KR20180062631A - Composition for cell regeneration comprising cells that hypersecreate growth factors, and at least one of a neural stem cells, neurons and GABAergic neurons - Google Patents

Abstract

The present invention relates to a cell regeneration composition excellent in biocompatibility. The cell regeneration composition is capable of promoting growth of in-vivo or in-vitro cells by containing a cell hypersecreating growth factors and at least one of a neural stem cell, a nerve cell, and a GABAergic nerve cell. In addition, the cell regeneration composition inhibits cell death and promotes growth of damaged nerve cells and nerve fibers, can be used as a therapeutic agent for neurological diseases even if cells in an intermediate stage of conversion is contained, and is able to prevent, ameliorate, or treat various neurological diseases and ischemic diseases. In addition, the cell regeneration composition barely causes side effects which may occur when embryonic stem cells or induced pluripotent stem cells are applied in vivo, and can be applied as a cosmetic composition.

Description

TECHNICAL FIELD The present invention relates to a composition for regenerating cells comprising at least one of cells of neural stem cells, neurons, and neural cells and hypersecretory growth factors , neurons and GABAergic neurons}

The present invention relates to a composition for cell regeneration comprising at least one cell of a neural stem cell, a neuron cell, and a neural cell, and a cell which hypersensitizes a growth factor.

Neurological damage and degenerative neurological disorders remain a major problem for clinicians and scientists because there is no clear treatment yet. Stem cells have the ability to self-replicate and differentiate into multiple lines of cells and are considered to be an effective source of cellular therapy. Human pluripotent stem cells, embryonic stem cells (ESCs) and inducible pluripotent stem cells (iPSCs), are potent candidates for regenerative therapy and are used in a wide variety of metabolic, genetic, and degenerative diseases, . However, the ethical problems related to embryonic stem cells, induced pluripotent stem cells have technical problems such as safety problems related to foreign cell reprogramming factors which may activate the carcinogenic pathway, slow reprogramming process and low efficiency, Clinical application of the cells remains a tubal defect.

Thus, adult mesenchymal stem cells have low cell proliferation and differentiation ability compared to embryonic stem cells and inducible pluripotent stem cells. However, adult mesenchymal stem cells have advantages such as self-conformity, high acquiring ability, and strong proliferative ability Which is becoming a more suitable supply source for regenerative medicine.

In this regard, attempts have been made to use adult mesenchymal stem cells as a source of various cells. In particular, various efforts have been made to differentiate into mesenchymal stem cells, neuronal cells or neural neural cells, There is not yet known a composition for cell regeneration comprising cells converted into neural stem cells, neurons, or cells of neuronal cells by conversion ratio.

Korean Patent Publication No. 2015-0014417

It is an object of the present invention to provide a composition for cell regeneration capable of promoting the growth of inner cells with invivo or invitro.

It is an object of the present invention to provide a composition for cell regeneration capable of preventing, ameliorating or treating neurological diseases.

It is an object of the present invention to provide a composition for cell regeneration that can be used as a therapeutic agent for neurological diseases even when cells in the intermediate stage of transformation are contained.

The object of the present invention is to provide a composition for cell regeneration which has a low probability of occurrence of side effects that may occur when embryonic stem cells or induced pluripotent stem cells are applied in vivo.

The object of the present invention is to provide a composition for cell regeneration which can be utilized as a cosmetic composition.

It is an object of the present invention to provide a composition for cell regeneration excellent in biocompatibility.

1. at least one selected from the group consisting of neural stem cells, neurons, and neural cells; And growth factor hypersecretory cells.

2. The composition for cell regeneration according to 1 above, wherein the neural stem cells, neurons, GABA neurons and growth factor hypersecretory cells are cells derived from mesenchymal stem cells.

3. The composition for cell regeneration according to item 1 above, wherein the neural stem cells, neurons, and neural cells are differentiated cells from adipose tissue-derived mesenchymal stem cells.

4. The composition for cell regeneration according to 1 above, wherein the growth factor-immunodeficient cells are differentiated from bone marrow-derived mesenchymal stem cells.

5. The composition for cell regeneration according to 1 above, wherein said composition for cell regeneration further comprises a cell expressing a marker expressed in at least one cell selected from the group consisting of astrocytes and rare prominent glue cells.

6. The composition for cell regeneration according to item 1 above, wherein the neural stem cells are symmetrically dividing cells.

7. The neural stem cell according to 1 above, wherein said neural stem cell expresses at least one selected from the group consisting of Nestin, Sox2, Sox1, Musashi-1, FoxG1, Nkx2.1, Pax6, Gli3, Vimentin, Tuj1, Emx1 and Ascl1. A composition for cell regeneration.

8. The method of claim 1, wherein the neuron expresses at least one selected from the group consisting of Tuj1, MAP2, FoxG1, Nkx2.1, Pax6, Dlx2, Dlx5, Lhx6, GAD67, SCN5A, GFAP, S100, Olig2 and NeuN Wherein the cell regeneration method comprises the steps of:

9. The method of claim 1, wherein the Gabbilian neuron comprises NKX2.1, LHX6, TuJ1, MAP2, MGE, Dlx2, Dlx5, GABRA1, GABRA2, GABRA5, CALB2, GAD65, GAD67, PSD95, NF-M and SYP Lt; RTI ID = 0.0 > 1, < / RTI >

10. The composition for cell regeneration according to item 1 above, wherein the Gabbee neuron has a radial neurite.

11. The composition for cell regeneration according to item 1 above, wherein said gabbial neuron exhibits a spontaneous inhibitory post-synaptic current upon treatment with a glutamate receptor blocker.

12. The composition for regeneration of cell according to 1 above, wherein said growth factor-secreting cells express at least one selected from the group consisting of GFAP, Po, S100, CNPase and p75NTR.

13. The method of claim 1, wherein the growth factor is selected from the group consisting of HGF (hepatocyte growth factor), vascular endothelial growth factor (VEGF), insulin-like growth factor binding protein-1, IGFBP-2, IGFBP- -6 and SCF (at least one selected from the group consisting of stem cell factor, VEGF and HGF).

14. The composition for cell regeneration according to 1 above, wherein the growth factor is at least one selected from the group consisting of VEGF and HGF.

15. The composition for cell regeneration according to claim 1, wherein the composition for regenerating the cells is selected from the group consisting of Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin 3 (NT-3), Neurotrophin 4/5 Insulin Like Growth Factor 2 (IGF 1), Insulin Like Growth Factor 2 (IGF), and Insulin Like Growth Factor 2 (FGF) 2), Glia cell line-derived neurotrophic factor (GDNF), TGF-b2, TGF-b3, Interleukin 6 (IL-6), Ciliary Neurotrophic Factor (CNTF) , A composition for cell regeneration.

16. A pharmaceutical composition for improving or treating ischemic diseases, which comprises the composition for cell regeneration of item 1 above.

17. A pharmaceutical composition for improving or treating neurological diseases comprising the composition for cell regeneration of 1 above.

18. The method of claim 16, wherein said ischemic disease is selected from the group consisting of trauma, rejection upon transplantation, ischemic cerebrovascular disorder, ischemic renal disease, ischemic lung disease, ischemic disease associated with infectious disease, ischemic disease of limb and ischemic heart disease Or a pharmaceutically acceptable salt thereof.

19. The method of claim 17 wherein said neurological disease is selected from the group consisting of stroke, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, traumatic central nervous system diseases, and spinal cord injury disease.

The composition for cell regeneration of the present invention can promote the growth of cells in vitro or in vivo.

The composition for cell regeneration of the present invention can exhibit an effect of increasing the growth of damaged nerve cells, nerve fibers, and the like and inhibiting cell death.

The composition for cell regeneration of the present invention can be used as a therapeutic agent for neurological diseases even if cells in the intermediate stage of transformation are contained.

The composition for cell regeneration of the present invention can prevent, ameliorate or treat various neurological diseases, ischemic diseases and the like.

The composition for cell regeneration of the present invention is less likely to cause side effects that may occur when embryonic stem cells or induced pluripotent stem cells are applied in vivo.

The cell regeneration composition of the present invention can be utilized as a cosmetic composition.

The composition for cell regeneration of the present invention is intended to provide a composition for cell regeneration excellent in biocompatibility.

1 is a diagram illustrating optimization of a neural stem cell cross-differentiation protocol using a small molecule inhibitor (SMI)
A: A schematic representation of the process from induction of adipocyte-derived mesenchymal stem cells to neural stem cells.
B: Scale bar: 100 μm.
C: Real-time PCR analysis of the characteristics of inducible neural stem cells. The vertical axis represents the relative amount of gene expression.
^* P <0.05, ^** P <0.01, ^*** P <0.001 The significance was compared with adipocyte-derived mesenchymal stem cells and ^† P <0.05, ^†† P <0.01, ^††† P <0.001 Probability is the value compared to the group not treated with low molecular inhibitor.
D: Quantification of induced neural stem cells by flow cytometry analysis of neural cell adhesion molecule (NCAM) using a fluorescence flow cytometer (FACS Caliber).
Figure 2 shows the characteristics of inducible neural stem cells cross-differentiated by treatment with a low-molecular inhibitor:
A: Detection of inducible neural stem cells expressing both Nestin and Sox2 through fluorescent immunoassay analysis.
B: Real-time gene amplification analysis was used to identify the neural stem cells and early neuronal cell markers (Sox1, Sox2, Nestin, Musashi-1, FoxG1, Nkx2.1, Pax6, Gli3, Vimentin, Tuj1, Emx1). The vertical axis represents the relative amount of gene expression.
^* P <0.05, ^** P <0.01, ^*** P <0.001 The significance is compared with the adipocyte-derived mesenchymal stem cells.
^† P <0.05, ^†† P <0.01, ^††† P <0.001 The significance was compared with the cells in step 1 and the probability of #P <0.05 was compared with the cells in step 2.
FIG. 3 is a diagram showing an experimental outline and morphology of inducing neurons:
A: Schematic representation of experiments for differentiation from adipocyte-derived mesenchymal stem cells into inducible neurons.
B: Indicates that the inducing neuron shows the same morphology as the mature neuron. Scale bar: 100 μm.
C: induced neural stem cells (iNSC), induced neural cells (iN)
^* P <0.05, ^** P <0.01, ^*** P <0.001 The significance was compared with the adipocyte-derived mesenchymal stem cells, and ^† P <0.05, ^†† P <0.01, ^††† P <0.001 The probability is the value compared to the induction neural stem cells.
FIG. 4 is a graph showing the characteristics of inducing neurons derived from adipocyte-derived mesenchymal stem cells:
A: Scale Bar: 20 μm.
B: The number of nerve cell progenitor cells, nerve cells, and guinea neurons expressing the glial cell marker was counted in at least three different areas. Percentage represents the proportion of the number of inducing neurons expressing the corresponding molecular marker among the DAPI-expressing inducible neurons corresponding to the total number of cells. (Mean value ± standard error of mean)
C: Electrophysiological recording samples were measured from inducing neurons having typical neuronal morphology. The sample image shows the induction of the action potential by current injection, and the current injection protocol is under the action potential record. Bottom recordings are representative spontaneous synaptic activities obtained from inducible neurons in a voltage clamped manner (-60 mV fixed), with a single amplified single current being shown below the consecutive record.
Figure 5 shows optimization of the cross-differentiation protocol to Gabbian neurons:
B: Scale bar: 20 μm.
C: The number of induced ganglion neurons expressing the medial ganglion bulge (MGE) cells and neuronal cell markers were counted in at least three different areas. The percentage indicates the proportion of the number of inducible neurons expressing the corresponding molecular marker among the DAPI-expressing inducible neurons corresponding to the total number of cells. (Mean value ± standard error of mean)
D: Scale bar: 100 μm.
Figure 6 shows the functional characteristics of the inducible neural cells:
^{A, B: * P <0.05} , ** P <0.01, *** P <0.001 significance probability value is compared with adipocyte-derived MSCs and ^{† P <0.05, †† P <} 0.01, ††† The probability of P <0.001 significance is the value compared to the induced neurons of the 25th day of in vitro culture.
C: Scale bar: 20 μm.
D: Number of inducible callus neurons expressing callus neuron cell markers were counted in at least three different areas. The percentage indicates the ratio of the number of inducible neurons expressing the corresponding molecular marker among the inducible neural cells expressing DAPI corresponding to the total number of cells (mean value ± standard error of mean).
E: The upper left panel shows the activity potential speech record of the induced calcium neurons recorded by current clamping method. Below the activity potential record is the current injection protocol.
The upper right panel shows that the application of the ramp protocol in inducible neurons induces a rapid current flow and depolarizes the fixed voltage. The voltage gradually increased from -100 mV to 0 mV for one second, and the dashed box is the enlarged current indicated by the asterisk.
A schematic of the experimental design is shown in the lower left panel.
FIG. 7 is a diagram showing microscopic and quantitative RT-PCR results of changes in cell morphology and Schwann cell marker gene after induction of growth factor-rich human stem cells in human mesenchymal stem cells:
A: Microscopic photographs of cell morphology observed during the induction of human bone marrow-derived mesenchymal stem cell (hMSC) into Schwann cells;
B: gel photographs of PCR products of Schwann cell specific genes (GFAP, PO, S100, CNPase and p75NTR) in differentiated hMSCs;
C: A graph ( ^* P <0.05, ^** P <0.001 versus uhMSC. ^† P <0.05 versus gfMSC) obtained by quantifying the mRNA level of each gene in a densitometry system from PCR results of Schwann cell specific genes.
8 is a diagram showing the expression of Schwann cell-labeled protein in gfMSC through a cell immunochemical analysis:
A: Cell-immunofluorescent staining micrographs of Schwann cell-labeled proteins (GFAP, Po, S100, CNPase and p75NTR) in differentiated hMSCs;
B: A graph showing the fluorescence intensity of each protein in the immunofluorescent staining micrograph ( ^* P <0.05, ^** P <0.001 versus uhMSC. ^†† versus gfMSC).
FIG. 9 is a diagram showing cell immunochemical analysis that the cell cycle is stopped due to the progress of differentiation in gfMSC:
A: Microphotographs stained with p75NTR (red) and bromodeoxyuridine (BrdU) (green) in uhMSC, gfMSC and gfMSC + GM by cell-mediated immunofluorescence staining;
B: a graph showing the proportion of BrdU-positive cells in the micrograph of A;
C: p75NTR (+) / BrdU in the micrograph of the A (-) a graph showing the percentage of cells ^{(* P <0.05, ** P} <0.001 versus uhMSC †† P <0.001 versus gfMSC.).
10 is a graph showing the results of analysis of secretion of various growth factors from gfMSC through a growth factor analysis array:
A: Growth factor microarray analysis results in medium, uhMSC-CdM and gfMSC-CdM;
B: a graph showing the relative expression level of each growth factor as a relative value to the positive control in the result of the above A;
C: The relative expression of each growth factor was compared with the amount detected in the medium ( ^* P <0.05, ^** P <0.001 versus Media Alone. ^† P <0.05, ^†† P <0.001 versus uhMSC- CdM);
D: A graph showing the results of ELISA for the expression of HGF in uhMSC, gfMSC and gfMSC + GM;
E: Graph ( ^* P < 0.05, ^** P < 0.001 versus CdM) showing the results of ELISA for the expression of VEGF in uhMSC, gfMSC and gfMSC + GM.
F: ELISA results of gfMSC confirmed HGF production in induction differentiation medium (SCIM) and normal growth medium (GM);
G: ELISA results of gfMSC confirming VEGF production in induction differentiation medium (SCIM) and normal growth medium (GM);
H: Expression of HGF and VEGF in uhMSC, gfMSC, gfMSC + SCIM, and gfMSC + GM by gel filtration assay by quantitative RT-PCR;
I: a graph in which the expression amount of HGF in the above H result is corrected to a relative amount to GAPDH;
J: A graph ( ^* P < 0.05, ^** P < 0.001 versus uhMSC) in which the expression level of VEGF in the above H result is corrected to a relative amount to GAPDH.
Fig. 11 shows the result of quantitative analysis of neurites and trypan blue staining, which promotes the growth and proliferation of neurite of Neuro2A cells co-cultured with gfMSC:
A: Simultaneous culture of Neuro2A cells and human mesenchymal stem cells;
B: Micrographs of neurites observed in Neuro2A cells co-cultured with gfMSC;
C: A graph showing the percentage of cells having at least one neuron in the Neuro2A cell of at least one cell diameter in the result of the above B;
D: a graph showing the sum of the neurite length per cell from the result of the B in Neuro2A cells ^{(* P <0.05, ** P} <0.001 versus Media † P <0.05, †† P <0.001 versus uhMSC # P <.. 0.05, ^# P < 0.001 versus gfMSC);
E: graph showing the average number of Neuro2A cells in each group staining Neuro2A cells co-cultured with gfMSC in trypan blue;
F: graph gfMSC the dyeing cocultured with Neuro2A cells with trypan blue shows the ratio of each group the Neuro2A cell death ^{(* P <0.05, ** P} <0.001 versus Media † P <0.05, †† P. <0.001 versus uhMSC. ^# P <0.05, ^## P <0.001 versus gfMSC).
FIG. 12 is a graph showing the results of tissue immunochemical staining and TUNEL analysis of the increase of spinal nerve growth and cell survival in a damaged spinal cord tissue section model of gfMSC transplantation:
A: Microscopic photographs of tissues immunoprecipitated spinal nerve growth in injured spinal cord segmental models of gfMSC transplantation;
B:.. Graph was standardized by the integrated optical density on the result to the control spinal cord tissue sections of the ^{A (* P <0.05, **} P <0.001 versus Control †† P <0.001 versus lysolecithin-treated slices (LPC) ## . P <0.001 versus LPC + uhMSC §§ P <0.001 versus LPC + gfMSC);
C: Microscopic photographs showing that the transplantation of gfMSC increased cell survival in damaged spinal cord tissue section model by TUNEL analysis; And
D: A graph showing the quantitative analysis of TUNEL-positive cells in the result of C above ( ^** P <0.001, versus Control. ^†† P <0.001 versus LPC. ^# P <0.05, ^## P <0.001 versus LPC + uhMSC).
FIG. 13 shows the results of quantitative analysis of neurites, trypan blue staining and tissue immunochemical staining of the effect of exogenous HGF and VEGF as neurotrophic factors in Neuro2A cells and damaged spinal cord tissue section model:
A: A graph showing the proportion of cells having at least one neuronal cell length longer than that of Neuro2A cells cultured with addition of recombinant HGF;
B is a graph showing the sum of neurite length per cell in Neuro2A cells cultured with addition of recombinant HGF;
C: a graph showing the percentage of cells having at least one neural prolate longer than the diameter of a cell in Neuro2A cells cultured with addition of recombinant VEGF;
D: Graph showing total neurite length per cell in Neuro2A cells cultured with recombinant VEGF ( ^* P <0.05, ^** P <0.001 versus untreated Neuro2A cells. ^† P <0.05, ^†† P <0.001 versus HGF And VEGF co-treated Neuro2A cells);
E: a microphotograph showing that extrinsic HGF and / or VEGF protein increases neurite outgrowth of injured spinal cord tissue sections;
F: A graph showing the relative integrated optical density (IOD) values of NF-M immunofluorescently stained nerve fibers according to the concentration of HGF and / or VEGF protein as a result of the above E standardized to a control spinal cord tissue section ( ^* P & ^** P <0.001 versus control. ^† P <0.05, ^†† P <0.001 versus ^lyso lecithin treated spinal cord tissue (LPC));
G: Growth Factor Differentiated from Human Bone Marrow-derived Mesenchymal Stem Cells [0060] It is a figure showing a mechanism in which human stem cells secrete HGF and VEGF from impaired spinal nerves and function as a neurotrophic factor.

The present invention relates to a composition for cell regeneration, which can promote growth of cells in an invivo or an invitro by including at least one cell of a neural stem cell, a neuron cell, and a neural cell and a hypersecretory growth factor, Neurons, nerve fibers, and the like, and can inhibit apoptosis. Even if cells in intermediate stages of conversion are contained, they can be used as therapeutic agents for neurological diseases, and can prevent, ameliorate or prevent various neurological diseases, ischemic diseases, The present invention relates to a composition for cell regeneration, which can be used as a cosmetic composition and is excellent in biocompatibility. The present invention relates to a composition for cell regeneration, which can be used as a cosmetic composition and has a low probability of occurrence of side effects that may occur when embryonic stem cells or induced pluripotent stem cells are applied in vivo.

Hereinafter, the present invention will be described in detail.

"Stem cell" refers to a cell capable of self-replicating and capable of differentiating into two or more cells, and "adult stem cells" Stem cells appear in the stage.

By "treatment" is meant preventing the recurrence of an epileptic seizure, and the term "symptom improvement" The severity of epileptic seizures, and the like.

The composition for cell regeneration of the present invention comprises at least one selected from the group consisting of neural stem cells, neurons, and neural cells; And growth factor hypersecretion cells.

Neural stem cells are cells that maintain a potency capable of producing neurons and glia cells. The neural stem cells may be symmetrically or asymmetrically cleaved, preferably symmetrically cleaved, but may not be limited thereto. When symmetrically cleaved, the neural stem cells divide to form two daughter neural stem cells or two precursor cells, and when they divide asymmetrically, the neural stem cells divide into one daughter neural stem cell and one Progenitor cells (e. G., Neuronal precursor cells or glioma precursor cells). The neural stem cells are different from known neural stem cells and are different from neural stem cells in the body or neural stem cells of the control group in terms of expression and growth rate, differentiation rate, The ability to do is better. And the neural stem cells can perform one or more cell division. Preferably 10 to 30 times, more preferably 40 times or more, more preferably 50 times or more, and most preferably, unlimited cell division can be performed. The neural stem cells may divide symmetrically or asymmetrically, and preferably the neural stem cells divide symmetrically.

The neural stem cells may be those expressing the following neuronal marker: ABCG2, ASCL1 / Mash1, beta-Catenin, BMI-1, Brg1, N-Cadherin, Calcitonin R, CD15 / Lewis X, CD133, CDUP1, 2, GFAP, Glut1, HOXB1, ID2, Meteorin, MSX1, IGF-I / NR2F1, CXCR4, FABP7 / B-FABP, FABP8 / M-FABP, FGF R2, FGF R4, FoxD3, Frizzled- , Musashi-1, Musashi-2, Nestin, NeuroD1, Noggin, Notch-1, Notch-2, Nrf2, Nucleostinum, Numb, Otx2, Pax3, Pax6, PDGFR alpha, PKC zeta, Prominin2, ROR2, RUNX1 / 4, Vimentin, ZIC1, Nestin, FoxG1, Nkx2.1, Gli3, Vimentin, RXR alpha / NR2B1, sFRP-2, SLAIN1, SOX1, SOX2, SOX9, SOX11, SOX21, SSEA- At least one of Nestin, Sox2, Sox1, Musashi-1, FoxG1, Nkx2.1, Pax6, Gli3, Vimentin, Tuj1, Emx1, Olig2 and Ascl1 may be expressed , Preferably Nestin, Sox2, Sox1, Musashi-1, FoxG1, Nkx2.1, Pax6, Gli3, Vimentin, Tuj1, Emx1, Olig2 and Ascl1 It may be one which expresses parts.

The nerve cell is the main cell that constitutes the nervous system. It is a cell which expresses the ion passage of the sodium passage and the potassium passage and can transmit the signal by an electric method unlike the other cell. The neuron is a new neuron different from neurons in the body and is required for cell survival and differentiation such as expression marker, proliferation rate, differentiation rate, adhesion rate, survival rate, environment adaptive ability, etc. The ability is better. In addition, since it is differentiated from mesenchymal stem cells, which are adult stem cells, there is little concern about the occurrence of side effects when applied to a living body. Due to the excellent properties, neurological diseases (such as brain tumor, peripheral neuropathy, Ellis's syndrome, Parkinson's disease, Alzheimer's disease, ), The effect is excellent.

The neurons are capable of expressing the neural stem cell markers described above and are also capable of expressing neural stem cell markers such as NSE, NeuN, DCX (Doublecortin), c-fos, Choline acetyltransferase, TH (tyrosine hydrdoxylase), Tau, Calbindin-D28k, Calretinin, NFP , NeuroD, PSA-NCAM, Tujl, MAP2, FoxGl, Nkx2.1, Dlx2, Dlx5, Lhx6, GAD67, SCN5A, Olig2, S100 and the like. At least one of Tuj1, MAP2, FoxG1, Nkx2.1, Pax6, Dlx2, Dlx5, Lhx6, GAD67, SCN5A, Olig2, S100 and NeuN, , Pax6, Dlx2, Dlx5, Lhx6, GAD67, SCN5A, GFAP, S100, Olig2 and NeuN.

GABA neurons are neurons that produce gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the central nervous system, and are used in cell therapy for brain and nervous system disorders such as stroke, traumatic brain injury, cerebral palsy, epilepsy and Huntington's disease It can serve as an excellent source of supply. The GABAergic neurons exhibit IPSC (spontaneous inhibitory post-synaptic current) upon treatment with a glutamic acid receptor blocker, and functionally disrupt IPSCs in the treatment of GABA receptor blocker. However, they have differentiation ability, survival rate, The frequency of expression of IPSC, and the expression marker are novel cells different from the body-derived neural cells or control cells.

The cell regenerating composition of the present invention comprising the above-described Ca2 + -activated neurons can prevent, ameliorate or treat epilepsy, bipolar disorder, asthma, anxiety disorder and the like, and can preferably prevent, ameliorate or treat epilepsy. Epilepsy is a chronic cerebellar disease characterized by seizure brain dysfunction caused by the discharge of transient neurons. Symptoms of epilepsy include epileptic seizures. Epileptic seizures can be divided into partial (focal, local) seizures, and generalized seizures. Partial seizures include, for example, simple partial seizures, complex partial seizures (e.g., automatism), seizures that develop from partial seizures to secondary seizures seizures evolving to secondarily generalized seizures. General seizures include, but are not limited to, seizures, atypical absence seizures, Myoclonic seizures, Clonic seizures, Tonic seizures, Tonic-clonic seizures, and Atonic seizures (Astatic).

GABA, GAD1, GAD2, GABA _B receptors 1 and 2, MAP2, Dlx2, Dlx5, GFAP, CALB2 and GABA receptors, which are capable of expressing the neural cell and / or neural stem cell markers described above. , GALB2, GABRA1, GABRA2, GABRA5, GAD65, GAD67, GADG1, GABRA1, GABRA2, GABRA5, GAD65, GAD67, PSD95, SYP and NF- , PSD95, SYP, and NF-M.

The growth factor hypersecretory cell may be an adult stem cell (for example, mesenchymal stem cell), and it is preferably, but not limited to, a Schwann-like cell in the lineage, Or Schwann cells.

IGFBP-2, IGFBP-4, IGFBP-6, SCF (IGFBP-1, IGFBP-2, IGFBP- stem cell factor, and the like, and may preferably overexpress HGF and VEGF, but the present invention is not limited thereto.

The growth factor-immunodeficient cells may express at least one marker selected from the group consisting of GFAP, Po, S100, CNPase, and p75NTR, and may preferably express all of them, but the present invention is not limited thereto .

The growth factor-secreting cells may secrete HGF and / or VEGF 10-fold more than the bone marrow-derived mesenchymal stem cells, but may not be limited thereto.

The growth factor hypersecretory cell can increase neurite outgrowth and cell growth and survival, and can be included in the cell regeneration composition of the present invention to increase the growth and survival rate of neural stem cells, neuron cells and neurons Therefore, it can exhibit a very excellent effect in the treatment of neurological diseases.

According to one embodiment of the present invention, neural stem cells, neurons, neural cells, and growth factor secretion cells may be derived from mesenchymal stem cells.

Mesenchymal stem cells are undifferentiated stem cells isolated from human or mammalian tissues and can be derived from various tissues. In particular, mesenchymal stem cells derived from umbilical cord, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose derived mesenchymal stem cells, muscle derived mesenchymal stem cells, nerve-derived mesenchymal stem cells, , Amniotic membrane-derived mesenchymal stem cells and placenta-derived mesenchymal stem cells, and techniques for separating stem cells from each tissue are well known in the art. Preferably in a human, a pig, a cow, a sheep, a rabbit, a mouse or a rat, more preferably a human, a pig or a mouse, more preferably a human bone marrow, cord blood or fat tissue . For example, mesenchymal stem cells may be adipose tissue-derived cells.

According to one embodiment of the present invention, the mesenchymal stem cells may be adipose-derived or bone marrow-derived mesenchymal stem cells, and preferably the neural stem cells, neuron cells and neurons are differentiated from adipose derived mesenchymal stem cells And the growth factor hypersecretory cell may be differentiated from bone marrow derived mesenchymal stem cells, but is not limited thereto.

Mesenchymal stem cells 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, α-MEM, Iscove's MEM, 199 medium, CMRL 1066, RPMI 1640, F12, F10, DMEM, a mixture of DMEM and F12, Way-mouth's MB752 / 1, McCoy's 5A, and MCDB series. The cells are cultured in the subculture until confluency of 70-80% is reached, then adherent cells are harvested and a portion of the harvested cells are used for subsequent passaging. 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 composition for cell regeneration of the present invention may include cells from mesenchymal stem cells to neural stem cells, neurons, and intermediate cells (undifferentiated cells) that differentiate into neural cells. For example, the undifferentiated cell may be a mesenchymal stem cell. The undifferentiated cells may express markers of known mesenchymal stem cells, neural stem cells, neuronal cells, and neural neural cells. The markers of adipose tissue-derived mesenchymal stem cells are ALCAM / CD166, Aminopeptidase Inhibitors, Aminopeptidase N / CD13, CD9, CD44, CD90 / Thy1, Endoglin / CD105, ICAM-1 / CD49, Integrin alpha 4 / 1, Integrin alpha 4 beta 7 / LPAM-1, Integrin alpha 5 / CD49e, Integrin beta 1 / CD29, MCAM / CD146, Osteopontin / OPN, PUM2, SPARC, VCAM-1 / CD106 Do not. Neural stem cells, nerve cells, and neoplastic cell markers may be, but are not limited to, the markers described above. The undifferentiated cells can improve the growth, survival rate, and differentiation rate of the cells in the cell or in vivo injected site contained in the composition for cell regeneration of the present invention. The composition for cell regeneration of the present invention includes the undifferentiated cells, thereby promoting the growth of the neural stem cells, neurons, and neural cells, improving the survival rate and improving the differentiation rate. It is possible to exhibit a better therapeutic effect. In addition, the undifferentiated cells can express markers expressed in at least one cell of astrocytes and rarely proliferating glial cells. The cell regeneration composition of the present invention may contain the undifferentiated cells in an amount of 30% or less, preferably 15% to 20%, more preferably 10%, and even more preferably 30% or less of 100% May include, but is not limited to, 5%. For example, 1%.

The cells included in the cell regeneration composition of the present invention are all derived from mesenchymal stem cells, which are adult stem cells, and can be used as a safe cell therapy agent because of low probability of cytotoxicity when applied in the body. When the composition for cell regeneration of the present invention is used for the prevention, improvement or treatment of neurological diseases, the number of neural stem cells, neuron cells, And can be variously changed. For example, neural stem cells, which may contain the number of growth factor-immunodeficient cells optimized for the rate of survival, survival rate of neural stem cells, neurons and / or neural cells, and survival rates, Neural cells, &lt; RTI ID = 0.0 &gt; Ca2 + &lt; / RTI &gt; The composition for cell regeneration of the present invention may exhibit a more excellent effect in preventing, ameliorating or treating neurological diseases. When the composition for cell regeneration of the present invention is applied to the treatment of neurological diseases, ischemic diseases, etc., it may be used without being limited thereto.

The composition for cell regeneration of the present invention can be applied to the treatment itself without the need to remove undifferentiated cells, and thus the undifferentiated cells are removed from the composition for cell regeneration including cells differentiated from embryonic stem cells or induced pluripotent stem cells It is very safe because the probability of occurrence of cancer, differentiation into unwanted cells, toxicity to surrounding cells, etc. is very low, and there is no need to remove it, so efforts and efforts In particular, the adipose tissue-derived mesenchymal stem cells can be mass-produced because they can be obtained in large quantities in vivo such as abdominal fat.

The cell regenerating composition of the present invention may further comprise one or more of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), and platelet- A cytokine-like peptide factor that induces differentiation, growth, etc. of nerve cells and nerve tissue.

The composition for cell regeneration of the present invention may further comprise an extracellular matrix. Derived from nature or artifacts (combination). Examples of the material include collagen, proteoclycan, fibronectin, hyaluronic acid, tenacin, enstatin, elastin, fibril and laminin, or fragments thereof. These extracellular matrices can be used in combination. For example, preparations from cells such as BD Matrigel (trademark) may be used.

The composition for cell regeneration of the present invention may further comprise laminin or a fragment thereof. The laminin is a protein having a heterotetrameric structure having one α-chain, β-chain and γ-chain, and is not particularly limited. For example, α-chain is α1, α2, α3, α4 or α5, Is? 1,? 2 or? 3, and? Chain is? 1,? 2 or? 3.

The composition for cell regeneration of the present invention may further comprise a neurotrophic factor. Neurotrophic factors are ligands to membrane receptors that play an important role in the survival and function of motor neurons. Neurotrophin 3 (NT-3), Neurotrophin 4/5 (NT-4/5), Neurotrophin 6 (NT-6), basic FGF , Insulin Like Growth Factor 2 (IGF 2), Glia cell line-derived Neurotrophic (HGF), Insulin Like Growth Factor 1 (IGF 1), Acidic FGF, FGF-5, Epidermal Growth Factor (EGF), Hepatocyte Growth Factor Factor (GDNF), TGF-b2, TGF-b3, Interleukin 6 (IL-6), Ciliary Neurotrophic Factor (CNTF) and LIF. Neurotrophic factors are commercially available, for example, from Wako and R & D systems, and can be readily obtained by forced expression into cells according to methods known to those skilled in the art.

The composition for cell regeneration of the present invention can be prepared in unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient or by inserting it into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents. Examples of the carrier include those conventionally used at the time of formulation such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone But are not limited to, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline solution, phosphate buffered saline It is not.

Examples of the suspending agent include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragacanth, carboxymethylcellulose sodium, and polyoxyethylene sorbitan monolaurate. Examples of the solution auxiliary agent include polyoxyethylene hardened castor oil, polysorbate 80, nicotinic amide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester and the like. Examples of the stabilizer include dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate and the like,

Examples of isotonic agents include D-mannitol, sorbitol and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, chlorocresol and the like,

Examples of the adsorption inhibitor include human serum albumin, lecithin, dextran, ethylene oxide-propylene oxide copolymer, hydroxypropylcellulose, methylcellulose, polyoxyethylene hydrogenated castor oil, polyethylene glycol,

Examples of the sulfur reducing agent include N-acetylcysteine, N-acetylhomocysteine, thioctanoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, Examples of the antioxidant include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, alpha -tocopherol, acetic acid, and the like, and those having sulfhydryl groups such as thioalkanoic acids of 1 to 7, Tocopherol, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium hydrogen sulfite, sodium sulfite, gallic acid triamyl, gallic acid propyl or ethylenediaminetetraacetate (EDTA) And chelating agents such as sodium phosphate, sodium metaphosphate and the like. Examples of the cryopreservation agent include, but are not limited to, DMSO and glycerol, and further inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate and sodium hydrogen carbonate; And organic salts such as sodium citrate, potassium citrate, sodium acetate and the like.

The cell regenerating composition of the present invention can be administered orally or parenterally, and is preferably a parenteral administration method, most preferably intravenous administration, intra-brain tissue (for example, hippocampus) administration. A suitable dosage of the cell regeneration composition of the present invention varies depending on such factors as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient It can be prescribed. Typical dosages of the pharmaceutical compositions of the invention are from 10 ² to 10 ¹⁰ cells per day on an adult basis.

The composition for cell regeneration may include, but is not limited to, neural stem cells, neurons, goby neurons, and growth factor hypersecretory cells differentiated from the mesenchymal stem cells obtained from the subject to be administered.

The subject to which the pharmaceutical composition of the present invention can be applied may be a vertebrate animal, preferably a mammalian animal, more preferably an ape-like animal such as a chimpanzee, a gorilla, a rat, a rabbit, a guinea pig, a hamster, , A laboratory animal such as a cat, a human, and the like, and most preferably, it may be an ape-like animal or a human, but the present invention is not limited thereto.

The composition for cell regeneration of the present invention can be applied to a neurological disorder that is a nervous system disorder and includes, for example, the central nervous system (brain, brainstem and cerebellum), peripheral nervous system (including cranial nervous system), and autonomic nervous system And the like). In particular, the neurological disease includes any disease in which the function of the neural ridge cell or Schwann cell is impaired, altered or prevented. (EAE), acute disseminated encephalomyelitis (ADEM), post-infectious or post-vaccination encephalomyelitis, peripheral neuropathy, schwannomatosis, Charcot-Marie Disease, Guillain-Barré syndrome Stroke, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis (Alzheimer's disease), chronic inflammatory dehydrative multiple neuropathy (CIDP) lateral sclerosis, traumatic central nervous system diseases, and spinal cord injury disease, but the present invention is not limited thereto.

The composition for cell regeneration of the present invention can be applied to ischemic diseases such as trauma, rejection at transplantation, ischemic cerebrovascular disorder, ischemic kidney disease, ischemic lung disease, ischemic diseases related to infectious diseases, ischemic diseases of extremities, Heart disease, and the like, but the present invention is not limited thereto.

The cell regeneration composition of the present invention may be a medium.

When the cell regeneration composition of the present invention is a medium, it can be prepared using a medium used for culturing animal cells as a base medium. Medium, 199 medium, EEME medium, αMEM medium, Dulbecco's modified Eagle's medium (DMEM) medium, Ham's F12 medium, RPMI medium 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), and mixed media thereof. The medium may contain serum, or it may be bloodless. If necessary, the medium may be, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum replacement of FBS at ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen) And may contain one or more serum substitutes such as insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolclycerol, and the like, and may include fats, amino acids, L-glutamine, Glutamax (Invitrogen) But may also contain one or more substances such as vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, nucleic acids (for example, Dibutyryl cyclic AMP (dbcAMP)). In addition, it may include antibiotics, growth factors, amino acids, inhibitors or the like, as well as fetal calf serum (FCS) or fetal bovine serum (FBS) Hydrocortisone, insulin, and the like, but are not limited thereto. In addition, neurotrophic factors can be added as appropriate. The culture medium obtained from the culture medium can be used for preventing, improving or treating neurological diseases, and can be used as a cosmetic composition.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. It is to be understood, however, that the following examples and experimental examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

< Example  1> derived from fat cells Intermediate lobe  Stem Cells ( hADSC ) Culture

In order to maintain adipocyte-derived mesenchymal stem cells, the cells are cultured in DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, and maintained at 37 ° C in a 5% CO ₂ incubator. After such a system out, removing the cells by treating a 0.25% trypsin-EDTA in a cell culture dish, put into 10㎝ ² tissue culture dish 8 X 10 ⁴ cells were subcultured in the range 4 days. The medium was changed into a new medium every two days, and the cells were allowed to pass no more than 9 passages for subculturing.

< Experimental Example  1> In adipose-derived stem cells Derived  Similarity Neural stem cell  Confirmation of Establishment of Crossing Differentiation Method

<1-1> Establishment of three-stage differentiation condition

In order to cross-differentiate adipocyte-derived mesenchymal stem cells into neural stem cells, 3% knock out serum replacement (KOSR) was added without any treatment and 3% KOSR treatment with low molecular weight inhibitor treatment. Experiments were conducted to establish the differentiation conditions.

Differentiation is largely divided into three stages. In step 1, 2 x 10 ⁵ cells of adipocyte-derived mesenchymal stem cells are placed in a 6 cm ² gelatin-coated dish and cultured for one day at 37 ° C in a 5% CO ₂ incubator. The medium was replaced with a pre-induction medium on the following day. The composition of the medium was 3% KOSR, 1% Penicillin / Streptomycin, 1% Glutamax, 3 mM D-glucose, 1% non-essential amino acid and 4 ng / ml bFGF (basic fibroblast growth factor) and maintained in a 37 ° C 5% CO ₂ incubator for 8 days. At this stage, the conditions were divided into groups treated with 10 袖 M SB431542, 0.1 袖 g / ml Noggin, 0.5 袖 M LDN 193289 and untreated groups as low molecular inhibitors and the differentiation into neural stem cells was compared.

Next, in the second step, the medium composition is changed to a neural induction medium containing 1% Glutamax, 3 mM D-glucose, 0.2 mM ascorbic acid (1: 1) in DMEM F-12: Neurobasal , 1 mM sodium pyruvate, 2% B27 and 1% N2, and cultured in a 5% CO ₂ incubator at 37 ° C for 5 days.

The third step is to replace the growth medium containing 20 ng / ml bFGF and 20 ng / ml EGF (epidermal growth factor) in the neural induction medium for 7 days at 37 ° C and 5% CO ₂ incubator. Then, in order to confirm that the neural stem cells are properly differentiated, wash the cells with PBS in PBS and wash them with single cells at 37 ° C for 3 to 4 minutes. A cover glass of 12 mm ² prepared by coating with a 6 cm ² dish and 50 μg / ml PLO / 5 μg / ml FN prepared by coating with 10 μg / ml Poly-L-ornithine (PLO) / 1 μg / ml Fibronectin And incubated at 37 ° C in a 5% CO ₂ incubator for 2-3 days. Cell differentiation was then analyzed. ² 6cm dish was compared to the target gene expression by the real-time PCR analysis, 4 well dish with a cover glass was observed for protein expression and expression shape through immunofluorescence staining analysis.

<1-2> Real time PCR  Induced through Neural stem cell  Differentiation-related genes Degree of expression  Confirm

To analyze the degree of gene expression of the differentiated neural stem cells, a cell scraper was used for sampling and total RNA was isolated according to the manual method using Trizol reagent. The isolated total RNA was synthesized as CDNA by reaction with M-MLV reverse transcriptase at 42 ° C for 1 hour. The gene expression was analyzed by SYBR Green gene expression assays using synthesized CDNA as a template. The target genes and primer sequences to be compared are shown in Table 1 below. The expression level of the target gene was standardized by endogenous GAPDH, and the comparison of gene levels was performed by comparing the Ct value of the measured gene. The Ct value was determined by calculating the CT value by subtracting the value of GAPDH from the value of the target gene [? Ct = Ct (target) -Ct (GAPDH)] at a cycle cycle in which the fluorescence level reached the threshold value, The gene was expressed as? CT of the relative value of the target gene GAPDH = 2? Ct.

Gene name Forward (F) & reverse (R) primer sequences Product size (bp) Gene Bank Accession SEQ ID NO: CALB2 F: 5'-CTCCAGGAATACACCCAAA-3 ' 20 BC015484.2 One R: 5'-CAGCTCATGCTCGTCAATGT-3 ' 2 Dlx2 F: 5'-GCACATGGGTTCCTACCAGT-3 ' 153 BC032558.1 3 R: 5'-TCCTTCTCAGGCTCGTTGTT-3 ' 4 Dlx5 F: 5'-CCAACCAGCCAGAGAAAGAA-3 ' 150 BC006226 5 R: 5'-GCAAGGCGAGGTACTGAGTC-3 ' 6 Emx1 F: 5'-AAGCGCGGCTTTACCATAGAG-3 ' 150 NM_004097.2 7 R: 5'-GCTGGGGTGAGGGTAGTTG-3 ' 8 FoxG1 F: 5'-AGAAGAACGGCAAGTACGAGA-3 ' 189 BC050072.1 9 R: 5'-TGTTGAGGGACAGATTGTGGC-3 ' 10 GABRA1 F: 5'-GGATTGGGAGAGCGTGTAACC -3 ' 66 BC030696 11 R: 5'-TGAAACGGGTCCGAAACTG-3 ' 12 GABRA2 F: 5'-GTTCAAGCTGAATGCCCAAT-3 ' 160 BC022488 13 R: 5'-ACCTAGAGCCATCAGGAGCA-3 ' 14 GABRA5 F: 5'-ATCTTGGATGGGCTCTTGG-3 ' 130 BC111979 15 R: 5'-TGTACTCCATTTCCGTGTCG-3 ' 16 GAD65 F: 5'-GGTGGCTCCAGTGATTAAAG-3 ' 165 M81882.1 17 R: 5'-TGTCCAAGGCGTTCTATTTC-3 ' 18 GAD67 F: 5'-AGGCAATCCTCCAAGAACC-3 ' 218 M81883.1 19 R: 5'-TGAAAGTCCAGCACCTTGG-3 ' 20 GFAP F: 5'-CAACCTGCAGATTCGAGAAA-3 ' 153 AF419299.1 21 R: 5'-GTCCTGCCTCACATCACATC-3 ' 22 Gli3 F: 5'-TGGTTACATGGAGCCCCACTA-3 ' 116 M57609.1 23 R: 5'-GAATCGGAGATGGATCGTAATGG-3 ' 24 LHX6 F: 5'-GGGCGCGTCATAAAAAGCAC-3 ' 108 BC103936 25 R: 5'-TGAACGGGGTGTAGTGGATG-3 ' 26 Map2 F: 5'-CGCTCAGACACCCTTCAGATAAC-3 ' 122 U01828.1 27 R: 5'-AAATCATCCTCGATGGTCACAAC-3 ' 28 Mash1 F: 5'-TGCACTCCAATCATTCACG-3 ' 146 NM_004316 29 R: 5'-GTGCGTGTTAGAGGTGATGG-3 ' 30 Musashi F: 5'-TTCGGGTTTGTCACGTTTGAG-3 ' 250 AB012851.1 31 R: 5'-GGCCTGTATAACTCCGGCTG-3 ' 32 Nestin F: 5'-CACCTGTGCCAGCCTTTCTTA -3 ' 170 NM_006617 33 R: 5'-TTTCCTCCCACCCTGTGTCT-3 ' 34 NKX2.1 F: 5'-GTGAGCAAGAACATGGCCC-3 ' 182 BC006221.2 35 R: 5'-AACCAGATCTTGACCTGCGT-3 ' 36 Olig2 F: 5'-GCTGCGACGACTATCTTCCC-3 ' 244 NM_005806.3 37 R: 5'-GCCTCCTAGCTTGTCCCCA-3 ' 38 Pax6 F: 5'-AGGTATTACGAGACTGGCTCC-3 ' 104 AY047583 39 R: 5'-TCCCGCTTATACTGGGCTATTT-3 ' 40 SCN5A F: 5'-GGATCGAGACCATGTGGGAC-3 ' 151 BC144621 41 R: 5'-GCTGTGAGGTTGTCTGCACT-3 ' 42 Sox1 F: 5'-AGATGCCACACTCGGAGATCA-3 ' 184 NM_005986 43 R: 5'-GAGTACTTGTCCTCCTTGAGCAGC-3 ' 44 Sox2 F: 5'-AGTCTCCAAGCGACGAAAAA-3 ' 141 NM_003106.3 45 R: 5'-GCAAGAAGCCTCTCCTTGAA-3 ' 46 TuJ1 F: 5'-GGCCTTTGGACATCTCTTCA -3 ' 241 BC000748.2 47 R: 5'-ATACTCCTCACGCACCTTGC -3 ' 48 Vimentin F: 5'-AGAACTTTGCCGTTGAAGCTG-3 ' 255 NM_003380.3 49 R: 5'-CCAGAGGGAGTGAATCCAGATTA-3 ' 50 GAPDH F: 5'-GTCAGTGGTGGACCTGACCT-3 ' 256 BC083511.1 51 R: 5'-CACCACCCTGTTGCTGTAGC -3 ' 52

<1-3> Immunofluorescent staining  Induced through Neural stem cell  Identification of differentiation-related protein expression

The induced neural stem cells maintained in 4 wells were fixed with 4% formaldehyde for 15 minutes and then washed twice with PBS (phosphate buffered saline) containing calcium ion and magnesium ion. Triton X-100, a surfactant, was diluted to 0.1% and treated twice every 10 min.

To prevent nonspecific antibodies from being detected, chlorine serum is diluted to 5% in 0.1% Triton X-100 / PBS and allowed to react for 1 hour. The primary antibodies differ in the type of antibody attached to the cells, and the target antibodies according to the proteins are shown in Table 2.

Antibody name Company (Cat. No.) Antibody name Company (Cat. No.) Anti-DLX2 Santa Cruz (sc-81960) Anti-NKX2.1 Merck Millipore (MAB5460) Anti-GABA Sigma-Aldrich (A2052) Anti-Olig2 From lab stocks (Gift of Harvard University) Anti-GAD Merck Millipore (AB1511) Anti-PAX6 Merck Millipore (MAB5554) Anti-GFAP Merck Millipore (MAB3402) Anti-PSD96 Merck Millipore (MABN68) Anti-MAP2 Merck Millipore (MAB3418) Anti-SlOOo Dako (Z0311) Merck Millipore (AB5622) Anti-NCAM BD bioscience (562794) Aanti-SOX2 Merck Millipore (AB5603) Anti-Nestin BD bioscience (611658) Anti-SYP Sigma-Aldrich (SAB4502906) Anti-NeuN Merck Millipore (MAB377) Anti-TuJl Bio Legend (PRB-435P) Anti-NFM Merck Millipore (AB1987)

The primary antibody was attached and reacted in a 4 ° C shaker for 16 hours. The secondary antibody was selected according to the host and wavelength of the primary antibody and the goat anti- (mouse IgG) -conjugated Alexa Fluor 555 (1: 200 dilution), goat anti- (mouse IgG) -conjugated Alexa (1: 200 dilution), goat anti- (rabbit IgG) -conjugated Alexa Fluor 555 (1: 200 dilution) and goat anti- (rabbit IgG) -conjugated Alexa Fluor 488 (1: 200 dilution) were used. Nuclei were stained with DAPI (1: 1000 dilution).

The stained samples were photographed and analyzed using a confocal laser-scanning microscope Carl Zeiss LSM700, and the above procedure was illustrated in FIG. 1A.

As a result, as shown in Fig. 1B to Fig. 1D, the induction neural stem cells showed a small size and a uniform shape in the neural stem cell cross-differentiation process in both the low-molecular inhibitor-treated group and the untreated group (Fig. 1B). However, as shown in Fig. 1C, mRNA expression of Nestin, Sox1, Pax6, Musashi-1, Vimentin, Olig2, Nkx2.1, FoxG1, Tuj1 and Ascl1, which are molecular markers of neural stem cells, But the expression of neural stem cell markers was significantly increased in the group treated with the low molecular inhibitor than in the untreated group (Fig. 1C). In addition, induction neural stem cells were quantified by flow cytometry analysis of neural cell adhesion molecule (NCAM) using a fluorescence flow cytometer (FACS Caliber) (FIG. 1D).

As shown in FIG. 2, detection of inducible neural stem cells expressing both Nestin and Sox2 was confirmed by fluorescence immunocytochemistry (FIG. 2A), and real-time gene amplification analysis was performed to follow the neural stem cell crossing differentiation process (Fig. 2B). The expression of the neuronal stem cells and early neuronal cell markers (Sox1, Sox2, Nestin, Musashi-1, FoxG1, Nkx2.1, Pax6, Gli3, Vimentin, Tuj1 and Emx1)

< Experimental Example  2> Confirmation of establishment of differentiation method from mature neurons in adipose-derived stem cells

Based on the method of differentiating the adipocyte-derived mesenchymal stem cells of Experimental Example 1 into neural stem cells, the cells having the characteristics similar to the neural stem cells were heterogeneously present in the three-step differentiation method The following experiments were conducted to differentiate these into mature neurons.

Specifically, 2 x 10 ⁵ adipocyte derived mesenchymal stem cells were placed in a 6 cm ² gelatin-coated dish and cultured in a 37 ° C 5% CO ₂ incubator for one day. The following day, pretreatment with 3% KOSR (knockout serum replacement), 1% Penicillin / Streptomycin, 1% Glutamax, 3 mM D-glucose, 1% non-essential amino acid and 4 ng / ml bFGF was added to the DMEM F- 10 μM SB431542, 0.1 μg / ml Noggin, and 0.5 μM LDN 193289 were added to the pre-induction medium and cultured in a 37 ° C 5% CO ₂ incubator for 6 days. In the second step, to give change to 2% B27 with 1% N2 the neural induction medium (neural induction medium) containing the incubated at 37 ℃ 5% CO ₂ the medium for 5 days. Then, in the last third step, the neural stem cells were replaced with growth medium containing 20 ng / ml bFGF and 20 ng / ml EGF in the neural induction medium for 5 days in a 5% CO ₂ incubator Lt; / RTI &gt; Then, in order to differentiate into mature neurons, the cells with the differentiation of the above three stages are washed with PBS, and 1X TryPLE select is treated with a single cell for 3 to 4 minutes at 37 ° C in an incubator. Then, 1 × 10 ⁶ cells / well in a 35 mm ² dish coated with PLO / FN and 1 × 10 ⁵ cells / well in a 4 well dish containing 12 mm ² cover glass were placed in a 37 ° C. 5% CO ₂ incubator Stabilize in the incubator for one day. The next day, the neural induction medium was replaced with medium containing 1 μM purmorphamine and 10 ng / ml brain-derived neurotrophic factor (BDNF), and the medium was replaced with fresh medium every 3 days in a 5% CO ₂ incubator at 37 ° C. for 12 to 14 days And cultured.

As a result, as shown in FIG. 3, the inducing neurons showed a morphology similar to mature neurons, as opposed to having a bipolar or multipolar neuron from a small cell body (FIG. 3B).

In addition, quantitative analysis using real-time gene amplification analysis revealed that Tuj1 and MAP2, which are molecular markers of neurons, and medial ganglionic eminence (MGE) -related transcription factors (FoxG1, (GAD67) and the sodium ion channel gene (SCN5A), the expression of genes related to mature neurons in the inducible neurons was remarkable. (Fig. 3C).

In addition, as shown in Fig. 4, in the induction neurons, neuronal progenitor cell markers (TuJ1, Pax6), mature neuron cell markers (NeuN / MAP2), astrocytic molecular markers (GFAP / S100) (Fig. 4A), and it was confirmed that the number of inducing neurons alone or commonly expressing such a molecular marker was quantitatively confirmed (Fig. 4B). In addition, FIG. 4c shows electrophysiological recording samples measured from inducible neurons having a typical nerve cell morphology, and shows the induction of action potential by sample image and current injection. The current injection protocol is shown below the action potential record, and the bottom record is a representative spontaneous synaptic activity obtained from inducing neurons in a voltage-clamped manner (-60 mV fixed). The magnified single current is shown below the recording in succession (FIG. 4C).

< Experimental Example  3> In adipose-derived stem cells Gabba Castle  Confirmation of the establishment of the method of differentiation into neurons

Likewise, a method of differentiating adipocyte-derived mesenchymal stem cells into pluripotent neural cells based on the three-stage differentiation method established in Experimental Example 1 was established. In order to increase the number of days to be cultured in the nerve matured medium, the number of days of neural stem cell crossing differentiation was shortened and then neural cell induction medium composed of purmorphamine and BDNF and neural cell maturation medium composed of dbcAMP and BDNF were used.

Specifically, 2 x 10 ⁵ cells of adipocyte-derived mesenchymal stem cells were placed in a 6 cm ² gelatin-coated dish and cultured in a 37 ° C 5% CO ₂ incubator for one day. On the next day, pre-induction medium of DMEM F-12 basic medium, 3% KOSR, 1% Penicillin / Streptomycin, 1% Glutamax, 3 mM D- glucose, 1% nonessential amino acid and 4 ng / ml bFGF, , 10 μM SB431542, 0.1 μg / ml Noggin, and 0.5 μM LDN193289 were added to the medium and cultured in a 37 ° C. 5% CO ₂ incubator for 6 days. After the culture of step 1 was completed, the cells were washed with PBS and treated with 1X TryPLE select for 3 to 4 minutes at 37 ° C in a single cell, and the cells were plunged into PLO / FN coated 35 mm ² dishes at 1 × 10 ⁶ cells In 1 × 10 ⁵ cells / well in a 4-well dish containing 12 mm ² cover glass, and stabilized in the incubator for one day at 37 ° C in a 5% CO ₂ incubator. Change to neural induction medium containing the second step of 2% B27 and 1% N2 and incubate for 5 days at 37 ° C in a 5% CO ₂ incubator. Then, in order to differentiate into neural progenitor cells, the neural induction medium, neuron differentiation condition, was changed to medium containing 1 μM purmorphamine and 10 ng / ml BDNF, and cultured for 10-14 days at 37 ° C. in a 5% CO ₂ incubator Change to new medium once every three days for same culture. After 10 to 14 days of differentiation into neurons, 50 μM dbcAMP and 20 ng / ml BDNF were added to the neural induction medium to induce further differentiation into Caucasian neurons and differentiation was added for 13-20 days.

As a result, as shown in FIG. 5, the inducible callus neurons exhibited numerous radial neurites extending from the neurite-like population, and many of the inducible callus neurons exhibited the medial ganglion elevation (MGE) NKX2.1, DLX2, LHX6 and TuJ1, MAP2, which are neuronal cell markers (FIG. 5B), and quantitatively confirmed the ratio of the number of inducible neural cells expressing these molecular markers alone or in common (Fig. 5C). In addition, the guinea pig neurons exhibited neurites extending from a neuron-like cell population and confirmed to express neurofilament-M (NF-M), a neurofilament molecular marker (Fig. 5D).

As shown in FIG. 6, the real-time gene amplification assay showed that the MAP2, MGE transcription factor (Dlx2, Dlx5), astrocyte (GFAP), calcium binding protein (CALB2), GABA (GABRA1, GABRA2, GABRA5) and GABA (GAD65, GAD67) gene expression were examined. Inducible gibbous neurons with a high number of differentiation days in most in vitro cultured 32 days showed a dramatic increase in the expression of mature gibberellic neurons in the in vitro cultured 25-day-old gibberellic neurons, (Fig. 6A and 6B). &Lt; / RTI &gt; GABA / MAP2, GAD / MAP2, PSD95 / MAP2, and PSD95 / SYP) were expressed by fluorescence immunocytochemistry (FIG. 6C) Which was confirmed quantitatively (Fig. 6D). In addition, as shown in the lower right of FIG. 6E, a spontaneous inhibitory post-synaptic current (IPSC) was observed when glutamic acid receptor blockers (50 μM APV and 20 μM CNQX) were added and treated with a 10 μM bicuculline GABAA receptor blocker (Fig. 6E).

< Experimental Example  4> derived from human bone marrow Intermediate lobe  As a stem cell that secretes a large amount of growth factors from stem cells, mesenchymal  stem cell; gfMSC)

The present inventors have found that human bone marrow-derived mesenchymal stem cells (hMSCs) purchased from Cambrex (USA) can be used as growth hormone releasing human mesenchymal stem cells (gfMSCs) Lt; / RTI &gt;

Specifically, the undifferentiated human bone marrow-derived MSCs (Untreated hMSC; uhMSC) is DMEM- low glucose medium, 10% FBS and 1% penicillin - 37 ℃ with the primary growth medium consisting of a mixture of streptomycin, 5% CO ₂ In a cell incubator. After incubation for 2 days in a cell incubator, the primary growth medium was cultured in primary differentiation medium (DMEM-low glucose medium, 10% FBS, 1% penicillin-streptomycin mixture, 1 mM? -Mercaptoethanol) (DMEM-low glucose medium, 10% FBS, 1% penicillin-streptomycin mixture, 0.28 g / ml) for 24 hours after washing the mesenchymal stem cells with PBS (phosphate buffer solution) After 3 days, the mesenchymal stem cells were washed with PBS and cultured in a third differentiation medium (DMEM-low glucose medium, 10% FBS, 1% penicillin- Human fibroblast growth factor, 5 ng / ml human platelet derived growth factor-AA, 200 ng / ml human fibroblast growth factor, ml herferiulin 1-beta1], and cultured for 8 days. At this time, The cell viability during the differentiation induction process was confirmed by trypan blue staining, and cells not in trypan blue staining (cells not in trypan blue staining Living cells).

As a result, as shown in FIG. 7A, uhMSC was observed to change into an elongated bipolar shape that secretes a large amount of growth factors together with morphological changes over time through induction of cell differentiation. Especially, it was changed into human stem cell (gfMSC) which secretes a large amount of growth factor up to 54% at the eighth day of differentiation and 80% or more at the day 12 differentiation, and it was observed that most of the cells were alive during the differentiation induction process. Approximately 50% of the stem cells cultured in the normal growth medium for 3 days after induction of differentiation maintained the shape of human stem cells secreting growth factors.

< Experimental Example  5> Confirmation of growth factor secretion of differentiated growth factor-rich human stem cells

<5-1> Analysis of Growth Factor Secretion of Differentiated Growth Factor Mass Secreted Human Stem Cells

The present inventors analyzed human growth factors secreted in gfMSC cells differentiated in Example 4 as follows.

Specifically, to prepare the conditioned medium (CdM), uhMSC and gfMSC were washed 4 times with PBS and cultured in serum-free Neurobasal medium (Gibco-BRL). After 18 hours of culture, each medium was collected and centrifuged at 1500 g for 5 minutes to remove cell debris. The supernatants (uhMSC-CdM and gfMSC-CdM) of each cell culture obtained after centrifugation were analyzed according to the manufacturer's instructions using a human growth factor array (RayBiotech). At this time, the medium used for the cell culture was also measured as a negative control. Briefly, the human growth factor array membrane was blocked with blocking buffer and 1 ml of conditioned medium was incubated overnight at 4 ° C. The membrane was then washed 3 times with Washing Buffer I and 2 times with Washing Buffer II for 5 minutes each at room temperature And washed. Then, 1 ml of diluted biotin-conjugated antibody was added, followed by incubation at room temperature for 2 hours. The membrane was washed again and finally incubated with diluted HRP-conjugated streptavidin for 2 hours at room temperature. The membrane was detected using a Lumino image analyzer, LAS-3000 (Fujifilm), and the intensity of the signal was quantitated with a density meter (Bio-Rad). Relative expression levels of each human growth factor were analyzed by comparing the staining intensity of each slot to the staining intensity of positive control (POS) in the same assay. The results of the above analysis are shown in FIG. 10B. The ratio (%) of the growth factor of uhMSC-CdM or gfMSC-CdM to the positive control is shown in FIG. 10C and the amount of growth factor (multiplication) of uhMSC-CdM or gfMSC- , And the experiment was independently repeated at least 3 times and expressed as mean ± standard error.

As a result, as shown in FIGS. 10A to 10C, it was confirmed that hGFhepatocyte growth factor, vascular endothelial growth factor (IGFBP-1), insulin-like growth factor binding protein-1 (IGFBP- -2, IGFBP-4, IGFBP-6 and SCF (stem cell factor) were more highly expressed.

&Lt; 5-2 > Differentiated Growth Factor Mass Secreted Human Stem Cells HGF  And VEGF  Yield analysis

The present inventors confirmed the difference in the amount of growth factors secreted by uhMSC and gfMSC in Example 5-1 through an enzyme-linked immunosorbent assay.

(Mouse anti-human VEGF antibody (MAB293), R & D Systems) or anti-HGF antibody (mouse anti-human HGF (MAB294), R & D Systems) or anti-VEGF antibody ELISA was performed using an ELISA kit for HGF or VEGF (RayBiotech). The experiments were repeated at least 3 times independently and expressed as mean ± standard error.

As a result, it was confirmed that the amounts of HGF and VEGF produced in gfMSC increased about 10 times and about 11 times, respectively, as compared to uhMSC, as shown in Figs. 10D and 10E.

<5-3> Differentiated Growth Factor Mass Secreted by Human Stem Cell Medium HGF  And VEGF  Yield analysis

The present inventors cultured the gfMSCs for 0, 1, 3 and 5 days in the induction differentiation medium (SCIM) and the normal growth medium (GM), respectively, in order to confirm how long the production of HGF and VEGF is maintained after induction of differentiation ELISA analysis was carried out in the same manner as in Example 5-2.

As a result, as shown in FIG. 11F and FIG. 11G, the secretion amount of HGF and VEGF gradually decreased with time, but it was confirmed that the secretion amount was still maintained higher than uhMSC (Control) even on the fifth day. We confirmed that the secretion volume of the induced differentiation culture was higher.

<5-4> Differentiated Growth Factor Mass Secretory Human Stem Cells HGF  And VEGF mRNA  Expression level analysis

The present inventors confirmed quantitative RT-PCR as to whether the expression of growth factors according to the differentiation of uhMSC identified in Examples 5-2 and 5-3 was controlled from the mRNA stage. Specifically, the sense primer (5'-atgctcatggaccctggt-3 ') shown in SEQ ID NO: 53 and SEQ ID NO: 54 in the group in which water, uhMSC, gfMSC or gfMSC were cultured in SCIM or GM for 1, (5'-gcctggccaagcttcatta-3 ') and a primer pair described in SEQ ID NO: 55 (5'-gccttgctgctctacctcca-3') and SEQ ID NO: 56 (5'-caaggcccacagggatttt- ) Using a primer pair for VEGF of the antisense primer described in Example 4-2.

As a result, when compared with uhMSC as shown in Figs. 11h to 11j, it was confirmed that the expression level of HGF and VEGF mRNA of gfMSC was maintained high until 5 days regardless of the kind of medium.

< Experimental Example  6> Effects of Differentiated Growth Factor Mass Secretory Human Stem Cells on Neurons

The present inventors carried out the following experiment in order to examine the effect of gfMSC, which is hypersecretive of differentiated growth factors from uhMSC, on neurons.

<6-1> Identification of growth of neurite outgrowth by differentiated growth factor-rich secretory human stem cells

Neuro2A cells (ATCC # CCL-131), a neuroblastoma, were cultured overnight in growth media on a 6-well plate coated with 2 μg / ml fibronectin, with 1.3 × 10 ⁵ cells per well. And Neuro2A cells were cultured with Neurobasal medium (0.5% FBS) with or without anti-HGF antibody or anti-VEGF antibody. Twenty-four hours before Neuro2A cells were plated, uhMSC and gfMSC were plated at a cell density of 5 × 10 ⁴ cells / cell in a 1.0-μm diameter cell culture insert and cultured in each medium. After 48 hours, the culture insert was washed 3 times with PBS and then transferred to a 6-well plate containing Neuro2A cells (Fig. 12A). In the case of negative control cells without cells, only the culture insert containing the Neurobasal strain containing 0.5% FBS was incubated under the same conditions as the experimental group. After uhMSC or gfMSC transferred to a 6-well plate containing Neuro2A cells was incubated for 48 hours, Neuro2A cells were analyzed using a phase contrast microscope. In addition, as a result of the analysis, the ratio of the cells having at least one neuron having a length of at least one cell diameter and the total number of neuron projections per cell in Neuro2A cells are shown in FIGS. 12C and 12D, respectively.

As a result, as shown in FIGS. 12B to 12D, the length of neurite was longer in Neuro2A cells co-cultured with gfMSC as compared to uhMSC, Lt; RTI ID = 0.0 &gt; VEGF &lt; / RTI &gt;

<6-2> Identification of growth and survival of nerve cells by differentiated growth factor-rich secretory human stem cells

The effect of gfMSC on neuronal growth and survival under the same conditions as in Example 6-1 was confirmed by trypan blue staining. The average number of Neuro2A cells and the proportion of killed Neuro2A cells in each group are shown in FIGS. 12E and 12F, respectively. Cell death was expressed as a percentage of cells stained with trypan blue staining (apoptotic cells). At least 700 cells in each group were selected from arbitrary regions and the experiment was independently repeated at least 3 times and expressed as mean ± standard error.

As a result, as shown in FIGS. 12E and 12F, the growth and cell death reduction of Neuro2A cells co-cultured with gfMSC were confirmed when compared with uhMSC, and the above effect was confirmed by HGF and / or VEGF &Lt; / RTI &gt;

< Experimental Example  7> Effect of Differentiated Growth Factor Mass Secretory Human Stem Cells on Damaged Spinal Cord Tissue

<7-1> Identification of neurite outgrowth of damaged spinal cord tissue by differentiated growth factor-rich secretory human stem cells

In order to confirm whether the effect of increasing neurite outgrowth of gfMSC in ex vivo, as confirmed in Examples 3-1 and 6-2 described above, the present inventors conducted the following experiment Respectively.

Specifically, spinal cord tissue sections were anesthetized with avertin at 16 days of Sprague-Dawley rats and lumbar spinal cord of rats was collected under sterile conditions. The nerve root and connective tissues were removed from the spinal cord using a Hank's balanced salt solution (Gibco-BRL) containing 6.4 mg / ml of cold glucose. The spinal cord was removed using a McIlwain tissue chopper (Mickle Laboratory Engineering) And then dehydrated and chopped neuronal tissue sections (LPC) were prepared. (Gibco-BRL), 25% Hank's balanced salt solution, 25% horse serum (Gibco-BRL), and 50% Eagle's minimum essential medium (Millipore) The membrane insert was placed in a 6-well plate containing 1 ml of medium containing 6.4 mg / ml glucose and 20 mM HEPES (Sigma-Aldrich). The tissue sections were cultured in a 5% CO ₂ incubator at 37 ° C, and the culture medium was changed twice a week. At 7 days of culture, spinal cord tissue sections were treated with 0.5 mg / ml lysophosphatidyl choline, lysolecithin for 17 hours. And the anti-HGF antibody or anti-VEGF antibody was replaced with fresh or untreated fresh medium. To transplant mesenchymal stem cells, uhMSC or gfMSC was centrifuged at 2,000 g for 3 min after trypsin treatment, and the precipitated cells were suspended in PBS, and the cells (3 x 10 ⁴ cells per 1.5 μl) Each cell was directly implanted into the ventral portion of each spinal cord tissue section using an aspirator tube assembly with a capillary pipette. Spinal cord tissue sections were fixed with 4% paraformaldehyde overnight at 4 ° C, and PBS containing 0.5% Triton X-100, 1% bovine serum albumin (BSA) was incubated at room temperature for 10 days Min to make it permeable. Tissue sections were incubated for 1 hour in PBS containing 0.1% Triton X-100 and 3% bovine serum albumin (BSA) and blocked with rabbit anti-neurofilament (NF) -M (1: 500, (1: 500, transplanted human mesenchymal stem cell stain, Chemicon) at 4 ° C overnight, and stained them. . The secondary antibody against anti-NF-M antibody was Alexa 546 antibody (red), and the secondary antibody against anti-human nuclear antibody was Alexa 488 antibody (green). z-Stack images were obtained using a laser scanning confocal microscope at 3-4 μm intervals. Neurofilament-M immunofluorescence was observed on a z-Stack laser scanning confocal microscope and quantified by morphometric image analysis (IOD) using Image J software (National Institutes of Health). The microscopic observation results are shown in FIG. 6A, and the relative integrated optical density (IOD) of the NF-M stained nerve fibers in the spinal cord tissue sections is shown in FIG. 6A. In Fig. 6A, the spinal cord tissue section (SC) and the implanted mesenchymal stem cells were marked with a borderline. The integrated optical density was normalized by a control spinal cord tissue section, and the bar graph represents the mean ± SE of at least 7 tissue sections.

As a result, as shown in Figs. 13A and 13B, when the uhMSC was transplanted to the LPC and the gfMSC were transplanted 3.5 times and 5.5 times, respectively, as compared with the case where only the LPC was transplanted, Induced LPC neurite outgrowth was significantly decreased when the antibody to HGF and / or VEGF was treated.

&Lt; 7-2 > Confirmation of inhibition of apoptosis of injured spinal cord tissue by differentiated growth factor-rich secretory human stem cells

The present inventors performed the following experiments to confirm that the cell death-suppressing effect of gfMSC confirmed in Examples 6-1 and 6-2 is the same in ex vivo.

Specifically, dehydrated chitosan sections (LPC) were prepared in the same manner as in Example 7-1. The four sections were carefully placed in a single membrane insert and incubated in 50% Eagle's minimum essential medium (Gibco-BRL), 25% Hank's balanced salt solution, 25% horse serum (Gibco-BRL), 6.4 mg / The membrane insert was placed in a 6-well plate containing 1 ml of medium containing mM HEPES (Sigma-Aldrich). The tissue sections were cultured in a 5% CO ₂ incubator at 37 ° C, and the culture medium was changed twice a week. On day 7 of culture, spinal cord tissue sections were treated with 0.5 mg / ml lysolecithin for 17 hours. And the anti-HGF antibody or anti-VEGF antibody was replaced with fresh or untreated fresh medium. To transplant mesenchymal stem cells, uhMSC or gfMSC was centrifuged at 2,000 g for 3 min after trypsin treatment, and the precipitated cells were suspended in PBS, and the cells (3 x 10 ⁴ cells per 1.5 μl) Each cell was directly implanted into the ventral portion of each spinal cord tissue section using an aspirator tube assembly with a capillary pipette. In each group, cell death mediated by lysolecithin was stained (red) with TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) using the In Situ Cell Death Detection Kit (Roche Diagnostics) Respectively. At this time, nuclei were observed with DAPI staining (blue). The above results were observed by a microscope and the number of TUNEL-positive cells in the lower part of the spinal cord tissue section was measured from the microscope photograph of the 100-fold magnification (arrowhead: TUNEL-positive cells) The degree of death is shown in a graph D in FIG. 6B. The bar graph represents the mean ± standard error of at least 5 tissue sections.

As a result, as shown in FIG. 14C and FIG. 14D, when compared with the case of transplanting uhMSC into LPC, it was confirmed that the cell death of the spinal cord tissue slice increased by lyso lecithin treatment was further reduced in the case of transplanting gfMSC . However, the inhibitory effect of the gfMSC on the cell death of the spinal cord tissue sections did not show any significant difference in antibody treatment to HGF and / or VEGF.

< Experimental Example  8> HGF  And VEGF  Effects on nerve cells and damaged spinal cord tissues

The inventors of the present invention found that the increase in neurite outgrowth and cell growth of gfMSC and the inhibition of neurite outgrowth and apoptosis of injured spinal cord tissue sections of gfMSC as described in Examples 6-1 to 7-2 were due to the growth factors secreted by gfMSC The following experiment was performed to confirm.

<8-1> HGF  And To VEGF  Of neurite outgrowth

We used a phase contrast microscope in Neuro2A cells cultured for 48 hours with 0, 12.5, 25, 50 and 100 ng / ml of recombinant HGF or VEGF protein (R & D Systems) The ratio of the cells having neurites and the total number of neurites per cell are shown in FIGS. 13A and 13B, respectively.

As a result, neurite length growth was highest in Neuro2A cells treated with 100 ng / ml HGF and 50 ng / ml VEGF as shown in Figs. 15A and 15B.

<8-2> HGF  And To VEGF  Of neuronal cell growth and survival

The present inventors stained the Neuro2A cells, which were cultured for 48 hours by adding 0, 12.5, 25, 50 and 100 ng / ml of recombinant HGF and / or VEGF protein (R & D Systems) Cell growth and viability. The total number of cells and the percentage of living cells are shown in Figures 13c and 13d, respectively. Cell viability was measured as percentage of cells (live cells) that were not stained with trypan blue staining. The experiments were repeated at least 3 times independently and expressed as mean ± standard error.

As a result, it was confirmed that the exogenous HGF and / or VEGF protein increased neurite outgrowth and cell growth of Neuro2A cells as shown in Figs. 15C and 15D.

<8-3> HGF  And To VEGF  Of increased neurite outgrowth in injured spinal cord tissue

The nerve fibers of the spinal cord tissue sections were stained with NF-M antibody and Alexa 546 antibody (red) in the same manner as in Example 7-1. The spinal cord tissue sections on the 7th day of culture were treated with 0.5 mg / ml lysolecithin for 17 hours and then treated with the recombinant HGF and / or VEGF protein at 0, 12.5, 25, 50 and 100 ng / And cultured in medium for 1 week. The relative relative optical density (IOD) values of the NF-M immunofluorescent stained nerve fibers in the spinal cord tissue sections were then measured in the same manner as in Example 7-1, and the integrated optical density was normalized to the control spinal cord tissue sections . The concentration unit (ng / ml) of the graph abscissa refers to the mixed concentration of HGF alone, VEGF alone, or HGF + VEGF. The bar graph represents the mean ± standard error of at least 5 tissue sections.

As a result, it was confirmed that exogenous HGF and / or VEGF protein increased neurite outgrowth of injured spinal cord tissue sections as shown in Figs. 16E and 16F.

Claims (19)

At least one selected from the group consisting of neural stem cells, neuronal cells, and neural neural cells; And
A composition for cell regeneration comprising growth factor hypersecretion cells.
The composition for cell regeneration according to claim 1, wherein the neural stem cells, neurons, GABA neurons, and growth factor hypersecretory cells are cells derived from mesenchymal stem cells.
The composition for cell regeneration according to claim 1, wherein the neural stem cells, neurons, and neural cells are differentiated from adipose tissue-derived mesenchymal stem cells.
The composition for cell regeneration according to claim 1, wherein the growth factor-secreting cells are differentiated from bone marrow-derived mesenchymal stem cells.
The composition for cell regeneration according to claim 1, wherein the composition for cell regeneration further comprises a cell expressing a marker expressed in at least one cell selected from the group consisting of astrocytes and rare prominent glue cells.
The composition for cell regeneration according to claim 1, wherein the neural stem cells are symmetrically dividing cells.
[3] The method according to claim 1, wherein the neural stem cells are at least one selected from the group consisting of Nestin, Sox2, Soxl, Musashi-1, FoxGl, Nkx2.1, Pax6, Gli3, Vimentin, Tujl, Emxl, / RTI &gt;
The neuronal cell of claim 1, wherein the neuron expresses at least one selected from the group consisting of Tuj1, MAP2, FoxG1, Nkx2.1, Pax6, Dlx2, Dlx5, Lhx6, GAD67, SCN5A, GFAP, S100, Olig2, A composition for cell regeneration.
The method according to claim 1, wherein the GABA is selected from the group consisting of NKX2.1, LHX6, TuJ1, MAP2, MGE, Dlx2, Dlx5, GABRA1, GABRA2, GABRA5, CALB2, GAD65, GAD67, PSD95, NF-M and SYP And expressing at least one selected.
The composition of claim 1, wherein the Gabbee neuron has a radial neurite.
The composition for cell regeneration according to claim 1, wherein the Gabbee neuron exhibits a spontaneous inhibitory post-synaptic current upon treatment with a glutamate receptor blocker.
The composition for cell regeneration according to claim 1, wherein the growth factor-secreting cell expresses at least one selected from the group consisting of GFAP, Po, S100, CNPase and p75NTR.
The method of claim 1, wherein the growth factor is selected from the group consisting of HGF (hepatocyte growth factor), vascular endothelial growth factor (VEGF), insulin-like growth factor binding protein-1 (IGFBP-1), IGFBP-2, IGFBP- And at least one selected from the group consisting of SCF (stem cell factor, VEGF, and HGF).
The composition for cell regeneration according to claim 1, wherein the growth factor is at least one selected from the group consisting of VEGF and HGF.
The cell regeneration composition according to claim 1, wherein the composition for cell regeneration is selected from the group consisting of Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin 3 (NT-3), Neurotrophin 4/5 (NT- Insulin Like Growth Factor 2 (IGF 2), Insulin Like Growth Factor 2 (IGF 2), Basic FGF, FGF-5, Epidermal Growth Factor (EGF), Hepatocyte Growth Factor Wherein the cell further comprises at least one selected from the group consisting of Glia cell line-derived neurotrophic factor (GDNF), TGF-b2, TGF-b3, Interleukin 6 (IL-6), Ciliary Neurotrophic Factor Composition for regeneration.
A pharmaceutical composition for improving or treating an ischemic disease comprising the composition for cell regeneration according to claim 1.
A pharmaceutical composition for improving or treating neurological diseases comprising the composition for cell regeneration according to claim 1.
[Claim 16] The method of claim 16, wherein the ischemic disease is selected from the group consisting of trauma, rejection upon transplantation, ischemic cerebrovascular disorder, ischemic renal disease, ischemic lung disease, ischemic disease associated with infectious disease, ischemic disease of limb and ischemic heart disease A pharmaceutical composition for prevention or treatment of ischemic diseases, which is any one.
18. The method of claim 17, wherein the neurological disease is selected from the group consisting of stroke, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, traumatic central nervous system disease central nervous system diseases, and spinal cord injury disease.

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