KR101591782B1 - Composition for inducing differentiation of mesenchymal stem cell and method for inducing differentiation - Google Patents

Abstract

The present invention relates to a technique for inducing the differentiation of mesenchymal stem cells into various cell types such as neurons, chondrocytes and bone cells using 5-Azacitidine and / or Trichostatin A .

Description

TECHNICAL FIELD The present invention relates to a composition for inducing differentiation of mesenchymal stem cells and a method for inducing differentiation,

The present invention relates to a technique for inducing the differentiation of mesenchymal stem cells into various cell types such as neurons, chondrocytes and bone cells using 5-Azacitidine and / or Trichostatin A .

Human mesenchymal stem cells are one of the adult stem cells that are free from cancer and ethical problems that are generally problematic in embryonic stem cells, as well as adipocytes, bone cells, cartilage cells, cardiac cells , Muscle cells, and nerve cells. In addition, since mesenchymal stem cells do not induce an immune response at the time of transplantation, they are becoming an effective means of cell therapy.

However, as a method of separating cells from the mesenchymal stem cells completely differentiated from the tissue of the donor and transplanting them into the patient, it is difficult to obtain enough cells to be supplied to the patient. Therefore, in order to solve such a problem of supplying cells, mesenchymal stem cells are isolated, cultured and propagated in vitro, and then induced to differentiate into cells of a desired lineage, and a technique of transplanting the differentiated cells into a patient is established Is studied.

For example, in recent years, it has been reported that mesenchymal stem cells have the potential to differentiate into glial cells, suggesting that mesenchymal stem cells can be used to treat diseases of the central nervous system (Li et al. Neurosci. Lett., 315, 67-70 (2001); and Chen, et al., Stroke 32, 1005-1011 (2001)). Thus, it is known that a growth factor or a hormone is used as a substance inducing differentiation from mesenchymal stem cells into neurons. However, in this case, there are problems in that various other cells besides neurons are generated (Sanchez- Et al., J. Neurosci. Res., 61, 364-370 (2000), and Deng., Et < RTI ID = 0.0 > al , Biochem. Biophys. Res. Comm., 282, 148-152 (2001)), there is a continuing need to develop a direct method for inducing differentiation of mesenchymal stem cells into neuronal cells.

As another example, studies have been conducted on techniques for inducing differentiation from mesenchymal stem cells into cartilage cells to produce cartilage as a tissue engineering or cell therapy agent. However, studies on the development of cartilage cells using mesenchymal stem cells Is not clearly presented.

In another example, bone marrow-derived adult stem cells were identified as osteogenic cells (Friedenstein AJ et al., Transplantation., 6: 230-247, 1968), and stem cells isolated from bone marrow were cultured to produce osteoblasts (Ohgushi H. et al., J. Biomed. Mater. Res., 48: 913-927, 1999). Therefore, a method of differentiating osteocytes from mesenchymal stem cells has been actively studied.

Accordingly, the inventors of the present invention have conducted studies to develop a technique for inducing large-scale differentiation of specific lineage cells from mesenchymal stem cells. As a result, 5-Azacitidine and / or Trichostatin A And that it functions to promote the differentiation from lobular stem cells into nerve cells, cartilage cells and bone cells. More specifically, 5-azacytidine or 5-azacytidine and tricostatin A are added to neuron, chondrocyte or osteoclast differentiation induction medium, which have been commonly used in the past, to produce mesenchymal stem cells It was found that the efficiency of differentiation from mesenchymal stem cells into neurons, chondrocytes and osteoblasts is greatly enhanced. Thus, the present invention has been completed.

An object of the present invention is to provide a technique for differentiating mesenchymal stem cells into neurons, chondrocytes or osteocytes using 5-Azacitidine and / or Trichostatin A .

More specifically, one object of the present invention is to provide a method for inducing the differentiation of mesenchymal stem cells into neurons, comprising (i) 5-azathiidine, or (ii) 5-azacytidine and tricostatin A To provide a composition.

It is still another object of the present invention to provide a method for inducing differentiation of mesenchymal stem cells into neurons using the composition.

It is still another object of the present invention to provide a composition for treating a neurological disease, which comprises the neural cell induced differentiation by the above method as an active ingredient.

Another object of the present invention is to provide a composition for inducing differentiation from mesenchymal stem cells into chondrocytes, comprising (i) 5-azathiidine or (ii) 5-azacytidine and tricostatin A .

It is another object of the present invention to provide a method for inducing differentiation of mesenchymal stem cells into chondrocytes using the composition.

Another object of the present invention is to provide a composition for treating cartilage diseases, which contains the differentiated chondrocyte as an active ingredient.

Another object of the present invention is to provide an artificial cartilage comprising differentiated chondrocyte cells by the above method.

Another object of the present invention is to provide a composition for inducing bone differentiation of mesenchymal stem cells, which comprises (i) 5-azathiidine, or (ii) 5-azacytidine and tricostatin A.

It is another object of the present invention to provide a method for inducing bone differentiation of mesenchymal stem cells using the composition.

It is still another object of the present invention to provide a composition for treating bone diseases, which comprises bone cells induced differentiation by the above method as an effective ingredient.

Another object of the present invention is to provide an artificial bone comprising bone cells induced differentiation by the above method.

The present invention provides a technique for differentiating mesenchymal stem cells into various lines of cells (neural cells, chondrocytes, and osteocytes) using 5-azacytidine and / or tricostatin A.

In the present invention, 5-azacytidine or 5-azacytidine and tricostatin A are added to the conventionally used neuron, chondrocyte or osteocytic differentiation induction medium to cultivate mesenchymal stem cells , It was confirmed that the efficiency of inducing differentiation from mesenchymal stem cells into neurons, chondrocytes and osteoblasts is greatly promoted.

In the present invention, "5-Azacitidine (5-AZA)" refers to 4-amino-1-β-D-ribofuranosyl-1,3,5-triazine- (1 H) -one), which is known to have DNA demethylation activity. The term " 4-amino-1 -? - D-ribofuranosyl-1,3,5-triazin-2 In addition, 5-azacytidine is also known as an anti-neoplastic drug that exhibits activity against leukemia, lymphoma and various solid tumors.

In the present invention, "trichostatin A (TSA)" refers to 7- (4- (dimethylamino) phenyl) -n-hydroxy-4,6-dimethyl- Amide (7- (dimethylamino) phenyl) -n-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide, which can be isolated from Streptomyces , It is a compound known to have action as a deacetylase inhibitor.

The 5-azacytidine and tracostatin A can be used in the form of salts derived from various pharmaceutically acceptable inorganic acids, organic acids, or bases. The compounds may be synthesized by a common chemical synthesis method known in the art or may be purchased commercially (for example, Sigma-Aldrich (St Louis, Mo, USA)).

The present invention is characterized in that 5- azacytidine and / or tracostatin A have a function of promoting the induction of differentiation from mesenchymal stem cells into neural cells, chondrocytes or bone cells.

In the present invention, for the purpose of inducing differentiation of mesenchymal stem cells into neurons, chondrocytes or osteocytes, the 5-azacytidine is added at a concentration of 2 μM, more preferably at a concentration of 500 nM to 10 μM Is preferably used.

In addition, for the purpose of inducing differentiation of mesenchymal stem cells into neurons, chondrocytes or osteocytes, the above-mentioned tracostatin A may be used together with 5-azacytidine, wherein the tracostatin A is 100-300 nM, more preferably at a concentration of 100 nM. When the concentration of 5-azacytidine and / or tracostatin A is too low, it is difficult to see the effect. When the concentration is too high, it becomes toxic. Therefore, it is preferable to confirm an appropriate concentration depending on the type of cell.

In the present invention, "mesenchymal stem cells" may be mesenchymal stem cells derived from various adult tissues such as bone marrow-derived, embryo-derived, cord blood-derived, other placenta, alveolar bone, muscle, May be derived from bone marrow.

In one embodiment, it is easy to separate mesenchymal stem cells from bone marrow using a concentration gradient difference using ficoll. The isolated bone marrow-derived mesenchymal stem cells are preferably cultured 2 to 5 times, preferably 3 to 4 times, most preferably 3 times.

Hereinafter, techniques for inducing the differentiation of mesenchymal stem cells into neurons, chondrocytes, and osteocytes will be described in more detail, respectively.

In one embodiment, the present invention provides a technique for inducing differentiation of mesenchymal stem cells into neuronal cells.

In a specific aspect, the present invention relates to a composition for inducing differentiation from mesenchymal stem cells into neurons, comprising (i) 5-azathiidine or (ii) 5-azacytidine and tricostatin A .

In another specific embodiment, the present invention relates to a method for inducing differentiation of mesenchymal stem cells into neurons using the composition.

In a preferred embodiment, the present invention relates to a method for culturing mesenchymal stem cells in the presence of (i) 5-Azacitidine, or (ii) 5-azathiidine and Trichostatin A To a method for inducing differentiation of mesenchymal stem cells into neural cells.

In the present invention, in order to induce differentiation of mesenchymal stem cells into neural cells, it is preferable that the 5-azacytidine is used at a concentration of 2 μM, more preferably 500 nM to 10 μM. Tracostatin A may also be used in combination with 5-azacytidine, wherein Tracostatin A is preferably used at a concentration of 100 to 300 nM, more preferably 100 nM. If the concentration of the above compounds is too low, sufficient induction of differentiation is hardly observed. If the concentration is too high, it may cause cytotoxicity. Therefore, it is preferable to select an appropriate concentration range.

In addition, in order to induce the differentiation of mesenchymal stem cells into neurons, in the case of using 5-azathiidine or 5-azacytidine and tricostatin A, mesenchymal stem cells are cultured in a normal neuronal differentiation medium These compounds may be used in the pre-treatment step, or may be used in combination with the medium components commonly used in neuronal differentiation in the art.

The nerve cell differentiation induction medium commonly used in the art includes, but is not limited to, butylated hydroxyanisole, dimethylsulfoxide, beta-mercaptoethanol, (BFGF), insulin, transferrin, progesterone, putrescine, sodium selenite, all-trans-retinoic acid, BDNF (Brain -derived Neurotrophic Factor).

More preferably, it is preferable to use a solution containing 50 to 150 μM butylhydroxyanisole, 1 to 3% dimethyl sulfoxide, 10 to 40 mM potassium chloride, 1 to 10 U / ml heparin, 10 to 30 ng / ml bFGF, ml insulin, 50-150 μg / ml transferrin, 10-40 nM progesterone, 50-150 μM putrescine, 10-50 nM selenite, 0.1-1 μM tretinoin, and 300-800 ng / ml BDNF .

In addition, the method of differentiating mesenchymal stem cells into neurons according to the present invention is characterized in that a basic-fibroblast growth factor (bFGF) is added to enhance the cell activity of mesenchymal stem cells and induce differentiation into neurons It is preferable to undergo a pretreatment process for about 12 to 24 hours. It is further preferred to induce neural differentiation in the medium containing 5-azathiidine, 5-azathiidine and tricostatin A, and other neuronal differentiation inducing substances for 5 hours or more after seeding the cells.

These differentiated neurons can be composed of various neurons such as neurons, glial cells, astrocyte, and oligodendrocytes.

According to the present invention, neurons differentiated from mesenchymal stem cells can be used as a cell therapy agent for the treatment of neurological diseases. Herein, neurological diseases that can be treated using neurons include Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotriphic lateral sclerosis and ischemic brain disease ; stroke), but the present invention is not limited thereto, and it can be used for treatment of various disorders caused by abnormal annihilation of nerve cells as well as movement disorders due to spinal cord injury. The composition of the present invention can treat the neurological diseases by inducing differentiation of neurons.

In another aspect, the present invention provides a technique for inducing differentiation of mesenchymal stem cells into cartilage cells.

In a specific aspect, the present invention relates to a composition for inducing differentiation from mesenchymal stem cells into chondrocytes, comprising (i) 5-azathiidine or (ii) 5-azacytidine and tricostatin A .

In another specific embodiment, the present invention relates to a method for inducing differentiation of mesenchymal stem cells into chondrocytes using the composition.

In a preferred embodiment, the present invention relates to a method for culturing mesenchymal stem cells in the presence of (i) 5-Azacitidine, or (ii) 5-azathiidine and Trichostatin A To a method for inducing differentiation of mesenchymal stem cells into cartilage cells.

In the present invention, in order to induce differentiation of mesenchymal stem cells into chondrocytes, it is preferable that the 5-azacytidine is used at a concentration of 2 μM, more preferably 500 nM to 10 μM. Tracostatin A may also be used in combination with 5-azacytidine, wherein Tracostatin A is preferably used at a concentration of 100 to 300 nM, more preferably 100 nM. If the concentration of the above compounds is too low, sufficient induction of differentiation is hardly observed. If the concentration is too high, it may cause cytotoxicity. Therefore, it is preferable to select an appropriate concentration range.

In addition, in order to induce differentiation of mesenchymal stem cells into chondrocytes, in the case of using 5-azacytidine or 5-azacytidine and trichostatin A, the mesenchymal stem cells are cultured in a normal chondrocyte differentiation medium These compounds may be used in the pre-treatment step, or may be used in combination with media components commonly used in chondrocyte differentiation in the art.

Examples of the chondrocyte differentiation induction medium commonly used in the art include, but are not limited to, insulin, transferrin, selenium, dexamethasone, L-ascorbate-2-phosphate, L-proline and sodium pyruvate can do. The insulin, the transferrin, and the selenium salt may be commercially available ITS (Gibco, USA).

More preferably, the insulin-transferrin-selenium is selected from the group consisting of 0.5 to 3% insulin-transferrin-selenium, 10 ^-6 to 10 ^-8 M dexamethasone, 10 to 100 μM L-ascorbate-2- phosphate, 10 to 100 μM L- DMEM / F-12 culture medium containing sodium pyruvate can be exemplified.

In addition, in the present invention, as a method of differentiating mesenchymal stem cells into chondrocytes, a three-dimensional culture method known in the art can be applied. Three-dimensional culture refers to a method of culturing a cell such that a cell monolayer is formed by cell growth, rather than a conventional monolayer culture, so that the cells are grown to form a three-dimensional solid form (for example, spherical or ellipsoidal form) , Preferably pelleted cultures can be applied. Pellet cultures are effective in maintaining the phenotype of chondrocytes, and they can easily aggregate cells through centrifugation to induce a junction effect between cells and cells to provide an extracellular environment similar to early cartilage tissue formation.

Such differentiated chondrocytes can be used as a cell therapy agent for treating various cartilage diseases. Herein, cartilage disease means a cartilage-related disease requiring cartilage differentiation and regeneration, and includes degenerative arthritis, rheumatoid arthritis or fracture.

In addition, the therapeutic composition containing the chondrocyte differentiated by the method of the present invention as an active ingredient may be directly injected into the joint of a patient according to a known method, or may be implanted together with a scaffold after three-dimensional culture (Kim, J., 30: 41, 2004), the severity of the disease or condition to be treated, the severity of the disease to be treated, It is preferable to control the number of cells to be administered considering various related factors such as route, patient's weight, age and sex.

In addition, a certain type of artificial cartilage can be obtained by using the conventional method of continuously culturing the cartilage cells differentiated by the method of the present invention in a certain type of scaffold, and using the artificial cartilage, The artificial cartilage can be manufactured.

In another aspect, the present invention provides a technique for inducing bone differentiation of mesenchymal stem cells.

In a specific embodiment, the present invention provides a method for the treatment of osteogenic differentiation of mesenchymal stem cells comprising (i) 5-Azacitidine, or (ii) 5-azathiidine and Trichostatin A To a composition for induction.

In another specific embodiment, the present invention relates to a method for inducing bone differentiation of mesenchymal stem cells using the composition.

In a preferred embodiment, the method comprises culturing mesenchymal stem cells in the presence of (i) 5-Azacitidine, or (ii) 5-azathiidine and Trichostatin A , And a method for inducing bone differentiation of mesenchymal stem cells.

In the present invention, the 5-azacytidine is preferably used at a concentration of 2 [mu] M, more preferably 500 nM to 10 [mu] M to induce bone differentiation of mesenchymal stem cells. Tracostatin A may also be used in combination with 5-azacytidine, wherein Tracostatin A is preferably used at a concentration of 100 to 300 nM, more preferably 100 nM. If the concentration is too low, it is difficult to induce a sufficient differentiation inducing effect. If the concentration is too high, it may cause cytotoxicity. Therefore, it is preferable to select an appropriate concentration range.

In addition, in order to induce bone differentiation of mesenchymal stem cells, 5-azacytidine, or 5-azacytidine and trichostatin A are used to induce bone formation in the bone marrow, The compounds may be used pretreated or may be used in conjunction with media components commonly used in the art for bone differentiation.

Examples of the bone differentiation inducing medium commonly used in the art include, but are not limited to, FBS or human serum, dexamethasone, L-ascorbate-2-phosphate, glycerophosphate and the like. More preferably, an α-MEM culture medium containing 7-12% FBS or human serum, 50-150 nM dexamethasone, 10-100 μM L-ascorbate-2-phosphate, and 5-20 mM glycerophosphate is added For example.

According to the present invention, osteogenic cells induced differentiation from mesenchymal stem cells can be used as a cell therapy agent for treating bone diseases. Here, the bone disease refers to a bone-related disease that requires bone differentiation and regeneration, and includes fracture, traumatic bone loss, bone defect, osteoporosis, and osteonecrosis.

The therapeutic composition comprising bone cells produced by the method of the present invention as an active ingredient may be directly injected into the affected part of the patient according to a known method, or may be implanted together with the scaffold after three-dimensional culture, It is preferable to control the number of cells to be administered in consideration of various factors such as severity of disease, route of administration, body weight of the patient, age and sex.

By using the conventional method of culturing the osteocytes differentiated by the manufacturing method according to the present invention in a certain type of scaffold, a certain type of artificial bone can be obtained, and the artificial bone or bone damage site The artificial bone used for forming the defective portion can be manufactured.

According to the present invention, it is possible to mass-induce mesenchymal stem cells into cells of a desired lineage such as nerve cells, cartilage cells and osteocytes in vitro, thereby solving the problem of conventional cell supply, As shown in FIG.

FIG. 1A shows the result of examining the immunophenotype of bone marrow-derived mesenchymal stem cells, and FIG. 1B shows a microscopic photograph of bone marrow-derived mesenchymal stem cells.
FIG. 2A shows the result of osteogenic differentiation of mesenchymal stem cells obtained by alizarin red staining. FIG. 2B shows the result of immunocytochemistry showing alkaline phosphatase. FIG. 2C shows the result of immunocytochemistry . Figure 2D shows the Western blot results confirming Osterix expression.
FIG. 3A shows the result of confirming the natural neuron marker NeuN in order to confirm the neural differentiation result of the mesenchymal stem cells, FIG. 3B shows the result of confirming astrocyte marker GFAP, FIG. 3C shows the result of oligodendrocyte ) Marker, which is a marker. Figure 3D shows Western blot results confirming the expression of MAP-2 and nestin.
Fig. 4A shows the result of saffranin-O staining to confirm the result of cartilage differentiation of mesenchymal stem cells, Fig. 4B shows the result of immunohistochemistry in which Type II collagen was confirmed, Fig. 4C shows a result of Western blot analysis Blot results.
FIG. 5A is a PCR result showing the expression of BDNF mRNA, a neuronal cell survival marker during induction of mesenchymal stem cell differentiation, FIG. 5B is a PCR result confirming the expression of SOX-9, a cartilage differentiation regulatory gene, and FIG. FIG. 5D is a PCR result confirming the expression of osteogenic differentiation marker Runx-2. FIG.
FIG. 6A shows the results of confirming HDAC activity of mesenchymal stem cells, and FIG. 6B shows results of confirming BDNF and Runx-2.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Sample Procurement

Bone marrow aspirate (BM Aspirate) was collected from five healthy donors who received approval and consent from the institution.

Example  2. Cell Isolation and Culture

Bone mononuclear cells (BM mononuclear cells) were separated from heparinized BM specimens using density gradient centrifugation. Cells were cultured in DMEM medium (Invitrogen, Grand Island, NY) supplemented with 10% FBS (Invitrogen) and 1% antibiotic, and the cultures were maintained at 37 ° C in a 5% CO 2 humidified atmosphere for 24 hours. Cells were washed with PBS to remove unattached materials. During the kidney period, the medium was changed twice a week. When 70-90% confluence was reached, the cells were isolated using 0.25 trypsin-1mM EDTA (Invitrogen), washed with PBS, counted, and replated.

Example  3. Flow cell  Bone marrow derived from analyzer Of mesenchymal stem cells  Immunophenotype test

Bone marrow-derived mesenchymal stem cells (BM MSC, 3rd pass) were incubated in ice for 30 min with primary stain, incubated at the appropriate concentration, and washed with PBS / 2% FBS. CD34 conjugated with Alopicoscien (APC; BD Pharmingen, San Diego, CA, USA), CD34 conjugated with PE (BD Pharmingen), fluorescent isothio cyanate (FITC; BD Pharmingen) Primary murine monoclonal antibodies were used for human HLADR conjugated with human CD45, human CD45 conjugated with PE (BD Pharmingen), human CD90 conjugated with PE (BD Pharmingen), and PE (BD Pharmingen) Respectively. Surface molecule staining was performed in a single step. Prior to analysis with Cytomics FC 500® (Beckman Coulter Inc., Fullerton, Calif.), Cell samples were washed twice with 2% FBS-containing HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Welgene Inc.) Respectively. Dead cells were stained with 1 μg / ml propidium iodide (PI: Sigma Chemical, St Louis, MO). The remaining cells were analyzed using CXP software (Beckman Coulter Inc.).

Example  4. 5- AZA  And TSA's Invitro  Treatment test

AZA and TSA were purchased from Sigma-Aldrich (St. Louis, MO, USA). In all in vitro experiments, AZA induced differentiation by treatment with 2 μM, while TSA treated with 100 nM final concentration induced differentiation, alone or together.

Example  5. Neurogenesis

The cultured mesenchymal stem cells were enzymatically separated and plated at 4,000 cells / cm ² in α-minimal essential medium (α-MEM, Invitrogen) medium containing 10% FBS. Twenty-four hours prior to induction, the medium was changed to whole-medium medium consisting of α-MEM, 10% FBS, and 10 ng / ml bFGF (Invitrogen). Cells were treated with 100 μM butylated hydroxyanisole, 2% dimethylsulfoxide, 25 mM KCl, 5 U / ml heparin, 20 ng / ml bFGF, 5 μg / ml insulin, 100 μg / ml transferrin , 20 nM progesterone, 100 μM putrescine, 30 nM sodium selenite, 0.5 μM all-trans-retinoic acid, and 500 ng / ml Brain-derived Neurotrophic Factor (Chemicon, Temecula , And CA) to induce neuronal differentiation. After induction, cells were maintained in NIM for 7 days.

Example  6. Osteogenesis  differentiation

For induction of osteogenic differentiation, mesenchymal stem cells were cultured in a specific induction medium containing 10% FBS, 100 nM dexamethasone, 50 μM L-ascorbate-2-phosphate, 10 mM glycerophosphate, and 1% antibiotic / 0.0 > a-MEM < / RTI > solution). Cells were incubated in a 12-well plate in the medium up to two weeks at a density of one per well 3 x 10 ⁵ cells. The medium was replaced every 3 days. On the 14th day, osteogenic differentiation analysis was performed. And cells were stained with Alizalin Red to indicate the calcium-deposited portion.

Example  7. Cartilage differentiation

For cartilage differentiation, 2.5 x 10 ⁵ mesenchymal stem cells were cultured in chondrogenic differentiation medium (CM; 1% insulin-transferrin-selenium, 10 ^-7 M L -dexamethasone, 50 μM L-ascorbate- In DMEM / F-12 containing L-proline and 1 mM sodium pyruvate). For pellet culture, 500 ul of the cell suspension was placed in a 15 ml polypropylene centrifuge tube and centrifuged for 5 minutes at 500 xg for bench-top centrifugation. Tubes were incubated in 95% air, 5% CO ₂ 37 ℃ under a humid atmosphere including 21 days. The cap of the tube was loosened to allow air to be exchanged, and the medium was changed every 3 days. After 21 days of culture, the pellet was removed and treated with Safranin-O to indicate glycosaminoglycan.

Example  8. Immunocytochemistry and immunohistochemistry

Immunohistochemistry and immunohistochemistry were performed to analyze neurogenesis and osteogenesis. Cell cultures were cultured in 12-well plates in neural or osteogenic medium. Plates were fixed with 4% formaldehyde for 10 minutes and placed in PBS containing 0.1% tryptone X-200 for 5 minutes to increase antibody permeability. And washed twice with PBS. Cells were cultured in 10% NGS (normal goat serum) and cultured in NeuN (Neuron Nuclei; Chemicon, Temecula, CA), glial fibrillary acidic protein (GFAP; Dako, Carpinteria, CA) and Tuj1 (Neuron-specific class III beta-tubulin ; Chemicon), osteocalcin (Chemicon) and osteopontin (Chemicon) and then incubated overnight with Alexa 488- or Alexa 594-labeled secondary antibody (Molecular Probes, Eugene, OR). The nuclei were stained with 4 ', 6-diamidino-2-phenylindole (DAPI, Molecular Probes). Fluorescence images were obtained using an LSM 510 meta-confocal microscope (Carl Zeiss, Jena, Germany).

To confirm cartilage differentiation, immunohistochemical analysis was performed using anti-collagen II (Chemicon) as the primary antibody. All cultures were performed in a humid chamber. Next, the divided surfaces were rinsed with PBS and incubated with the secondary antibody labeled with Alexa 488. After 3 washes in PBS, samples were incubated with DAPI for 5 minutes. Finally, the divided surfaces were confirmed using an LSM510 meta-confocal microscope.

Example  9. Western Blat  analysis

Western blot analysis was performed to see the expression of specific differentiation-related proteins (bone formation: osteocalcin and osteopontin; neural formation: MAP-2, nestin; cartilage formation: collagen type II (COL2A1, Chemicon)). Cells were rinsed with PBS and lysed in ice with RIPA buffer for 30 minutes. The same amount of protein extract was fractionated by 10% SDS-PAGE and transferred to BioTrace ^TM NT Nitrocellulose Transfer Membrane. After blocking with TBS-T (10 mM Tris; 150 mM Nacl, 0.05% Tween-20, Immunobioscience, USA) containing 5% nonfat milk powder (Becton, Franklin Lakes, NJ USA), the blot was incubated at room temperature for 2 hours Incubated with a specific primary antibody, washed, and incubated with secondary antibody for 1 hour at room temperature. Blots were developed using SuperSignal® West Pico Chemiluminescent Substrate (Pierce, Merdian Rd, Fockford, IL USA) according to the kit protocol and blots were assayed using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ).

Example  10. HDAC  activation Assay

Total protein lysates were quantitated using a standard Bradford protein assay (Bio-Rad) and deacetylase activity was measured using a colorimetric Histone Deacetylase Assay Kit (Millipore, Billerica, MA, USA) Respectively. The assay is to detect the p-nitroanilide based on the color change due to the p-nitroanilide released when the active factor solution is incubated with the deacetylated substrate. Colorimetric detection is measured at 405 nm using a spectrophotometer. Hela cell nuclear extract was used as a positive control, and the percent inhibition of the treated cells was compared with the untreated control group.

Example  11. Bisulfite - Pyrosequencing  analysis

The present inventors performed bisulfite-pyrosequencing analysis to quantitatively measure the degree of methylation in the CpG region of BDNF, Runx-2 and collagen type II in mesenchymal stem cells. Genomic DNA was extracted from mesenchymal stem cells using Qiagen QIAamp DNA Mini kit (Qiagen, Valencia, Calif.). DNA was analyzed using a Nanodrop 1000 spectrophotometer (Thermo Scientific; Wilmington, DE). For pyrosequencing, genomic DNA (500 ng) was treated with sodium bisulfate using EZ DNA Methylation Kit according to the manufacturer's instructions to convert unmethylated cytosine to uracil and methylated cytosine without modification. The bisulfate-converted DNA (50 ng) was PCR-amplified using PyroMark PCR Kit (Qiagen) with 25 μM reaction volume and forward and reverse primers, respectively. Each reaction contained 2.5 μM Coral Load Concentrate (Qiagen) to check for amplification products on agarose gels. One primer of each primer pair was conjugated with biotin at the 5 'end, and pyrosequencing was performed on a Pyromark Q96MD pyrosequencing instrument (Qiagen) to allow single strand retention using streptavidin beads. Sequencing analysis was validated using an internal control (non-CpG cytosine in the target methylated sequence region).

Example  12. Statistical Test

All quantitative data were expressed as group mean and standard deviation. Statistical analysis was performed using the Mann-Whitney U-test using SPSS software (SPSS, Chicago, IL, http://www.spss.com ) and the significance was set at p <0.05.

Experiment result

1. Bone marrow derived Of mesenchymal stem cells  Morphological and Immunophenotypic Characteristics

Mesenchymal stem cell cultures were successfully propagated in all healthy donors. Immunophenotyping analysis in the third passage confirmed that the cultures were composed of a homogeneous population of cells positive for CD73, CD90 surface antigen and negative for CD14, CD34, CD45 and HLADR (FIG. 1A). The MSC had a spindle-like morphology (Fig. 1B).

2. 5- AZA  And TSA  Goal by Multipotential  increase

The osteogenic potential of 5-AZA and TSA by mesenchymal stem cells was examined by treating 5-AZA and TSA alone or in combination in osteogenic medium. As a result of alizarin red staining, it was confirmed that when 5-AZA was treated alone and 5-AZA and TSA were treated together, calcium deposition of mesenchymal stem cells was increased compared to the control group. No significant changes were observed in the group treated with TSA alone. As a result of immunocytochemistry to confirm osteocalcin and osteopontin protein expression as the osteogenic differentiation markers, 5-aza alone treatment group and 5-aza and TSA combination treatment group as well as the above alizarin red staining result (Fig. 2B, C, D), Western blotting was performed to quantify the expression of these proteins in the bone differentiation process. As a result, the combination of 5-aza and TSA showed that osteocalcin and osteopontin (Fig. 2E). These results indicate that the combination of 5-AZA alone or 5-aza and TSA increases the osteogenic differentiation process of mesenchymal stem cells.

3. 5- AZA  And TSA  Nervous Multipotential  increase

The neural differentiation potential of mesenchymal stem cells by 5-AZA and TSA was examined by treating 5-AZA and TSA alone or in combination with neural induction medium. Immunoblotting and immunoblotting were performed to demonstrate the possibility of neural differentiation of 5-AZA and TSA by mesenchymal stem cells. MAP protein expression, which is a neurogenesis marker, Tuji1, a natural neuron marker NeuN, an astrocyte marker GFAP marker, and an oligodendrocyte marker, was confirmed using immunofluorescence. As a result, a significant increase in the expression of the neural differentiation markers was observed in the 5-AZA single treatment group and the 5-aza and TSA combination treatment groups as compared with the control group. In particular, GFAP and MAP-2 showed significant expression in the 5-aza and TSA combination treatment groups (Fig. 3A-D). Similar results were also confirmed in Western blot experiments. MAP-2 and nestin were found to have the highest protein accumulation in the 5-aza and TSA combination treatment groups, and increased in the 5-aza alone treatment group (Fig. 3E).

4. 5- AZA  And TSA  Cartilage by Multipotential  increase

The ability of 5-AZA and TSA to differentiate mesenchymal stem cells into cartilage was tested by treating 5-AZA and TSA alone or in combination with cartilage differentiation induction medium. As a result, it was confirmed that the ability of mesenchymal stem cells to differentiate into cartilage cells was increased in 5-AZA alone treatment group and 5-AZA and TSA combination treatment group (FIG. 4). In the results of saffranin-O staining, 5-AZA single treatment group and 5-AZA and TSA combination treatment group showed higher differentiation ability than untreated group (Fig. 4A). In addition, immunohistochemistry showed that the 5-AZA single treatment group and the 5-AZA and TSA combination treatment group promoted the synthesis of Col2A1 (FIG. 4B). Immunoblotting for the quantification of Col2A1 protein expression further supported the results of the saffranin-O staining and immunocytochemical staining (Fig. 4C, D)

5. 5- AZA  And TSA  Expression of Major Adjuvants by Differentiation

The major regulatory gene expression of mesenchymal stem cell differentiation was confirmed by real time PCR. First, the expression of BDNF mRNA, a neuronal survival marker, was increased three-fold in the 5-AZA single treatment group compared with the control group, and increased five-fold in the 5-AZA and TSA combination treatment group (FIG. 5A). Next, the expression of SOX-9, a regulatory gene for cartilage differentiation, was increased in both the single treatment group of 5-AZA and TSA and the combination treatment group. Compared with the untreated group, expression was increased 4-fold in the 5-AZA-treated group, 3.37-fold in the TSA-treated group, and 8.24-fold in the 5-AZA and TSA-treated group. Next, in the case of Osterix and Runx-2 expression in which bone formation can be confirmed, gene expression was increased in the 5-AZA single treatment group of both genes. Specifically, Osterix gene was 4.5 times and Runx-2 The gene was found to be 3-fold increased. In the 5-AZA and TSA combination treatment groups, osterix gene expression increased 7.8-fold and Runx-2 gene expression increased 4.23-fold.

6. 5- AZA  And TSA  On by HDAC  Reduction in activity

In order to confirm that HDAC activity is effectively inhibited in 5-AZA and TSA-treated mesenchymal stem cells, the present inventors confirmed the total HDAC activity in 5-AZA and TSA-treated mesenchymal stem cells. 5-AZA and TSA, alone or in combination, showed reduced levels of HDAC activity compared to the control group. This tendency was remarkable in the TSA alone treatment group (Fig. 6A). These results indicate that 5-AZA and TSA effectively inhibit HDAC activity in mesenchymal stem cells.

Claims (7)

(5-Azacitidine) and 100-300 nM of Trichostatin A (500 [mu] M to 10 [mu] M)
A composition for inducing differentiation from mesenchymal stem cells into chondrocytes.
The composition of claim 1, wherein said mesenchymal stem cells are derived from bone marrow.
delete delete The composition according to claim 1, further comprising at least one member selected from the group consisting of insulin, transferrin, selenium, dexamethasone, L-ascorbate-2-phosphate, L-proline and sodium pyruvate.
The method of claim 1, wherein, 0.5 ~ 3% (v / v) insulin-transferrin-selenium, 10 ^-6 ~ 10 ^-8 M dexamethasone, 10 ~ 100μM L- ascorbate-2-phosphate, 10 ~ 100 μM L- Proline and 0.5 to 3 mM sodium pyruvate.
Culturing the mesenchymal stem cells in vitro in the presence of 500 nM to 10 μM of 5-Azacitidine and 100 to 300 nM of Trichostatin A,
A method for inducing differentiation of mesenchymal stem cells into cartilage cells.

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