KR100683198B1 - A process for differentiating a neural progenitor cell to a dopaminergic neural cell and a culture medium therefor - Google Patents

Abstract

A process for differentiating a neural progenitor cell to a dopaminergic neural cell, and a culture medium therefor are provided to improve differentiation efficiency from a mesenchymal stem cell to the dopaminergic neural cell by using specific medium. The process for differentiating the neural progenitor cell to the dopaminergic neural cell comprises the steps of: (a) culturing the neural progenitor cell in N2 medium containing 20-100 ng/ml of fibroblast growth factor 8(FGF 8), 200-500 ng/ml of sonic hedgehog(SHH), 100-300 muM of ascorbic acid(AA) and 10-20 ng/ml of brain-derived neurotrophic factor(BDNF); and (b) culturing the cultured product in N2 medium containing 100-300 muM of ascorbic acid(AA), 10-20 ng/ml of brain-derived neurotrophic factor(BDNF), 2-20 ng/ml of glial cell-derived neurotrophic factor(GDNF) and 0.5-2 mM of dibutyryl cyclic adenosine monophosphate(dcAMP).

Description

Method for differentiating a neural progenitor cell to a dopaminergic neural cell and a culture medium therefor}

1 is a photograph of barton jelly-derived mesenchymal stem cells stained with hematoxylin and eosin (A and C are 40 magnification and B and D are 100 magnification).

Figure 2 is a photograph showing the morphology of the bartone jelly-derived mesenchymal stem cells and the results of immunophenotyping (immunophenotyping).

Figure 3 is a colony formation picture of bartone jelly-derived cord blood mesenchymal stem cells.

4 is a photograph observing the morphology of Barton jelly-derived mesenchymal stem cells during differentiation into neurons. A is the shape of cells before differentiation, B is the shape of cells when the cells are incubated for 24 hours in a culture medium added with 20 ng / ml bFGF and EGF, and C is a 1.5-day photo grown in neuronal differentiation medium.

5 is a photograph of immunocytochemical staining using antibodies specific to neurons, astroglia, and oligodendrocytes. (A) It has a positive response to β -tubulin III, a specific antibody of neurons, and has a neuron-like form in which axons and dendrites extend. (B) These cells showed positive plaques to GFAP, a specific antibody of astroglia. (C) It shows a positive response to Gal-C, an antibody specific for oligodendrocytes, and shows a characteristic form of oligodendrocytes.

Figure 6 is a graph showing the differentiation rate of neuronal cell differentiation induced in Barton jelly-derived mesenchymal stem cells. Barton jelly-derived mesenchymal stem cells were grown in neural differentiation medium for 1.5 days and then the differentiation rate of neurons, astroglia, and oligodendrocytes was measured. Diagonal bars: neuronal differentiation, coarse diagonal bars: astrocyte differentiation, dashed bars: oligodendrocyte differentiation.

7 is a diagram showing the expression patterns of various neuronal genes in the differentiation stage of mesenchymal stem cells.

FIG. 8 is a photograph showing immunocytochemical staining using specific antibodies of dopaminergic and cholinergic neurons in cells grown in neuronal differentiation medium. Green fluorescence represents β -tubulin III (A, D), a neuronal specific antibody, and red fluorescence represents TH (B), a specific antibody for dopaminergic neurons, and ChAT (E), a specific antibody of cholinergic neurons. Indicates. C and F are two overlapping pictures.

Figure 9 is a photograph of the immunocytochemical staining using a dopaminergic neuron-specific antibody after 20 days culture in dopaminergic nerve induction medium. Phase contrast micrographs show (A, E, I) as the axons and dendrites extend very thinly with the cell body rounded. Green fluorescence represents neuronal cell specific antibody β -tubulin III (B, F, J) and red fluorescence represents TH (C, G, F) specific antibody for dopaminergic neurons. D, H, L are two overlapping pictures.

10 is a graph showing the differentiation rate after inducing barton jelly-derived mesenchymal stem cells with dopaminergic neurons for 20 days. The dopaminergic neuron differentiation rate of cells (B) grown in dopaminergic neuron induction medium was 9 times higher than the dopaminergic neuron differentiation rate induced in (A) neuron differentiation induction medium. In contrast, the differentiation rate of cholinergic neurons decreased when cultured in neuronal differentiation induction medium.

FIG. 11 is an immunohistochemical staining photograph of human barton jelly-derived mesenchymal stem cells transplanted into brain sections of rats at 5 days of age and differentiated into dopaminergic neurons. (A) Barton jelly-derived mesenchymal stem cells of humans labeled with PKH26, (B) TH, a specific antibody to dopaminergic neurons, and (C).

The present invention relates to a method for differentiating neural progenitor cells into dopaminergic neural cells and a medium used therefor.

Dopaminergic neurons (DA) play an important role in the functioning of various brain tissues, including motor control and reward pathways. Dopaminergic nerves are present in various parts of the brain, and the most important pathways are those that extend from the substantia nigra and ventral tegmental areas to the forebrain. The development of dopaminergic nerves involves complex combinations of various transcription factors and signals to regulate the acquisition and maintenance of neurotransmitter-specific traits. Initially, sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF 8) are involved, and Lmx1b and nurr1 transcription factors also play an important role in differentiation of dopaminergic neurons. Pitx3 transcription factor and retinod synthase, Aldh1, are also important markers of developing dopaminergic neurons and their role has not been elucidated.

It is known from various studies that various cells differentiate into dopaminergic neurons under appropriate culture conditions. Human embryonic stem cells have been found to be capable of differentiation into dopaminergic neurons by sequentially processing stromal feeder cells and factors related to differentiation (Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J). , et al. Derivation of midbrain dopamine neurons from human embryonic stem cells.Proc Natl Acad Sci US A. 2004, 101: 12543-8). Several methods of differentiating from dorsal mesenchymal mesenchymal stem cells to dopaminergic neurons have been reported. Their culture medium contains 10% fetal calf serum (FCF), interleukin-1 (IL-1), IL-11, LIF based on FGF-2 and glial cell conditioned media. And glial cell-derived neurotrophic factor (GDNF) were added and cultured in a hypoxic environment (Studer L, Csete M, Lee SH, Kabbani N, Walikonis L, Wold B, Mckay R. Enhanced). proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen.J Neurosci. 2000 Oct 1; 20 (19): 7377-83). Using similar methods, human mesenchymal stem cells have been reported to differentiate into dopaminergic neurons (Storch A, PaulG, Csete M, Boehm BO, Carvey PM, Kupsch A, Schwarz J. Long-term proliferation and dopaminergic differentiation of human mesencephalic neural precursor cells.Exp Neurol. 2001 Aug; 170 (2): 317-25).

On the other hand, Fu et al. Have disclosed a method for obtaining mesenchymal stem cells from properly isolated Barton jelly, differentiated them in stepwise medium to differentiate into dopamine neurons (Stem Cells, 2005 Aug 11, Conversion of Human). Umbilical Cord Mesenchymal Stem Cells in Wharton's Jelly to Dopaminergic Neurons in Vitro Potential Therapeutic Application for Parkinsonism). Method for differentiating into dopaminergic neurons according to Fu et al. Is mesenchymal stem cells incubated for 3-6 days in 10% FBS-DMEM medium (step 1), 6-6 days in NCM medium (step 2), Culturing by adding SHH (500 ng / ml) and FGF8 (100 ng / ml) based on NCM medium or 10% -FBS-DMEM medium (step 3). Here, NCM medium (Neuronal Conditioned Medium) is extracted from the brain of Sprague-Dawley rats 7 days old, and added 15% FBS-DMEM to until 15 times the grinding (single cell) Then, after 5 days incubation in a 37 ℃ 5% CO ₂ incubator to recover the medium is prepared. However, the method of Fu et al. Is not only capable of infecting stem cells by pathogens of rats by using NCM medium using animal cells, but also having a low rate of dopaminergic neuronal differentiation at only 12.7%. There is this.

Therefore, there is a need in the art for a method of differentiating mesenchymal stem cells (MSCs), which are a kind of adult mesenchymal stem cells, into high dopaminergic neurons.

Therefore, the present inventors conducted a study to develop a method for differentiating mesenchymal stem cells into dopaminergic neurons, and neural precursor cells from mesenchymal stem cells derived from wharton's jelly. progenitor cells) were obtained, and when cultured in a specific medium, they found that they differentiate into dopaminergic neurons with high differentiation rate.

Accordingly, an object of the present invention is to provide a method for differentiating neuroprogenitor cells into dopaminergic neurons.

It is also an object of the present invention to provide a medium for inducing differentiation into dopaminergic neurons.

According to one aspect of the invention, (a) neural progenitor cells (fibroblast growth factor 8, FGF 8); Sonic hedgehog (SHH); Ascorbic acid (AA); Culturing in N2 medium comprising brain-derived neurotrophic factor (BDNF) (primary culture), and (b) culturing the culture obtained in step (a) AA; BDNF; Glia cell-derived neurotrophic factor (GDNF); And culturing neural progenitor cells into dopaminergic neural cells, comprising culturing in a N2 medium containing dibutyryl cyclic adenosine monophosphate (dcAMP) (secondary culture). Methods of making are provided.

In the method of differentiating neuronal progenitor cells into dopaminergic neurons, the primary culture N2 medium is 20 to 100 ng / ml of FGF 8; 200-500 ng / ml SHH; 100-300 uM AA; And 10-20 ng / ml BDNF, wherein the secondary delivery N2 medium comprises 100-300 uM AA; BDNF at 10-20 ng / ml; GDNF at 2-20 ng / ml; And 0.5 to 2 mM dcAMP. In addition, more preferably, the primary embryo culture N2 medium is 100 ng / ml of FGF 8; 200 ng / ml SHH; 200 uM AA; And 20 ng / ml BDNF, wherein the secondary culture N2 medium comprises 200 uM AA; 20 ng / ml BDNF; 20 ng / ml GDNF; And 1 mM of dcAMP.

In the present invention, N2 medium used as a basal medium to induce differentiation into dopaminergic neurons is DMEM / F-12 (Dulbecco's modified Eagle's medium // F-12), which supports growth of a wide range of animal cell lines. Has Therefore, although described herein as N2 medium, the use of a medium having the same characteristics as N2 medium as the base medium should be construed as being included within the scope of the present invention.

In the method for differentiating neural progenitor cells into dopaminergic neural cells, cells obtained by a known production method can be used as the neural progenitor cells. However, neural progenitor cells obtained from adult stem cells, i.e., mesenchymal stem cells, which do not cause bioethics problems may be preferably used, in particular from wharton's jelly that the mesenchymal stem cells can be properly separated. The obtained one can be preferably used. That is, as the neuronal precursor cells that can be used in the present invention, cells obtained from mesenchymal stem cells derived from wharton's jelly may be preferably used.

Barton's jelly is a protective tissue that surrounds the vessels of the umbilical cord, and consists of mucous connective tissue and is known to be rich in mesenchymal stem cells (Mitchell KE, Weiss ML, Mitchell BM, Martin P, Davis D, et al. Matrix cells from Wharton's jelly form neurons and glia.Stem Cells. 2003, 21: 50-60).

In order to obtain mesenchymal stem cells from the Barton jelly and prepare them as neural progenitor cells, methods disclosed in US Patent Publication Nos. 2004/0136967 and 2005/0148074 may be used, or Stem Cells, 2005 Aug 11 (Conversion of Human Umbilical Cord Mesenchymal Stem Cells in Wharton's Jelly to Dopaminergic Neurons in Vitro Potential Therapeutic Application for Parkinsonism.

The neural progenitor cells used in the present invention are preferably obtained from Barton jelly-derived mesenchymal stem cells. That is, the neural progenitor cells (a ') bartone jelly-derived mesenchymal stem cells β-mercaptoethanol; Non-essential amino acids (eg L-alanine; L-asparagine; L-aspartic acid; L-glutamic acid; glycine; L-proline; and L-serine); Glutamine; And cultured in a dedifferentiation medium containing fetal bovine serum (FBS), and (b ') the epidermal growth factor (EGF) of the culture obtained in step (a'); Basic fibroblast growth factor (bFGF); And N2 supplement (for example, insulin; human transferrin; progesterone; putrascine; and selenite).

In the above, the dedifferentiation medium is 0.001% β-mercaptoethanol; 1X nonnessential amino acid; 2 mM glutamine; And DMEM medium comprising 20% of FBS, and the N2 medium used in step (b ') comprises 20 ng / ml of EGF; 20 ng / ml bFGF; And 1 × of N2 supplement.

According to another aspect of the present invention, a medium for differentiating dopaminergic neural cells of neural progenitor cells is provided.

That is, the present invention is fibroblast growth factor 8 (FGF 8); Sonic hedgehog (SHH); Ascorbic acid (AA); And N2 medium including brain-derived neurotrophic factor (BDNF), and ascorbic acid (AA); Brain-derived neurotrophic factor (BDNF); Glia cell-derived neurotrophic factor (GDNF); And dopaminergic cells of neural progenitor cells selected from the group consisting of N2 medium (B) comprising dibutyryl cyclic adenosine monophosphate (dcAMP). neural cell) differentiation medium.

The N2 medium (A) was 20-100 ng / ml of FGF 8; 200-500 ng / ml SHH; 100-300 uM AA; And 10-20 ng / ml BDNF, preferably 100 ng / ml FGF 8; 200 ng / ml SHH; 200 uM AA; And 20 ng / ml of BDNF.

In addition, the N2 medium (B) is 100 to 300 uM AA; BDNF at 10-20 ng / ml; GDNF at 2-20 ng / ml; And 0.5 to 2 mM dcAMP, preferably 200 uM AA; 20 ng / ml BDNF; 20 ng / ml GDNF; And 1 mM of dcAMP.

In addition, as a medium for differentiating dopaminergic neurons of neural progenitor cells according to the present invention, the N2 medium (A) is 100 ng / ml FGF 8; 200 ng / ml SHH; 200 uM AA; And 20 ng / ml BDNF, wherein the N2 medium (B) is 200 uM AA; 20 ng / ml BDNF; 20 ng / ml GDNF; And 1 mM of dcAMP.

According to the present invention, neural progenitor cells, in particular neural progenitor cells obtained from mesenchymal stem cells derived from Barton jelly can be differentiated into dopaminergic neurons at high differentiation rate in vitro, and thus, differentiate into dopaminergic neurons. When transplanted, it can be used as a treatment for diseases caused by dopaminergic neuronal degeneration (eg, Parkinson's syndrome).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the invention and not to limit the invention.

Reference Example 1. RT-PCR

In the following example, RT-PCR was performed as follows. Total RNA was extracted from each experimental group using Tri-reagent (MRC), and 5 using oligo-dT primer (Promega) μg of total RNA was synthesized with cDNA. Reverse transcription was carried out using M-MLV reverse transcriptase (Promega) for 1 hour at 42 ℃, 5 minutes at 99 ℃. cDNA was amplified in 35 cycles using Tag polymerase (Taq polymerase, Promega). Amplified cDNA was isolated from 1% agarose gel and confirmed by ethidium bromide (EtBr) staining. The sequence and the size of the primer is shown in Table 1.

Reference Example 2 Immunocytochemistry

In the following examples, immunocytochemical staining was performed as follows. Cover slips of adult stem cells were cultured in a solution containing 10% normal goat serum (Sigma, USA) in PBS containing 0.2% Triton X-100. Room temperature treatment for 1 hour blocked the antibody to bind only to specific proteins. Β-tubulin III, an antibody against neurons (1: 400, Sigma, USA), glial fibrillary acidic protein (1: 500, Dako Cytomation, Denmark), and oligodendrocytes Gal-C (galactocerebroside, 1:50, Chemicon International, USA), the antibody against dopaminergic neurons TH (1: 250, Vector Lab.), ChAT (antibody against cholinergic neurons) 1: 100, Chemicon) was treated with a primary antibody and reacted at 4 ° C. for 24 hours. Each tissue was washed three times with phosphate buffered saline (PBS) for 15 minutes three times, followed by fluorescence-labeled secondary antibody (Cy3-conjugated goat anti-rabbit IgG) 1: 1000, Amersham), FITC-conjugated goat anti rabbit IgG (FITC-conjugated goat anti rabbit IgG) (1: 128, Sigma) was reacted at 4 ° C for 1 hour. The negative control group was subjected to the same procedure except for the primary antibody section of each group. Fluorescently labeled cells were observed under a fluorescence microscope (Carl Zeiss Axioskop2 +, Germany), and the degree of differentiation of dopaminergic neurons was determined by labeling β-tubulin III + cells and TH + cells and measuring only double fluorescently labeled cells to confirm the degree of differentiation. The degree of differentiation of cholinergic neurons was determined by labeling β-tubulin III + cells and ChAT + cells to measure the degree of differentiation by measuring only fluorescently labeled cells.

Example 1 Isolation and Culture of Human Barton Jelly-Derived Mesenchymal Stem Cells

Barton jelly was separated from the altar, and the altar was collected by Ms. Gynecology (Gimhae). The umbilical cord was put into a sterile PBS and transported to a laboratory within 2 hours after delivery and immediately used for the experiment. The collected umbilical cord was washed three times in sterile PBS and then the veins and arteries were removed, and the remaining connective tissue was treated with antibiotics (100 μg / ml of penicillin, 10 μg / ml of streptomycin, and amphotericin B 250 μg / ml, Sigma Transfer to Dulbecco's modified essential media (DMEM, Gibco Co., Ltd.). The small pieces were placed in DMEM containing 20% fetal bovine serum (fetal bovine serum, FBS, Gibco) and incubated for 7-10 days at 37 ℃, 5% CO ₂ incubator. The isolated Barton's jelly cells were cultured in DMEM (low gloucose) with 10% FBS at a concentration of 1 × 10 ⁵ cells / cm ² at 37 ° C. and 5% CO ₂ , and after 3 days of initial culture, Cells attached to the bottom were isolated and passaged at weekly intervals. The cell shapes isolated from the obtained Barton jelly contained mesenchymal cells and fusiform cells each having a spindle shape or a radiation shape (FIG. 1).

Example 2 Immunophenotyping

In order to confirm that the Barton jelly isolated in Example 1 is a mesenchymal stem cell, immunophenotyping was performed. The cultured cells were fixed at 4 ° C. in 4% paraformaldehyde for 2 hours. After washing with PBS, the reaction was performed in 3% hydrogen peroxide and then blocked with 2% bovine serum albumin (BSA). Prepared cells were reacted overnight at 4 ° C. by diluting one of the following antibodies to PBS solution containing 2% BSA in dilution factor: fibronectin (1: 100), ICAM-1 (1: 200), type I collagen (1:50), TRA-1-60 (1: 150), bimentin (1: 200). After completion of the reaction, the cells were reacted with biotinylated goat anti-mouse IgG (anti-rabbit IgG) antibodies. After the reaction, cells Stained with diaminobenzidine and embedded in Canada balsam. The negative control was reacted with PBS instead of the primary antibody and the same procedure was performed.

As a result of immunophenotyping, Barton's jelly cells showed positive responses to antigens specific for mesenchymal stem cells (FIG. 2). Barton jelly cells were observed to form colonies when cultured at low concentration (approximately 1 × 10 / ml) by adding epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). 3).

Example 3 Induction of Neuronal Differentiation of Barton Jelly-derived Mesenchymal Stem Cells

In order to confirm that Barton jelly-derived mesenchymal stem cells differentiate into neurons, Barton jelly cells were differentiated into neurons through three stages of media (de-differentiation medium, pluripotent medium, and neural differentiation medium). Barton jelly-derived mesenchymal stem cells isolated and cultured were trypsinized and collected in a culture dish coated with poly-D-ornithine / laminin at 1 × 10 ⁴ cells / cm. Cultured at a density of ² and cultured at 0.001% β-mercaptoethanol, 1 × nonnessential amino acid (composition (mg / L ): L-alanine 890.00; L-asparagine 1,320.00; L-aspartic acid 1,330.00; L -Glutamic acid 1,470.00; glycine 750.00; L-proline 1,150.00; and L-serine 1,050.00), 2 mM glutamine, 20% FBS added in DMEM medium (low glucose) for 1 to 3 days (dedifferentiation medium). De-differentiated stem cells were treated with 20 ng / ml EGF, 20 ng / ml bFGF, 1X N2 supplement (N2 supplement, Gibco Inc. (ug / ml): insulin 500; human transferrin 10,000; progesterone 0.63; putrascine 1,611 And incubated in N2 medium containing Selenite 0.52)) for 24 hours to differentiate into neural progenitor cells (starch), and then in N2 medium containing 1 × N2 adjuvant, 2% DMSO, 200 uM BHA. After time differentiation, 3-7 days in N2 medium (neural differentiation medium) with 1X N2 supplement, 2% DMSO, 200uM BHA, 25mM KCl, 2mM valproic acid, 1uM hydroxycortison Further incubation.

By treating as described above, human Barton jelly-derived mesenchymal stem cells caused a morphological change similar to that of neurons (FIG. 4). In order to confirm that the morphologically changed cells were neurons, immunocytochemical staining (see Example 2) was performed. Β-tubulin III, a neuron-specific antibody, GFAP, a specific antibody of astrocytes, and Gal-C, an antibody against oligodendrocytes, showed positive responses (FIG. 5). The differentiation rate of bartone jelly-derived mesenchymal stem cells in humans was about 40% for neurons, about 15% for astroglia and about 12% for oligodendrocytes (Fig. 6).

As a result of performing RT-PCR according to Reference Example 1 to confirm the neural differentiation of human Barton jelly-derived mesenchymal stem cells (FIG. 7), the stem cell markers SCF and Stat-3 were expressed only in dedifferentiation medium. Nestin used as a neuronal progenitor specific marker was strongly expressed in starch medium and decreased in differentiation medium. Expression of neuronal markers NeuroD1, GFAP and MBP were strongly expressed in differentiation medium. These results indicate that Barton jelly cells have the characteristics of stem cells after incubation in dedifferentiated medium, and have the characteristics of neural progenitor cells when cultured in predifferentiated medium, and then differentiated into neurons when cultured in neural differentiation medium.

Example 4 Induction of Dopaminergic Neuron Differentiation

Bartone jelly-derived mesenchymal stem cells were isolated and cultured in a poly-D-ornithine / laminin-coated culture dish at a density of ¹ × ^{10 4} cells / cm ² . Cultured and dedifferentiated for 1 to 3 days in DMEM medium (low glucose) added with 0.001% β-mercaptoethanol, 1 × nonnessential amino acid, 2 mM glutamine and 20% FBS (dedifferentiation medium) ). De-differentiated stem cells were treated with 20 ng / ml EGF, 20 ng / ml bFGF, 1X N2 supplement (N2 supplement, Gibco Inc. (ug / ml): insulin 500; human transferrin 10,000; progesterone 0.63; putrascine 1,611 And Selenite 0.52)) were added to N2 medium (starch) for 24 hours to differentiate into neural progenitor cells. 100 ng / ml fibroblast growth factor 8 (FGF 8, R & D), 200 ng / ml sonic hedgehog (SHH, R & D), 200 uM ascorbic acid (ascorbic acid, AA, Sigma G), incubated in N2 medium with 20 ng / ml brain-derived neurotrophic factor (BDNF, Gibco) for 7-9 days, 200uM AA, 20 ng / ml BDNF, 20 ng N2 medium containing / ml of glia cell-derived neurotrophic factor (GDNF, Gibco), 1 mM dibutyryl cyclic adenosine monophosphate (dcAMP, Sigma) Differentiation was induced into dopaminergic neurons in.

Human Barton jelly-derived mesenchymal stem cells were induced in neuronal differentiation medium and then immunohistochemical staining was performed using TH, an antibody specific for dopaminergic neurons, and ChAT, an antibody specific for cholinergic neurons. Reference Example 2). The cells positive for these antibodies were 6.3 ± 1% and 4.2 ± 0.8%, respectively (FIG. 8). After inducing differentiation of these cells in dopaminergic neuronal differentiation medium, 49 ± 4.9 of the cells showed positive response to β-tubulin antibody and 75.2 ± 5.4% of the positive cells responded positively to TH. (FIG. 9). It can be seen that the dopaminergic neuronal differentiation rate in the dopaminergic neuronal differentiation medium is significantly higher than in the normal neuronal differentiation medium (FIG. 10).

Example 5 Brain Tissue In Vitro

Five-day-old rats were sacrificed by cervical dislocation immediately and brain tissues were immediately extracted and cut into 400 μm thickness using a biomicrocutter (1500, The Vibratome Company). Each tissue was placed in a 0.4 μm Millicell culture insert (Millipore, Mass., USA) and placed in a 6-well culture dish containing 1 ml of culture medium. The cells were incubated for 3 days before transplanting cells at 37 ° C., 5% CO ₂ incubator. The culture medium consists of 100 ml basal Eagle medium (with Earle's salts), 50 ml Earle's balanced salt solution (Gibco), 50 ml heat-inactivated horse serum (Gibco), 1 ml 200 mM L-glutamine, and 2 ml 50% glucose (Sigma).

PKH26 was used to label mesenchymal stem cells for transplantation into rat brain sections. After thawing and washing the frozen cells to remove DMSO, the survival rate was tested with 0.4% trypan blue solution, and then diluted in 1 ml diluent solution (Sigma, USA) to 1 × 10 ⁷ cells / ml. Diluted. After binding to the cell membrane and mixing well with 2μl of red fluorescent 4μM PKH26 (Sigma, USA), incubated at room temperature for 5 minutes and washed three times with PBS. Labeled cells can be observed through a rhodamine filter.

Fluorescently labeled cells were prepared in 1 × 10 ⁴ cells / 5 μl in DMEM medium and then transplanted into brain sections. The transplanted brain sections were incubated for 10 days in a 37 ° C., 5% CO ₂ incubator. At this time, dopaminergic differentiation medium was used to induce the transplanted cells to differentiate into dopamine neurons. Ten days later, immunocytochemical staining (see Example 2) was performed with the antibody against dopaminergic neurons (TH). Fluorescently labeled cells were observed under a fluorescence microscope (Carl Zeiss Axioskop2 +, Germany), and the degree of differentiation into dopaminergic neurons was determined by measuring only PKH26 and TH double fluorescently labeled cells.

As a result of the treatment (Fig. 11), the transplanted mesenchymal stem cells were alive in the brain tissue, some of them can be confirmed through the immunocytochemical staining method to differentiate into neurons.

The method of differentiating neural progenitor cells into dopaminergic neural cells can differentiate neural progenitor cells into dopaminergic neurons with high differentiation rate.

Claims (15)

(a) neuronal progenitor cells (fibroblast growth factor 8, FGF 8); Sonic hedgehog (SHH); Ascorbic acid (AA); And culturing in N2 medium comprising brain-derived neurotrophic factor (BDNF) (primary culture), and (b) the culture obtained in step (a) was AA; BDNF; Glia cell-derived neurotrophic factor (GDNF); And culturing in a N2 medium comprising dibutyryl cyclic adenosine monophosphate (dcAMP) (secondary culture), Method of differentiating neural progenitor cells into dopaminergic neural cells. The method of claim 1, wherein (a) the neuronal progenitor cells at 20-100 ng / ml FGF 8; 200-500 ng / ml SHH; 100-300 uM AA; And culturing in N2 medium comprising 10-20 ng / ml BDNF (primary culture), and (b) 100 to 300 uM AA of the culture obtained in step (a); BDNF at 10-20 ng / ml; GDNF at 2-20 ng / ml; And culturing in N2 medium containing 0.5 to 2 mM of dcAMP (secondary culture), Method of differentiating neural progenitor cells into dopaminergic neurons. The method of claim 1, wherein the primary culture N2 medium is 100 ng / ml of FGF 8; 200 ng / ml SHH; 200 uM AA; And 20 ng / ml of BDNF. The method of differentiating neural progenitor cells into dopaminergic neurons. The method of claim 1, wherein the secondary culture N2 medium is 200 uM AA; 20 ng / ml BDNF; 20 ng / ml GDNF; And 1 mM of dcAMP. The method of differentiating neural progenitor cells into dopaminergic neurons. The method of claim 1, wherein the primary culture N2 medium is 100 ng / ml of FGF 8; 200 ng / ml SHH; 200 uM AA; And 20 ng / ml BDNF, wherein the secondary culture N2 medium contains 200 uM AA; 20 ng / ml BDNF; 20 ng / ml GDNF; And 1 mM of dcAMP. The method of differentiating neural progenitor cells into dopaminergic neurons. 6. The dopaminergic nerve according to any one of claims 1 to 5, wherein the neural progenitor cells are cells obtained from wharton's jelly-derived mesenchymal stem cells. How to differentiate into cells. The method of claim 6, wherein the neural progenitor cells (a ') Barton jelly-derived mesenchymal stem cells were β-mercaptoethanol; Nonnessential amino acid; Glutamine; And culturing in a dedifferentiated medium containing fetal bovine serum (FBS), and (b ') epidermal growth factor (EGF) of the culture obtained in step (a'); Basic fibroblast growth factor (bFGF); And neural progenitor cells, which are cells obtained by culturing in N2 medium containing an N2 supplement, into dopaminergic neurons. The method of claim 7, wherein the dedifferentiation medium is 0.001% β-mercaptoethanol; 1X nonnessential amino acid; 2 mM glutamine; And DMEM medium containing 20% of FBS. 8. The method of claim 7, wherein the N2 medium used in step (b ') comprises 20 ng / ml EGF; 20 ng / ml bFGF; And 1 × of N2 supplement (N2 supplement). The method of differentiating neural progenitor cells into dopaminergic neurons. Fibroblast growth factor 8 (FGF 8) at 20-100 ng / ml; 200-500 ng / ml sonic hedgehog (SHH); Ascorbic acid (AA) of 100-300 uM; And N2 medium (A) comprising 10-20 ng / ml of brain-derived neurotrophic factor (BDNF), and Ascorbic acid (AA) of 100-300 uM; Brain-derived neurotrophic factor (BDNF) of 10-20 ng / ml; 2-20 ng / ml glia cell-derived neurotrophic factor (GDNF); And N2 medium (B) comprising 0.5-2 mM dibutyryl cyclic adenosine monophosphate (dcAMP). A medium for differentiating dopaminergic neural cells (dopaminergic neural cells) of neural progenitor cells selected from the group consisting of. delete The method of claim 10, wherein the N2 medium (A) is 100 ng / ml FGF 8; 200 ng / ml SHH; 200 uM AA; And 20 ng / ml BDNF. delete The method of claim 10, wherein the N2 medium (B) is 200 uM AA; 20 ng / ml BDNF; 20 ng / ml GDNF; And 1 mM of dcAMP. The method of claim 10, wherein the N2 medium (A) is 100 ng / ml FGF 8; 200 ng / ml SHH; 200 uM AA; And 20 ng / ml BDNF, wherein the N2 medium (B) is 200 uM AA; 20 ng / ml BDNF; 20 ng / ml GDNF; And 1 mM of dcAMP.

Images