers SMA and desmine were detected (A of FIG. 11 and A of FIG. 12).

<8-3> Reverse transcriptase PCR ( Reverse Transcriptase PCR ) Analysis to identify differentiation

According to the manufacturer's instructions, total RNA was isolated from cells differentiated as described above using TRI-reagent (TRI-reagent; Molecular Research Center, Cincinnati, OH, USA). RNA was denatured at 70 ° C. for 10 minutes and placed on ice for 5 minutes, and cDNA was prepared using reverse transcriptase (Invitrogen) and random hexamers. In addition, specific marker genes (for mouse-derived samples: SMAα, SMAγ, Caldesmon and Smootheiln, for human-derived samples: MYH11 Smootheiln) was amplified. In the reverse transcriptase PCR reaction, the primer for the SMAα gene was used as a forward primer (5'-CCAAATCATTCCTGCCCAAA-3 ') as shown in SEQ ID NO: 39 and a reverse primer (5'-GTTGCTAGGCCAGGGCTACA-3') as shown in SEQ ID NO: 40). The primer for the SMAγ gene was used as a forward primer (5'-CCTCTTCCAGCCTTCCTTCA-3 ') as shown in SEQ ID NO: 41 and a reverse primer (5'-CAAATCTTTGCGGATGTCAATG-3') as shown in SEQ ID NO: 42. Primers for the Caldesmon gene were used as forward primers (5′-TGGAACTTGACCCCAACTTATACAG-3 ′) and reverse primers (5′-GATGACATCGGTGATCTTCAAATC-3 ′) as set forth in SEQ ID NO: 43. A forward primer (5'-CCGCATTAACGAATGGCTAAC-3 ') as depicted at 45 and a reverse primer (5'-GTTCCGCTTGCCAGATACATC-3') as depicted in SEQ ID NO: 46 were used and Smoothelin As primers for the gene, a forward primer (5'-CGCCCTCGCACGATTG-3 ') as shown in SEQ ID NO: 47 and a reverse primer (5'-CAGCTTGCGCTCCTCATATTC-3') as shown in SEQ ID NO: 48 were used. The PCR reaction was incubated at 95 ° C. for 2 minutes, followed by 30 cycles of 20 seconds at 95 ° C., 10 seconds at 60 ° C., and 4 seconds at 70 ° C. After the PCR reaction as described above, the reaction was analyzed by 1.5% agarose gel electrophoresis, and visualized by EtBr (ethidium bromide) staining method. In the reverse transcriptase-RCR experiment, a GAPDH gene using a forward primer (5'-ATGACTCCACTCACGGCAAAT-3 ') as set out in SEQ ID NO: 49 and a reverse primer (5'-GGGTCTCGCTCCTGGAAGAT-3') as set out in SEQ ID NO: 50 was used. It was used as a control.

As a result, it was confirmed that the specific marker genes were not expressed or less expressed in MSCs, whereas SMAα, SMAγ, Caldesmon, MYH11 and Smootheiln genes were remarkably expressed in the cells differentiated as in <8-1>. C of 11 and C) of FIG.

<8-4> real time PCR ( Real - Time PCR ) Analysis to identify differentiation

7500 using SYBR ^(R) Green PCR Master Mix (Takara, Otsu, Japan) containing cDNA corresponding to 33 ng of total RNA and a final concentration of 300 nM using the cDNA prepared in <8-3>. Specific marker genes (for mouse-derived samples: SMAα, SMAγ, Caldesmon, and Smootheiln, for human-derived samples: MYH11 and via a 7500 Real-time PCR System; Applied Biosystems, Foster City, CA, USA) Smootheiln) was amplified. In the real-time PCR reaction, a primer for the SMAα gene was used as a forward primer (5'-CCAAATCATTCCTGCCCAAA-3 ') as shown in SEQ ID NO: 39 and a reverse primer (5'-GTTGCTAGGCCAGGGCTACA-3') as shown in SEQ ID NO: 40. For primers for the SMAγ gene, a forward primer (5'-CCTCTTCCAGCCTTCCTTCA-3 ') as shown in SEQ ID NO: 41 and a reverse primer (5'-CAAATCTTTGCGGATGTCAATG-3') as shown in SEQ ID NO: 42 were used. Primer was used as the forward primer (5'-TGGAACTTGACCCCAACTTATACAG-3 ') as shown in SEQ ID NO: 43 and the reverse primer (5'-GATGACATCGGTGATCTTCAAATC-3') as shown in SEQ ID NO: 44, primers for the Caldesmon gene is SEQ ID NO: 45 A forward primer (5'-CCGCATTAACGAATGGCTAAC-3 ') and a reverse primer (5'-GTTCCGCTTGCCAGATACATC-3') as shown in SEQ ID NO: 46 were used. The primer for the ruler was a forward primer (5'-CGCCCTCGCACGATTG-3 ') as shown in SEQ ID NO: 47 and a reverse primer (5'-CAGCTTGCGCTCCTCATATTC-3') as shown in SEQ ID NO: 48. The PCR reaction was incubated at 95 ° C. for 4 minutes, followed by 40 cycles of 15 seconds at 95 ° C., 20 seconds at 60 ° C., and 34 seconds at 72 ° C. For analysis of the data, cycle threshold values were determined through automated threshold analysis, and the calculated cycle threshold values were exported to Microsoft Excel. In addition, as described by the manufacturer (Applied Biosystems), the relative expression of each target mRNA was calculated using a relatively cyclic threshold method.

As a result, in the mouse-derived sample, the expression level of SMAα was increased about 8-fold, the expression level of SMAγ was increased 15-fold, and the expression of Caldesmon in the cells differentiated as in <8-1> compared to MSCs. The amount was confirmed to increase about 2 times, the expression level of Smootheiln increased about 1.5 times (B of Figure 11). In addition, in the case of human-derived samples, it was confirmed that the expression level of MYH11 was increased by about 4 times and that of Smootheiln was increased by about 2.5 times in the cells differentiated as in <8-1> compared to MSCs (Fig. 12 B).

Therefore, it can be seen that SKPs-derived MSCs are differentiated into myocytes by the method of <8-1>.

<110> Seoul National University <120> Method inducing differentiation from skin derived precursor cells          to neural and mesenchymal lineage cells and differentiation          inducing composition used pretty <130> 10P-02-45 <160> 50 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Peripherin forward primer <400> 1 ccactctgtc ccgcctagaa 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> peripherin reverse primer <400> 2 tgcaggtctc gaagttcctc tt 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TuJ1 forward primer <400> 3 tccgcctgcc ttttcgtctc ta 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TuJ1 reverse primer <400> 4 agttgccgct ggggtctatg c 21 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GFAP forward primer <400> 5 catgccacgc ttctccttgt ctc 23 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GFAP reverse primer <400> 6 gctcgctcgc ccgtgtct 18 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S100beta forward primer <400> 7 cctttcagga tgatcgcttt g 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S100beta reverse primer <400> 8 atggacagca gagggagcaa 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALP forward primer <400> 9 gtgccctgac tgaggctgtc 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ALP reverse primer <400> 10 ggatcatcgt gtcctgctca c 21 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cbfa1 forward primer <400> 11 gccttcaagg tggtagccc 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cbfa1 reverse primer <400> 12 cgttacccgc catgacagta 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Bone sialoprotein forward primer <400> 13 caccccaagc acagactttt g 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bone sialoprotein reverse primer <400> 14 tcctcgtcgc tttccttcac 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Osteocalcin forward primer <400> 15 ccgggagcag tgtgagctta 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Osteocalcin reverse primer <400> 16 tagatgcgtt tgtaggcggt c 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Osteopontin forward primer <400> 17 gatgccacag atgaggacct c 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Osteopontin reverse primer <400> 18 ctgggcaaca gggatgacat 20 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CEBPalpha forward primer <400> 19 tcagaccaga aagctgagtt gtg 23 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CEBPalpha reverse primer <400> 20 tggtccccgt gtcctccta 19 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LPL forward primer <400> 21 catcgagagg atccgagtga a 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LPL reverse primer <400> 22 tgctgagtcc tttcccttct g 21 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARgamma forward primer <400> 23 aactgccgga tccacaaaaa 20 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PPARgamma reverse primer <400> 24 gcccaaacct gatggcatt 19 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Aggrecan forward primer <400> 25 tgagtgaaac cacctctgca tt 22 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Aggrecan reverse primer <400> 26 gacgcctcgc cttcttga 18 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ATF3 forward primer <400> 27 cctcctgggt cactggtatt tg 22 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ATF3 reverse primer <400> 28 tccgatggca gaggtgttta t 21 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Collagen type II alpha 1 forward primer <400> 29 ccggccctca aggatttcaa 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Collagen type II alpha 1 reverse primer <400> 30 tcccagcttc accgtcgtca 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ihh forward primer <400> 31 ctgcccaggc taagctcctt 20 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ihh reverse primer <400> 32 gatttccccc tccatgcaa 19 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sox5 forward primer <400> 33 aacagtccac cacccaaaag c 21 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox5 reverse primer <400> 34 aggtgggtga tgcaggtgat 20 <210> 35 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Sox6 forward primer <400> 35 caccaggaag agacttacga attaga 26 <210> 36 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Sox6 reverse primer <400> 36 cgcgcctcct gaatgg 16 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sox forward primer <400> 37 ttacccactt gtggccaatc a 21 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Sox9 reverse primer <400> 38 gcttgctctg aagagggttt aaaa 24 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SMAalpha forward primer <400> 39 ccaaatcatt cctgcccaaa 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SMAalpha reverse primer <400> 40 gttgctaggc cagggctaca 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SMAgamma forward primer <400> 41 cctcttccag ccttccttca 20 <210> 42 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SMAgamma reverse primer <400> 42 caaatctttg cggatgtcaa tg 22 <210> 43 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> MYH11 forward primer <400> 43 tggaacttga ccccaactta tacag 25 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MYH11 reverse primer <400> 44 gatgacatcg gtgatcttca aatc 24 <210> 45 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Caldesmon forward primer <400> 45 ccgcattaac gaatggctaa c 21 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Caldesmon reverse primer <400> 46 gttccgcttg ccagatacat c 21 <210> 47 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Smoothelin forward primer <400> 47 cgccctcgca cgattg 16 <210> 48 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Smoothelin reverse primer <400> 48 cagcttgcgc tcctcatatt c 21 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 49 atgactccac tcacggcaaa t 21 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 50 gggtctcgct cctggaagat 20

Claims (20)

Skin-derived precursor cells (SKPs) are serum-free containing brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and nerve growth factor (NGF). Induction of differentiation of peripheral nerve cells from skin-derived progenitor cells (SKP), comprising culturing in a medium.
Culturing skin-derived precursor cells (SKP) in a serum-free medium containing dibutyryl cyclic AMP (dbcAMP), bFGF and neuregulin Including, Schwann cells differentiation induction method from skin-derived progenitor cells (SKP).
The method of claim 1 or 2, wherein the skin-derived progenitor cells (SKPs) are derived from a back skin of a mouse or a human foreskin.
The method of claim 1 or 2, wherein the serum-free medium is serum-free DMEM / F12.
The method of claim 1 or 2, wherein the culturing is carried out in a culture dish coated with polyornithine and laminin.
Mesenchymal stem cells (MSCs) are media containing beta-glycerol phosphate (β-glycerol phosphate), dexamethasone, ascorbic acid and fetal bovine serum (FBS). Inducing osteoblast differentiation from mesenchymal stem cells (MSCs) comprising culturing.
Culturing mesenchymal stem cells (MSCs) with a medium containing transforming growth factor-β (TNF-β), ascorbic acid and fetal bovine serum (FBS). Method of inducing chondrogenic differentiation from mesenchymal stem cells (MSCs).
Mesenchymal stem cells (MSCs) were identified as 1-methyl-3-isobutylxanthine, dexamethasone, insulin, indomethacin and fetuses. Method of inducing adipocyte differentiation from mesenchymal stem cells (MSCs), comprising culturing in a medium containing serum (fetal bovine serum, FBS).
Myogenic cells from mesenchymal stem cells (MSCs) comprising culturing mesenchymal stem cells (MSCs) in a medium containing fetal bovine serum (FBS) in a culture dish. ) Differentiation induction method.
10. The method of any one of claims 6-9, wherein said mesenchymal stem cells (MSCs) are derived from skin-derived progenitor cells (SKPs).
10. The method of claim 6, wherein the medium is alpha-minimal essential medium (α-MEM).
10. The method according to any one of claims 6 to 9, wherein the culturing is carried out in a culture dish coated with polyornithine, laminin and fibronectin.
The method of claim 10, wherein the mesenchymal stem cells (MSCs) are produced by the steps of 1) to 3) below.
1) removing skin-derived progenitor cells (SKPs) and diluting with DMEM / F12 medium containing EGF, bFGF and B27;
2) DMEM / F12 medium containing EGF, bFGF and B27 in a culture dish coated with diluted skin-derived progenitor cells (SKPs) of step 1) with polyornithine, laminin and fibronectin. Culturing with; And
3) replacing the medium of step 2) with an alpha-minimal essential medium (α-MEM) containing fetal bovine serum (FBS) without a growth factor.
The method of claim 13, wherein the skin-derived progenitor cells (SKPs) are derived from a back skin of a mouse or a human foreskin.
For inducing differentiation of peripheral nerve cells from skin-derived progenitor cells (SKP), including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and nerve growth factor (NGF) Composition.
Composition for inducing differentiation of Schwann cells from skin-derived progenitor cells (SKP), including dibutyryl cyclic AMP (dbcAMP), bFGF and neuregulin.
Osteogenic cells from mesenchymal stem cells (MSCs), including beta-glycerol phosphate, dexamethasone, ascorbic acid and fetal bovine serum (FBS) Composition for inducing differentiation of a).
For inducing differentiation of chondrogenic cells from mesenchymal stem cells (MSCs), including transforming growth factor-β (TNF-β), ascorbic acid and fetal bovine serum (FBS) Composition.
1-methyl-3-isobutylxanthine, including dexamethasone, insulin, indomethacin and fetal bovine serum (FBS), Composition for inducing differentiation of adipogenic cells from mesenchymal stem cells (MSCs).
Composition for inducing differentiation of myogenic cells from mesenchymal stem cells (MSCs), including fetal bovine serum (FBS).

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