KR100659779B1 - Osteogenic differentiation of human mesenchymal stem cells using histone deacetylase inhibitors - Google Patents

Abstract

The present invention relates to osteoinduction of mesenchymal stem cells (MSC) by histone deacetylase (HDAC) inhibitors and to a method of treating HDAC inhibitors.

According to the present invention, it can be seen that pretreatment of the HDAC inhibitor significantly increases the effect of inducing osteoblast differentiation in mesenchymal stem cells. Therefore, HDAC inhibitors can be prepared on the basis of promoting the differentiation of bone cells by HDAC inhibitors, and can be used to develop bone regeneration technology using bone differentiation inducing agents.

Description

Osteodifferentiation-inducing compositions of mesenchymal stem cells containing histone deacetylase inhibitors and methods for increasing bone differentiation using the same {Osteogenic differentiation of human mesenchymal stem cells using histone deacetylase inhibitors}

1 is a result of measuring the degree of bone differentiation by treatment with valproic acid (VPA), a kind of HDAC inhibitor in mesenchymal cells.

Figure 2 is a result showing that the differentiation into osteoblasts by the pretreatment of VPA in mesenchymal stem cells.

Figure 3 is the result of measuring the action of inhibiting the differentiation of fat by VPA premedication in mesenchymal stem cells.

FIG. 4 shows the effect of another HDAC inhibitor, tracostatin A (TSA), and valproic acid-like derivatives on the differentiation of mesenchymal stem cells into osteoblasts, and the degree of bone differentiation of zalpromide without HDAC inhibitory activity. This is the result of checking.

5 is a result of confirming the change in gene expression confirmed by Microarray by real time PCR.

6 is a result confirming the effect of VPA on the proliferation of mesenchymal stem cells.

Figure 7 is a result showing the increase in the biological skull regeneration ability in VPA pretreated cells.

The present invention relates to the control of bone differentiation, and more particularly, to an HDAC inhibitor, a derivative compound thereof, and a bone differentiation-promoting composition containing the same, which are first identified in the mesenchymal stem cell (MSC).

The DNA of the chromosome has a very compact structure called chromatin. Gene expression in the nucleus is partly controlled by a group of protein complexes that release from these tight nucleosomal particles to accessible nucleosomal particles. Mechanisms of such packing and unpacking processes include acetylation and deacetylation of histone proteins that make up the nucleosome core. One nucleosome core particle contains an octamer of histone proteins (H2A, H2B, H3 and H4). Acetylated histone proteins allow transcription factors to access DNA templates for expression. Several known steroid receptor coactivators have this histone acetyltransferase (HAT) activity. In contrast, histone deacetylases (hereinafter referred to as HDACs) are chromatin remodeling factors that deacetylate histone proteins and may act as transcriptional repressors.

Valproic acid (VPA; 2-propyl-pentanoic acid) is a short-chain fatty acid that was first developed as an anticonvulsant and is currently used to treat a variety of psychiatric disorders. The mechanism of action of VPA on these diseases is not well known, but several mechanisms are thought to be involved (Johanessen, 2000; Gurvich et al, 2002). VPA is known to inhibit histone deacetylase (HDAC) at therapeutic doses (Phiel et al, 2001; Gottlicher et al, 2001). Acetylation of the amino terminus of histones affects the structure of chromatin to determine histone codes affecting gene expression (Strahl et al, 2000). When the histone acetyltransferrease acts as a transcription factor complex and the histone is acetylated, the DNA has a more open form, which generally increases the expression of the gene. Conversely, deacetylation of histones by HDAC converts the conformation of chromatin into closed form, leading to inhibition of the transcriptional process. Thus, inhibition of HDAC will promote transcriptional processes. In addition, VPA has been reported to inhibit neuronal growth by inducing inositol deficiency (Williams et al, 2002).

It has been reported that HDAC inhibitors inhibit cell growth and induce differentiation of various cancer cells (Marks et al, 2001; Cinatl et al, 1996; Blaheta & Cinatl, 2002; Gottlicher et al, 2001). In actual clinical trials (Marks et al, 2001; Kramer et al, 2001; Gore et al, 2000; Cheson et al, 2000). VPA has also been reported to inhibit the differentiation of embryonic stem cells into cardiomyocytes (Na et al, 2003) and to inhibit the differentiation of human fat cells (Lagace & Nachtigal, 2004).

Mesenchymal stem cells (MSCs) are easily proliferated in vitro and are capable of differentiating into various cell types (fat cells, chondrocytes, muscle cells, bone cells) [Caplan, AI 1991. J. Orthop. Res. 9, 641-650; Beresford, JN, Bennett, JH, Devlin, C., Leboy, PS, and Owen, ME (1992). J. Cell Sci . 102, 341-351; Pittenger, MF, Mackay, AM, Beck, SC, Jaiswal, RK, Douglas, R., Mosca, JD, Moorman, MA, Simonetti, DW, Craig, S., and Marshak, DR (1999). Science 284, 143-147; Patricia, AZ, Min, Z., Hiroshi, M., Jerry H., William, FJ, Adam, JK, Prosper, B., Peter LH, and Hedrick MH 2001. Tissue engineering. 7, 211-227. Thus, mesenchymal stem cells can be used as useful targets in gene therapy and cell therapy, and understanding the differentiation mechanism of mesenchymal stem cells plays an important role in understanding the early differentiation of various cells [Banfi, A., Muraglia, A., Dozin, B., Mastrogiacomo, M., Cancedda, R., and Quarto, R. 2000. Exp. Hematology . 28, 707-715.

In the present invention, it was demonstrated that HDAC inhibitors in mesenchymal stem cells can regulate the bone differentiation process of mesenchymal stem cells, in particular, the effect of pretreatment before differentiation alone. Therefore, the present invention is a patent for the control of bone differentiation of HDAC inhibitors and provides a basis for the development of bone differentiation control and bone therapy using the same.

Therefore, the main object of the present invention is to provide a composition for promoting osteoblast differentiation of mesenchymal stem cells (MSC) containing HADC inhibitor.

Another object of the present invention is to use in the development of a therapeutic agent for bone-induced related bone diseases containing the composition of the present invention.

Another object of the present invention to provide a method for increasing bone differentiation of mesenchymal stem cells using the composition of the present invention.

In order to achieve the object of the present invention, the present invention provides a composition for inducing bone differentiation of mesenchymal stem cells (MSC) comprising a histone deacetylase (HDAC) inhibitor as an active ingredient.

In the compositions of the present invention, HDAC inhibitors have been widely used in conventional anticancer therapies as agents for inhibiting deacetylation of histones. Examples include short-chain fatty acids such as 4-phenylbutyrate and valproic acid, suveroylanilide hydroxamic acid (SAHA), pyroxamides, hydroxamic acid such as tracostatin A, oxamplatin and CHAPS, trapoxine, api Cyclic tetrapeptides such as cydines and depsipeptides (also known as FK-228 or FR 901228), and benzamides such as MS-275 (Kouraklis G. and Theocharis S., Current Medicinal Chemistry "Histone Deacetylase Inhibitors and Anticancer Therapy", 2002, vol. 2, no. 4, pp. 477-484). The present invention has revealed a novel use of the HDAC inhibitor as an osteoblast differentiation promoter.

In the composition of the present invention, preferably, the HDAC inhibitor provides a composition for inducing bone differentiation, characterized in that the valproic acid of Formula 1 or a derivative thereof:

[Formula 1]

Valproic acid (VPA) is 2-propylpentanoic acid [(CH ₃ CH ₂ CH ₂ ) ₂ CHCOOH], which has been used for several decades as a stabilizer and anticonvulsant for the treatment of mental disorders. 400-1000 mg has been used (Johannessen CU. MNeurochem Int 200037: 103-110; Gurvich N, Klein PS. Pharmacol Ther 200296: 45-66.). The present invention includes such valproic acid as well as derivatives thereof that exhibit HDAC inhibitory potency in vivo or in vitro. The derivatives include various pharmaceutically acceptable inorganic or organic salts.

In the composition of the present invention, the valproic acid or a derivative thereof is preferably contained at a concentration of 1-4 mM, more preferably at a concentration of 2-4 mM. Too low a concentration does not have an effect. Too high a concentration is toxic, so you should check the appropriate concentration according to the type of cell.

In the composition of the present invention, the HDAC inhibitor provides a composition for inducing bone differentiation, characterized in that trachostatin of Formula 1 or a derivative thereof:

[Formula 2]

Trichostatin (TSA) is 7- (4- (dimethylamino) phenyl) -n-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadieneamide (7- (4- ( dimethylamino) phenyl) -n-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide (C ₁₇ H ₂₂ N ₂ O ₃ ), histone deacetylase isolated from Streptomyces. The present invention includes such tracostatin as well as derivatives thereof that exhibit HDAC inhibitory activity in vivo or in vitro, including various pharmaceutically acceptable inorganic or organic salts.

In the composition of the present invention, the trichostatin or its derivatives are preferably contained at a concentration of 300-900 uM, more preferably at a concentration of 500-800 uM. If the concentration is too low, it will be difficult to see the effect. If the concentration is too high, it will be toxic, so you should check the proper concentration according to the cell type.

In order to achieve the other object of the present invention, the present invention is a bone comprising a step of treating any agent to the mesenchymal stem cells (MSC) and measuring the degree of osteoblast differentiation in the mesenchymal stem cells (MSC) It provides a method for screening a Histone deacetylase (HDAC) inhibitor having a cell differentiation control function.

In the method of the present invention, preferably, in the first step, the cultured mesenchymal stem cells of the human body are separated from adipose tissue and bone marrow and cultured in a multi-well culture vessel such as 6, 12, 24, 46, or 96. Bone differentiation is induced with or without HDAC inhibitor. In the second step, the effect of HDAC inhibitor is measured by measuring the degree of bone differentiation by the same method as alizarin S staining.

In order to achieve the other object of the present invention, the present invention is to separate the mesenchymal stem cells from fat or bone marrow, pre-treatment with differentiation-induced differentiation-induced differentiation of isolated mesenchymal stem cells (HDAC) inhibitor, and pre-treated Provided is a method for increasing bone differentiation of stromal stem cells comprising culturing the mesenchymal stem cells in a bone differentiation medium.

In the method of the present invention, preferably, the HDAC inhibitor is valproic acid or trachostatin, more preferably the HDAC inhibitor is 1-3 in the case of valproic acid. In the case of mM or trachostatin it is characterized in that it is contained in a concentration range of 300-800 uM.

In the method of the present invention, the bone differentiation-inducing medium may be a medium commonly used for bone differentiation in the art, but preferably 8-12% FBS, 48-52 μg / ml ascorbic acid, 1.3-1.7 mg / It is characterized in that the α-MEM medium containing ml β-glycerophosphate, 0.8-1.2 x10 ^-8 M dexamethasone, more preferably the same composition α-MEM medium containing 8-12% human serum instead of animal serum It is done.

In order to achieve the another object of the present invention, the present invention provides a cell therapy composition containing osteoblast differentiated mesenchymal stem cells (MSC) in vitro in accordance with the present invention as an active ingredient.

The composition of the present invention, mesenchymal stem cells pretreated with an HDAC inhibitor in vitro can be used for various cell treatment methods. [Lee JA, Parrett BM, Conejero JA, Laser J, Chen J, Kogon AJ, Nanda D, Grant RT, Breitbart AS. 2003. Ann Plast Surg. 50 (6): 610-7.] In this case, cells treated with HDAC inhibitors in vitro can be mixed with various bioadjuvant such as collagen or hydroxyapatite scaffold and surgically implanted into bone tissue without induction of differentiation. The cell therapy composition of the present invention may further contain collagen or bone support as an auxiliary component in addition to the bone differentiated mesenchymal stem cells. Differentiated stem cells in the composition of the present invention may vary depending on the type of disease, route of administration, age and sex of the patient, and degree of disease, but preferably 1-5 x 10 ⁷ cell / cm ³ for the average adult. Administer the scaffold.

In the composition of the present invention, the cell therapy composition is characterized in that it is used for the treatment of bone diseases. Here, the bone disease refers to a disease related to bone that requires bone differentiation and regeneration, and includes traumatic bone injury such as fracture and bone defect occurring after surgery. The composition of the present invention can treat the bone disease through osteoinduction.

Specifically, in the present invention, VPA in bone mesenchymal stem cells isolated and cultured from adipose tissue and bone marrow increased bone differentiation depending on concentration and inhibited fat differentiation. The same effect was observed on mesenchymal stem cell differentiation by two or four days before differentiation induction. VPA inhibited the proliferation of mesenchymal stem cells at concentrations above 2 mM. Tracostatin A (TSA), another inhibitor of histone deacetylase (HDAC), also increased mesenchymal differentiation of mesenchymal stem cells, but valpromid, which is structurally similar to VPA but without the inhibitory action of HDAC, was significant in bone differentiation. No effect was observed.

As a result of gene expression analysis using microarray, the expression changes of various genes were observed in VPA treated cells. In particular, mRNA levels of genes involved in inducing bone differentiation such as BMP-2 and Runx2 were increased and confirmed by real-time PCR.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Isolation of Mesenchymal Stem Cells from Fat and Bone Marrow

Stromal stem cells were isolated from the fat and bone marrow obtained from patients undergoing femoral replacement and liposuction at Pusan National University Hospital. Ficoll is added to bone marrow to separate stromal stem cells from bone marrow, centrifuged at 3500 rpm for 30 minutes, and the supernatant, a mononuclear cell layer, is washed with HBSS. 58% low glucose DMEM, 40% MCDB-201 or F-12, 1x insulin-transferrin-selenium, 1x linoleic acid-BSA, 10 ^{-8 in a} fibronectin-coated culture vessel after centrifugation M dexamethasone, 10 ^-4 M ascorbic acid 2-phosphate, is cultured in a culture solution of 100 U penicillin, 1,000 U streptomycin, 2% FCS, 10 ng / ml EGF (Daewoong pharmaceutical). The separated cells are plated in a dish to remove unattached cells after 24 hours. Adipose tissues are obtained from patients undergoing subcutaneous fat removal surgery in plastic surgery to separate stromal stem cells from adipose tissue. After washing the separated adipose tissue with HBSS, cut the adipose tissue using tweezers and scissors and treat 0.075% collagenase at 37 ^o C for 30 minutes. 58% crude glucose DMEM, 40% MCDB-201 or F-12, 1x insulin-transferrin-selenium, 1x linoleic acid-BSA, 10 ^-8 M dexamethasone, 10 ^-4 M After resuspending by adding a medium consisting of ascorbic acid 2-phosphate, 100 U penicillin, 1,000 U streptomycin, 2% FCS, 10 ng / ml EGF (Sigma Chemical Co), and then passing through a 100 μm nylon mesh. The isolated cells are plated in a dish to remove unattached cells after 24 hours. As a result of confirming the expression of the mesenchymal stem cell surface antigens isolated as described above by flow cytometry, there was no significant difference in the surface antigen expression between mesenchymal stem cells cultured with human serum or fetal bovine serum ( See the previously filed Korean Patent Application No. 2004-24900).

Example 2: BPA-induced Bone Differentiation and Adipogenesis Inhibition in Mesenchymal Stem Cells

Adipose and bone marrow stromal cells (mesenchymal stem cells) were treated with 0.5 to 3 mM valproic acid for the first three days of differentiation to assay the effect on VPA's bone differentiation. Bone differentiation induction was cultured in bone differentiation induction medium (α-MEM medium containing 10% FBS or human serum, 50 μg / ml ascorbic acid, 1.5 mg / ml β-glycerophosphate, ^10-8 M dexamethasone). After 3 days of VPA treatment at 0.5, 1, 2, and 3 mM of adipocytes and bone marrow stromal cells, bone differentiation medium was added to control cells (cells not treated with VPA) and induced differentiation for a week. The degree was taken to compare with the control group. 1 is a result of measuring the degree of bone differentiation by treatment of valproic acid (VPA), a kind of HDAC inhibitor in mesenchymal cells. When the cells differentiate into osteocytes, calcium carbonate is formed on the cell surface, which is stained red by Alizarin Red S. From Figure 1 it can be seen that the cells treated with VPA 0.5, 1, 2, 3mM to the adipocytes and bone marrow stromal cells, respectively, compared to the control group to promote bone differentiation.

Induction of differentiation into bone cells is incubated for 2 weeks in α-MEM medium containing 10% FBS or human serum, 50 μg / ml ascorbic acid, 1.5 mg / ml β-glycerophosphate, ^10-8 M dexamethasone. Differentiation into bone cells is confirmed by Alizarin Red S staining (Lee DH, Kang SK, Lee RH et al. J Cell Physiol 2004; 198: 91-99). VPA showed a concentration-dependent promoting effect of bone differentiation. In order to confirm whether the effect of the VPA is the same by pretreatment, pretreatment with 1 mM VPA for 1, 2, or 4 days was confirmed that the degree of bone differentiation increased with the pretreatment period. Figure 2 is a result showing that differentiation into osteoblasts by pretreatment of VPA in mesenchymal stem cells. Figure 2 is an experiment to determine whether the same concentration of VPA 1mM to treat the adipocytes and bone marrow stromal cells affect bone differentiation according to the treatment period. Therefore, after the treatment period was set to 1, 2 or 4 days, the degree of differentiation was confirmed in the bone differentiation media. As a result, after the same treatment with VPA 1mM it was confirmed that the difference in the degree of differentiation according to the pretreatment period.

As a result of observing the effect of VPA on the degree of fat differentiation, the degree of inhibition was not significant but it was confirmed that the degree of fat differentiation was suppressed in a concentration-dependent manner. Induction of differentiation into adipocytes is incubated for 3 weeks in α-MEM medium containing 1% FCS, 10 ^-7 M dexamethasone and 6 ng / ml insulin. Adipocytes are identified by staining with Oil red O. Figure 3 is the result of measuring the differentiation inhibitory action by VPA pretreatment in mesenchymal stem cells. In FIG. 3, the differentiation ability into fat other than bone differentiation was tested in the same manner as in FIG. 1, and it was confirmed that the differentiation into fat inhibited the differentiation of fat as opposed to the result of bone differentiation. Experiments were performed for 3 days with VPA at 0.25, 0.5, 1, 2, and 3mM in adipocytes and bone marrow stromal cells, respectively, and then differentiated for 1 week by adding adipose differentiation medium like the control group (cells not treated with VPA). After induction, the degree of differentiation was compared with the control group. When cells differentiate into osteocytes, a lipid drop occurs on the cell surface, which is stained by oil red O. In other words, the cells treated with 0.5, 1, 2, and 3 mM of VPA in the adipose and bone marrow matrix cells, respectively, compared to the control group, can be seen that the differentiation of adipose differentiation.

Example 3 Induction of Bone Differentiation by Trachostatin, an HDAC Inhibitor, in Mesenchymal Stem Cells

In order to confirm whether increased bone differentiation by VPA is related to the inhibition of HDAC, the effect of valpromide and another HDAC inhibitor, tracostatin, which is a similar derivative of Kyabavalproic acid and has no HDAC inhibitory effect, was the same as that of Example 2. It was observed by the method. After 3 days pretreatment with tracostatin 75, 150, 300, 500 nM and 0.5, 1, 2, 3 mM of valpromide in adipocytes and bone marrow stromal cells, the bone differentiation induction medium was induced. The degree of bone differentiation was observed. As a result, the pretreatment with tracostatin increased the bone differentiation capacity in the fat matrix cells in a concentration-dependent manner, but the valpromide did not show a significant effect. 4 shows the differentiation of tracostatin A (TSA), a valproic acid-derived derivative, and the degree of bone differentiation of Kambabapromide without HDAC inhibitory activity in mesenchymal stem cell differentiation into osteoblasts. This is the result of confirming the effect. In FIG. 4, the treatment of tracostatin A (TSA) at 75,150,300,500 nm promoted bone differentiation in a concentration-dependent manner. However, when treated with valpromide 0.5,1,2,3mM, no significant effect was observed. These results suggest that HDAC may play an important role in inducing bone differentiation, as it promotes bone differentiation similar to HDAC inhibitors tracostatin A (TSA) and valproic acid.

Example 4 Genes Showing Bone Differentiation Induction among Genes Changed by VPA Pretreatment

Microarray analysis was performed to reveal the mechanism of bone differentiation induced by VPA. Affymetrix U133 PLUS 2.0, a cDNA microarray that recognizes more than 47,000 transcripts, was used to analyze the genes changed by VPA pretreatment according to the Affymetrix protocol. Microarray analysis of the mesenchymal stem cells and the VPA-treated mesenchymal stem cells mRNAs showed that the mesenchymal stem cells treated with VPA increased or decreased compared to the mesenchymal stem cells. Tables 1 and 2 show results of functional classification of genes whose expression is regulated by VPA.

TABLE 1

 Functional classification of downregulated genes by valproic acid in hATSCs

TABLE 2

 Functional classification of upregulated genes by valproic acid in hATSCs

As a result of microarray analysis, BMP-2, Runx2, osteopontin, etc., which are involved in bone differentiation, were significantly increased, and the expression of genes involved in cell cycle was decreased. In order to confirm the expression changes of these genes, the analysis of real-time PCR revealed that the expression of BMP-2 and Runx2 was increased by 30 and 4 times. 5 is a result of confirming the change in gene expression confirmed by microarray by real-time PCR. LightCycler (Roche) was used in accordance with the protocol of LightCycler Software Version 3 provided by Roche. ( Rajeevan MS, Ranamukhaarachchi DG, Vernon SD, Unger ER: Methods 2001; 25: 443-451 ) Figure 5a shows that in order to analyze the expression level of BMP-2 and Runx2, the same amount of cDNA must be analyzed first. To determine the amount of cDNA produced from total RNA of mesenchymal stem cells and mesenchymal stem cells treated with VPA, cDNA levels were compared using β-actin (house keeping gene) primers. . As a result, it was confirmed that the cDNA amounts of the two samples (total RNA of mesenchymal stem cells and mesenchymal stem cells treated with VPA) diluted to 1/10000 match. Figure 5b shows the expression level of the Runx2 gene based on the results of a Runx2 gene expression diluted to 1/10000 of mesenchymal stem cells treated with VPA runx2 gene diluted to 1/10000 of mesenchymal stem cells The expression of Runx2 gene was increased in mesenchymal stem cells treated with VPA as a result supporting the microarray analysis. 5C is a result of confirming the expression level of the BMP-2 gene based on the result of a As a result supporting microarray analysis, BMP-2 gene expression diluted to 1/10000 of mesenchymal stem cells treated with VPA was higher than BMP-2 gene expression diluted to 1/10000 of mesenchymal stem cells. Mesenchymal stem cells treated with VPA were found to increase the expression of BMP-2 gene.

Example 5: Action on Cell Proliferation by VPA Pretreatment

The pretreatment of VAP affected the proliferative capacity of adipocytes. Adipocytes were treated with low concentrations (5103 cells / well) of VPA 1, 2, and 3 mM, and then separated into single cells with trypsin / EDTA after 48 hours. The number of cells was measured. 7 is a result confirming the effect of VPA on the proliferation of mesenchymal stem cells. In FIG. 7, the proliferation ability of the control and the adipocytes treated with VPA 1,2,3 mM was measured by the method described above, and the number of cells was expressed as a percentage. As a result, it was observed that pretreatment of VPA significantly inhibited the proliferation of cells in a concentration-dependent manner.

Example 6 Increase of In Vivo Osteodifferentiation by VPA Pretreated Cells

To determine whether mesenchymal stem cells treated with VPA promote bone regeneration in vivo, bone regeneration was performed by adding collagen, fat matrix cells and pre-treated fat stromal cells to VPA in the skull defect (4 mm) model of mice. As a result of the evaluation, it was confirmed that bone formation was significantly increased in VPA-treated adipocytes. Mesenchymal stem cells treated as control and mesenchymal stem cells treated with 1 mM VPA for 2 days were planted in collagen cut to a size of 4 mm in diameter and 1 mm thick to 1 × 10 ⁶ cells / 10 μl serum-free α-MEM, respectively, for 30 minutes. Incubated. The culture was implanted into a cranial (4 mm) model of mice and 10 mg / kg of cyclosporine was administered every two days to suppress immune responses. Five weeks after transplantation, the mice were sacrificed to separate the skulls, and the separated skulls were decalcified and embedded in pparaffin. After cutting to 10㎛ thickness hematoxylin and iosin staining was confirmed whether the bone regeneration. 8 is a diagram showing that adipocytes treated with VPA promote bone regeneration in vivo. Arrows indicate bone tissue formed from transplanted adipocytes. It can be seen that the adipocytes treated with VPA promote bone regeneration in the cranial defects of mice rather than only the mesenchymal stem cells.

As described above, according to the present invention it can be seen that HDAC inhibitors in human mesenchymal stem cells (including MSC) significantly promote differentiation into bone cells. Therefore, based on the mechanism by which differentiation into osteoblasts is promoted by these drugs, HDAC inhibitors can be prepared and used for the development of therapeutic agents for bone diseases. That is, the present invention may provide the use of HDAC inhibitors in the treatment of bone regeneration and bone-related diseases.

Claims (12)

A bone differentiation-inducing composition of mesenchymal stem cells (MSC) comprising a histone deacetylase (HDAC) inhibitor as an active ingredient, wherein the HDAC inhibitor is a valproic acid of Formula 1 below Theft Composition: [Formula 1]

. delete The composition of claim 1, wherein the valproic acid is contained at a concentration of 1-4 mM. delete delete delete Separating mesenchymal stem cells from fat or bone marrow, pretreatment of isolated mesenchymal stem cells with differentiation-inducing histone deacetylase (HDAC) inhibitors, and culturing the pretreated mesenchymal stem cells in bone differentiation-inducing media A method for increasing bone differentiation of stromal stem cells, comprising the step of: HDAC inhibitor is valproic acid. delete 8. The method of claim 7, wherein the valproic acid is contained in a concentration range of 1-3 mM. The method of claim 7, wherein the bone differentiation-inducing medium comprises 8-12% FBS or human serum, 48-52 μg / ml ascorbic acid, 1.3-1.7 mg / ml β-glycerophosphate, 0.8-1.2 x10 ^-8 M dexamethasone Bone differentiation increasing method characterized in that the α-MEM medium. A cell therapeutic composition containing bone marrow differentiated mesenchymal stem cells (MSC) according to claim 9 or 10 as an active ingredient in vitro, wherein the cell is used for the treatment of bone diseases. Therapeutic composition. delete

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