KR101548616B1 - Method of Inducing Mesenchymal Stem Cell to Differentiate into GABAergic Neuronal Cell and Compostion Comprising BDNF for Inducing Differentiation into GABAergic Neuronal Cell - Google Patents

Abstract

The present invention relates to a method of differentiating mesenchymal stem cells into GABAergic neurons and a composition for inducing GABAergic neural differentiation of mesenchymal stem cells comprising BDNF.
The method of differentiating the mesenchymal stem cells of the present invention into GABA neurons differentiates the mesenchymal stem cells into GABA neurons in a large amount by using BDNF, It can serve as an excellent source of therapy.
In addition, the composition for inducing differentiation of GABA neurons in mesenchymal stem cells comprising BDNF of the present invention massively differentiates mesenchymal stem cells into GABA neurons in vitro and in vivo, so that mesenchymal stem cell transplantation It can be expected to be used for treatment.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for differentiating mesenchymal stem cells into GA B A nerve cells and a method for inducing the differentiation of GA B A neurons including BDNF into a GABAergic Neuronal Cell and Differentiating Compounds BDNF for Inducing Differentiation into GABAergic Neuronal Cells }

The present invention relates to a method for differentiating mesenchymal stem cells into GABAergic neurons and a composition for inducing GABAergic neuron differentiation including BDNF.

Gamma aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and is widely distributed in the cranial nervous system, making up about 70% of all synapses (Schwartz RD, Biochem Pharmacol. , 37: 3369-75 (1988)). However, GABAergic cells in areas such as the hippocampus can be degenerated in cerebral nerve diseases such as stroke (Nishino and Borlongan, Prog Brain Res. , 127: 461-76 (2000)). Drugs such as GABA agonists, calcium antagonists, glutamate antagonists, and antioxidants have been or have been developed as neuroprotective therapies (Stutzmann et al, CNS Drug Rev. , 8: 1-30 (2002); Rocelle et al., J Neurochem , 77: 353-71 (2001); Blezer et al., Eur J Pharmacol , 444: 75-81 (2002)), GABA-mediated drugs are also considered to be particularly effective in treating cranial nerve system disorders. Cell therapy may also be a useful tool for comprehensive treatment of primary and secondary damage caused by various brain injuries. For example, as an attempt to transplant a dopaminergic neuron into a patient with Parkinson's disease (Grisolia, Brain Res Bull , 57: 823-6 (2002)). Transplantation of GABAergic neurons may be an ideal therapy if GABAergic neurons are damaged in patients with cerebral nervous system such as stroke. One method of generating GABAergic cells from immature neurons has been reported (US Patent No. 6,602,680). Rubenstein et al. Reported the production of GABAergic cells by increasing the activity of DLX genes (eg, DLX1, DLX2, DLX5) in immature neuron cells. In addition, a method of inducing the differentiation of GABAergic cells from mouse embryonic stem cells has been reported (Hancock et al., Biochem Biophys Res Commun., 271 (2): 418-21 (2000); Westmoreland et al., Biochem Biophys Res Commun. , 284 (3): 674-80 (2001)). This method did not produce GABA neurons with high efficiency enough to perform cell therapy. Therefore, it is required to develop a method for inducing the differentiation of functional GABA neurons in a larger amount for cell therapy for functional recovery in cerebral neurological diseases.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made intensive researches for the development of a method capable of effectively inducing and mass-producing GABAergic neurons in vitro or in vivo, which can be used for cell therapy of brain neurological diseases. As a result, The present inventors have succeeded in mass-differentiating stem cells into GABAergic neurons, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a method of differentiating mesenchymal stem cells into GABAergic neurons.

Another object of the present invention is to provide a composition for inducing the differentiation of mesenchymal stem cells containing BDNF as an active ingredient into GABAergic neurons.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a method for producing a mesenchymal stem cell comprising: (a) obtaining mesenchymal stem cells; And (b) culturing the mesenchymal stem cells in a medium containing BDNF (brain-derived neurotrophic factor) to differentiate into GABAergic neurons, the mesenchymal stem cells are transfected with GABA (gamma-aminobutyric acid) Lt; RTI ID = 0.0 > cells. ≪ / RTI >

The present inventors have made intensive researches for the development of a method capable of effectively inducing and mass-producing GABAergic neurons in vitro or in vivo, which can be used for cell therapy of brain neurological diseases. As a result, The present inventors have succeeded in mass-differentiating stem cells into GABAergic neurons, thereby completing the present invention.

Stem cells are divided into two types: pluripotent stem cells and multipotent stem cells, including embryonic stem cells (ES) and embryonic germ cells (EG). The mesenchymal stem cells of the present invention are a kind of pluripotent stem cells. The mesenchymal stem cells of the present invention may be derived from bone marrow, umbilical cord blood, or adipose tissue, but are not limited thereto. The mesenchymal stem cells of the present invention are capable of differentiating into various cell types such as osteoblasts, cartilage cells, adipocytes, muscle cells, fibroblasts and bone marrow matrices which form bone as adult stem cells. Self-renewal capability.

The mesenchymal stem cells of the present invention exhibit at least one immunological characteristic selected from the group consisting of cell surface antigens CD14 +, CD31 +, CD45 +, CD34-, CD133- and CD166-.

Preferably, the mesenchymal stem cells used in the present invention can be obtained from the bone marrow, umbilical cord blood, or adipose tissue of a mammal. More preferably in human, pig, cow, sheep, rabbit, mouse or rat, more preferably human, pig or mouse, most preferably human bone marrow, cord blood or fat tissue .

The mesenchymal stem cells used in the present invention can be obtained in the manner described below. For example, the fat tissue is cut and the cut fat tissue is directly cultured to outgrow the cells. Then, the grown cells are subcultured to obtain mesenchymal stem cells. The medium used in the culturing may be any medium conventionally used for culturing animal cells. For example, Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), MEM (Stanner, CP et al., Nat . New Biol . 230: 52 (1971)), Iscove's MEM (Iscove, N. et al, J. Exp Med 147:... 923 (1978).....), 199 medium (Morgan et al, Proc Soc Exp Bio Med ., 73: 1 (1950) ), CMRL 1066, RPMI 1640 (Moore et al, J. Amer Med Assoc 199:.... 519 (1967)....), F12 (Ham, Proc Natl Acad Sci USA 53: 288 (1965)), F10 (Ham, RG Exp . Cell Res . 29: 515 (1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1959)), a mixture of DMEM and F12 (Barnes, D. et al., Anal . Biochem . 102:. 255 (1980) ), Waymouth's MB752 / 1 (Waymouth, C. J. Natl Cancer Inst . 100: 115 (1959)) and the MCDB series (Ham, RG et al., In (1987)), McCoy's 5A (McCoy, TA, et al., Proc . Soc . Exp . Biol . Vitro 14: 11 (1978)) and the like can be used.

The cells are cultured in the subculture until they exhibit 70-80% confluency, then adherent cells are harvested and a portion of the harvested cells are used for the next subculture. Through the above process, mesenchymal stem cells having the ability to differentiate into mesodermal tissues such as osteoblasts, adipocytes and chondrocytes and stem cell characteristics are produced.

The present inventors confirmed the possibility of inducing differentiation of mesenchymal stem cells (bone marrow, umbilical cord / umbilical cord blood, adipocyte-derived) into GABA neurons in vitro and their differentiation efficiency in vitro. That is, differentiation induction and culturing techniques were established to exploit the potential for differentiation into GABAergic neurons and optimal culture conditions (cell growth factors, differentiation-related factors) necessary for differentiation and to use them as cell therapy agents. In addition to the phenotype of the cells, biochemical and molecular biologic characteristics were analyzed in terms of functionality, and evaluation criteria were established to determine whether they could be effectively applied as a cell therapy for neurological diseases.

In a preferred embodiment of the invention, mesenchymal stem cells induce differentiation in DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) medium. It is possible to use HG-DMEM (High glucose-Dulbecco's modified eagle media) medium. However, when DMEM / F12 is used as a differentiation medium, it can produce GABA neurons in a large amount due to more efficient differentiation.

In the step of culturing and differentiating the mesenchymal stem cells of the present invention, BDNF is used as a neurophysiologist. When cultured in the above-mentioned culture medium, it is possible to differentiate into GABA neurons, and it is possible to produce GABA neurons in a large amount due to effective differentiation.

BDNF (brain-derived neurotrophic factor) is a glycoprotein of the nerve growth factor family of proteins, and calculates a neurotrophic factor that is processed from a large precursor promotes the survival of neuronal cell populations (Jones KR et al., Proc . Natl. Acad . Sci USA, 87: 8060-8064 (1990)). All of these neurons are located in or directly in the central nervous system.

Although it is preferred to use native BDNF in the present invention, modified BDNF molecules, analogs and recombinant BDNF molecules that perform the same function as natural BDNF molecules can also be used.

In a preferred embodiment of the invention, the amount of human brain-derived neurotrophic factor (BDNF) is added at 20-100 ng / ml based on the culture medium and cultured.

In a preferred embodiment of the invention, BDNF as a neurotrophin can be used with EGF (epidermal growth factor), FGF-2 (fibroblast growth factor-2) or mixtures thereof as cell proliferation factors. When the cell growth factor is additionally added to the medium, differentiation of mesenchymal stem cells into GABAergic neurons can be induced more efficiently.

GABAergic neurons produced in large quantities by the above method can be used as an excellent source of cell therapy for brain neurological diseases such as stroke, traumatic brain injury, cerebral palsy, epilepsy and Huntington's disease can do.

In a preferred embodiment, the GABAergic neuron is implanted at a site of lesion at a concentration of 10 cells / μl to 1 × 10 ⁷ cells / μl, more preferably 1 × 10 ₃ The cell / ㎕ to 1 × 10 ⁶ implanted in the site of the lesion to the cell / ㎕1 concentration.

In the case of an experiment using a mouse animal model, a total of 1 x 10 ⁴ cells to 1 x 10 ⁶ cells derived from GABA neurons can be transplanted into the lesion-side striatum.

According to another aspect of the present invention, there is provided a composition for inducing the differentiation of mesenchymal stem cells, which comprise BDNF (brain-derived neurotrophic factor) as an active ingredient, into GABA (gamma-aminobutyric acid) neurons.

In one embodiment, the composition is used to differentiate mesenchymal stem cells into GABAergic neurons in vitro as described above. The present invention of the present invention can be used for cell therapy by implanting GABA neurons which are grown in vitro using a composition containing BDNF as an active ingredient, in a site where a lesion is present.

 In another embodiment, the composition is used to differentiate mesenchymal stem cells into GABAergic neurons in vivo. More specifically, after the mesenchymal stem cells are transplanted into the lesion site, the composition of the present invention may be continuously injected into the transplantation site to effectively generate GABAergic neurons in vivo. This is because the method of transplanting mesenchymal stem cells into GABA neurons in a lesion site requiring GABA neurons prior to differentiation is possible, and thus neural regeneration and function recovery can be expected through mesenchymal stem cell transplantation therapy for refractory neurological diseases have.

In a preferred embodiment, the mesenchymal stem cells before the differentiation are implanted at a site of lesion at a concentration of 10 cells / μl to 1 × 10 ⁶ cells / μl, more preferably 1 × 10 ₃ Cells / ㎕ to 1 × 10 ⁵ cells are implanted in / ㎕1 concentration.

In order to differentiate the transplanted mesenchymal stem cells into GABAergic neurons, the composition for inducing differentiation into GABAergic neurons of the present invention should be continuously administered into the transplantation site to create a floating environment for cell proliferation and differentiation.

In another preferred embodiment, the composition of the present invention is continuously injected into the mesenchymal stem cell transplantation site for 2 to 30 days.

 The injection method is not limited. However, from the technical point of view, it is preferable to continuously administer the composition of the present invention into the ventricle using various osmotic pumps depending on the administration dose, administration rate, and administration period. Currently commercially available osmotic pumps include Alzet osmotic pumps and Medtronic's pump systems. The administration of the composition of the present invention using the osmotic pump may be performed at a rate of 0.01 to 10 μl / hr, 30 days, and the discharge volume is 1 쨉 l to 1 ml. It is preferable that the osmotic pump is pre-positioned at 37 degrees from the day before the operation so that the composition for inducing differentiation of GABA neurons into the GABA neurons of the present invention immediately after mesenchymal stem cell transplantation.

In a preferred embodiment, the composition of the present invention additionally comprises EGF (epidermal growth factor), FGF-2 (fibroblast growth factor-2) or a mixture thereof as a cell growth factor and the amount of BDNF is 1- 1000 mu g / ml.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a method for differentiating mesenchymal stem cells into GABAergic neurons and a composition for inducing GABAergic neuronal differentiation of mesenchymal stem cells comprising BDNF.

(Ii) The method of differentiating the mesenchymal stem cells of the present invention into GABAergic neurons involves massively differentiating mesenchymal stem cells into GABA neurons using BDNF, Can serve as an excellent source of cell therapy therapy.

(iii) The composition for inducing GABAergic neuronal differentiation of the mesenchymal stem cells comprising BDNF of the present invention massively differentiates mesenchymal stem cells into GABA neurons in vitro and in vivo, It can be expected to be used for cell transplantation therapy.

Fig. 1 shows the appearance of each mesenchymal stem cell derived from bone marrow (BM), umbilical cord blood (UCB), umbilical cord (UC) and adipose tissue (AT).
Fig. 2 shows the cell phenotype of each mesenchymal stem cell derived from bone marrow, umbilical cord blood, umbilical cord, and adipose tissue.
FIG. 3 shows the results of experiments to confirm the ability of human mesenchymal stem cells to differentiate into bone, fat, and cartilage.
FIG. 4 shows the expression of GABAgic neuron gene (GAD67, GAT-1) and neural progenitor cell gene (Nestin) after treatment of human adipose stem cells with various media.
FIG. 5 is a photograph showing the expression of GABA neuron labeled protein (GABA) and mature neuron labeled protein (βⅢ tubulin) by immunofluorescence after treating various adipocytes in human adipose stem cells (red: GABA, green : βⅢ tubulin, blue: nucleus of the cell).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

Example

Example 1: Isolation and culture of human mesenchymal stem cells

The present inventors have already succeeded in isolating and culturing mesenchymal stem cells derived from bone marrow, umbilical cord / umbilical cord, and adipose tissue. Adel Alhadlaq et al., Stem Cells and Development, 13 (4): 436-448 (2003); J. A. Romanov et al., Bulletin of Experimental Biology and Medicine, 140 (1): 138-143 (1994); DT Covas et al., Braz J Med Biol Res, 36 (9): 1179-1183 (2005)) to separate and culture human mesenchymal stem cells from bone marrow, umbilical cord / umbilical cord, and adipose tissue, respectively, and its appearance is shown in Fig.

The obtained multipotential stem cells showed cell surface antigens CD14 +, CD31 +, CD45 +, CD34-, CD133-, CD166-, and were able to differentiate into mesodermal tissues such as osteoblasts, adipocytes and chondrocytes. The cell phenotype of each mesenchymal stem cell derived from bone marrow, umbilical cord blood, umbilical cord, adipose tissue is shown in Fig. 2, and the experimental results of verifying the ability of human mesenchymal stem cells to differentiate into bone, Respectively.

Example 2 Induction of In Vitro GABA Neuronal Differentiation

2-1. Induction of GABAergic neuron differentiation using HG-DMEM medium and BDNF

The human adipose-derived stem cells cultured in Example 1 were inoculated into a cell culture container at a concentration of 5000 cells / cm ² . After attaching the cells, they were cultured in HG-DMEM medium (Gibco) containing 20% FBS (Fetal Bovine Serum) for 24 hours at 37 ° C in a 5% CO 2 atmosphere. After incubation, the cells were washed twice with PBS (phosphate buffered saline) and the differentiation medium was added. The differentiation medium was prepared by adding 200 μM BHA (Butylated Hydroxyanisole) (Finetech), 5 mM potassium chloride (KCl), 2 mM valproic acid (Alfa Chem), 10 μM forskolin to HG- 50 ng / ml of brain-derived neurotrophic factor (BDNF) (Sigma-aldrich) was added to 1 μM hydrocortisone (Spectrum) and 5 μg / ml insulin (Alfa Chem). The cells were incubated for 24 hours at 37 ° C in a 5% CO2 atmosphere.

2-2. HG - DMEM  On the badge BDNF  without GABA Induce sexual neuron differentiation

GABAergic neuron differentiation was induced in the same manner as in 2-1 except that BDNF was not used in the differentiation medium.

2-3. DMEM / F12 medium and BDNF use with GABA Induction of sexual neuron differentiation

GABAergic neuron differentiation was induced in the same manner as in 2-1 except that DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) medium (Gibco) was used as the differentiation medium instead of HG-DMEM.

2-4. DMEM / F12 medium BDNF  without GABA Induce sexual neuron differentiation

GABAergic neuron differentiation was induced by the same method as above 2-3 except that BDNF was not used in the differentiation medium.

Example  3. RT - PCR through GABA Identification of sexual neural differentiation markers

For the RT-PCR (Reverse Transcription-Polymerase Chain Reaction) analysis, 1 ml of Invitrogen was treated and dissolved in each of the cells induced to differentiate into neurons according to 2-1 to 2-4 for 5 minutes, 200 [mu] l of chloroform per 1 ml of the sol was treated and centrifuged at a speed of 12,000 rpm for 15 minutes. From this, the supernatant containing RNA was separated and transferred to a new tube, and 500 μl of isopropanol per 1 ml of trizol was added to induce RNA precipitation. The RNA precipitates were washed with 75% cold ethanol and dried at room temperature. RNA was dissolved in tertiary distilled water containing DEPC (diethyl pyrocarbonate) and used as a template for the reverse transcription reaction. The RNA thus prepared was placed at 65 DEG C for 5 minutes to remove the secondary structure. The reverse transcription reaction solution contained 5 μl of 6 × reverse transcriptase buffer (LAROVA), 2 μl of 2.5 mM dNTP (LAROVA), 1 μl of oligodeptide (500 ng / μl, LAROVA), 0.5 μl of RNase inhibitor (LAROVA) 2 μg of template RNA and 0.1 μl of reverse transcriptase (350 U / μl, TaKaRa) and titrated with DEP-treated sterilized tertiary distilled water to a total reaction volume of 30 μl.

The reverse transcription reaction was carried out at 44 ° C for 50 minutes, and the cDNA was synthesized by inactivating the reverse transcriptase at 94 ° C for 5 minutes to prepare a template cDNA to be used for the subsequent PCR. The primers used for PCR were primer pair (forward direction: 5'-CGAGGACTCTGGACAGTAGAGG-3 ', reverse direction: 5'-GATCTTGAGCCCCAGAGTTTTCTG-3') for the GAD67 gene specific for the Caucasian neuron, ; 5'-gccctgaccactccagttta -3 'for the nestin gene, which is a factor expressing early in neural differentiation (reverse direction, 5'-ACGTGTCTTCGCTGGGCTG-3', reverse direction 5'-TGGCTGATGGCTCTGTCTTC-3 ' ; 5'-ggagtcctggatttccttcc -3 ') and a primer pair (5'-ATCTCCATCTTCCAGGAGCG-3', reverse direction; 5'-CCTGCTTCACCACCTTCTTG-3 ') for correcting the concentration through GAPDH was used.

To the reaction tube, 0.5 μl of each primer (10 pmole), 2 μl of 2.5 mM dNTP mixture, 2.5 μl of 10 × Taq DNA polymerase buffer solution (containing magnesium chloride), 2 μl of template cDNA and 0.1 μl of Taq DNA polymerase (KOMA biotech) Was added to each well, and 25 μl of the total reaction volume was titrated with sterilized distilled water. PCR was carried out under the reaction conditions for each pair of primers. The PCR for the amplification of DNA is first denatured at 94 ° C for 5 minutes, denatured at 35 ° C for 30 seconds at 94 ° C, and then annealed at 48 ° C to 62 ° C for 30 seconds and at 72 ° C for 1 minute. After the last cycle, the cells were amplified at 72 ° C for 7 minutes. At this time, the annealing temperatures of the primers for the respective genes are GAD67 58 deg. C, GAT-1 67 deg. C and Nestin 57 deg. The amplified DNA product was electrophoresed on 2% agarose gel, stained with ethidium bromide, and band was confirmed using Gel Doc XR (BIO-RAD). The results are shown in FIG. 4 (line 1: Line 2: The above 2-1 cells were used as a sample. Line 3: The above-mentioned 2-4 cells were used as a sample. Line 4: The above 2-3 cells were used as a sample , Control (control): undifferentiated stem cells are used as a sample).

As a result, the expression of GAD67 and GAT-1, which are GABAgic neuron markers, was highest in the group treated with BDNF in the DMEM / F12 medium (2-3 above), and the expression of the neural progenitor cell nestin was decreased Respectively. In other words, when BDNF was treated in the DMEM / F12 medium to induce differentiation, it was confirmed that a large amount of GABAergic neurons could be produced. In the undifferentiated adipose tissue stem cells, the control group did not express the marker.

Example  4. In cell-mediated immunocytochemistry  by GABA Identification of sexual neural differentiation markers

In order to perform immunofluorescence staining, first, isolated adipose-derived stem cells were attached to a chamber slide, and then differentiated into GABA neuronal differentiation according to Example 1. After induction of differentiation, the chamber slides were first rinsed with PBS, and the cell samples of 2-1 to 2-4 were fixed at room temperature with 10% formalin solution for 15 minutes, respectively. Then, it was permeabilized with PBS solution containing 0.2% Triton X-100 for 5 minutes at room temperature and blocked with PBS containing 2% FBS at room temperature for 30 minutes to prevent nonspecific binding. The GABA / β III tubulin primary antibody specific for GABA neurons was diluted 1: 100 in PBS and reacted at 4 ° C. for 18 hours: polyclonal GABA antibody (Sigma), monoclonal βIII tubulin antibody (chemicon ).

After the primary antibody reaction, the cells were washed three times with PBS, and then reacted with FITC (Fluorescein isothiocyanate) and rhodamine-labeled secondary antibody for 30 minutes at room temperature. After washing twice with PBS, DAPI (4 ', 6-diamidino-2-phenylindole) was diluted 1: 1000 in PBS for 10 min at room temperature and washed twice with PBS The immunofluorescent cells were evaluated by observation with a fluorescence microscope and the results are shown in Fig.

As a result, in the group treated with BDNF in DMEM / F12 (above 2-3), the expression of mature neuron-labeled protein βIII-tubulin and GABA-labeled neural cell protein GABA was the highest. In other words, when BDNF was treated in DMEM / F12 medium to induce differentiation, it was confirmed that GABAergic neurons could be mass produced. But not in the control group, which was an undifferentiated adipose tissue stem cell.

Example  5. In vivo GABA Induction of sexual neuron differentiation

1 × 10 ⁴ cells to 1 × 10 ⁶ cells of mesenchymal stem cells were transplanted into the lesion-side striatum, and the neurophysiologist and cell proliferative factor were transferred to an Alzet osmotic pump (model 2002; discharge rate 0.5 μl / hr, discharge period 14 days, (Durect, Cupertino, Calif.) For 2 weeks. The Cannula (Brain Infusion Kit) was inserted into the right brain chamber using stereotaxic coordinates, and the osmotic pump was inserted into the subcutaneous tissue behind the back. The osmotic pump was pre-positioned at 37 degrees Celsius from the day before the operation, so that the proliferative or nutritional factors could be released immediately.

That is, by administering epidermal growth factor (EGF) or fibroblast growth factor-2 (FGF-2) as a cell proliferation factor, the proliferation of the transplanted cells is maintained and a neurotrophic brain- Induced neurotrophic factor BDNF (1 ㎍ / ml - 1,000 ㎍ / ml).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

Methods for differentiating mesenchymal stem cells into GABA (gamma-aminobutyric acid) neurons comprising the following steps:
The isolated mesenchymal stem cells are cultured in DMEM / F12 medium containing BDNF (brain-derived neurotrophic factor) to differentiate into GABA neurons.
delete The method according to claim 1, wherein the mesenchymal stem cells are obtained from bone marrow, umbilical cord blood, or adipose tissue.
The method according to claim 1, wherein the amount of BDNF is 20-100 ng / ml based on the medium.
delete delete delete delete delete A composition for inducing the differentiation of mesenchymal stem cells into GABA (gamma-aminobutyric acid) neurons in vitro, comprising BDNF (brain-derived neurotrophic factor) as an active ingredient.

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