Pharmaceutical composition for preventing or treating nervous system diseases comprising high-efficiency stem cells expressing TIMP-1, MCP-1, GROalpha and IL-6, and method for selecting high-efficiency stem cells

Abstract

The present invention relates to a method for selecting an efficient stem cell for treating a nervous system disease using TIMP-1, MCP-1, GROalpha, and IL-6, and a pharmaceutical composition for preventing or treating a nervous system disease. According to the present invention, a specific combination of markers comprising TIMP-1, MCP-1, GROalpha and IL-6 can be used as a biomarker for selecting stem cells having excellent therapeutic (ameliorating) potential in the treatment of nervous system diseases. In addition, the use of stem cells exhibiting increased expression or activity of TIMP-1, MCP-1, GROalpha, and IL-6 factors or the use of substances that modulate (particularly up-regulate) these factors in stem cells has a significant therapeutic effect on nervous system diseases due to high efficacy (potency) and efficiency. In addition, the invention provides higher treatment potential of allogeneic stem cell transplantation.

Description

Technical Field

[0001] The present invention relates to a method for selecting highly efficient stem cells for preventing or treating nervous system diseases by using TIMP-1, MCP-1, GROα and IL-6, and a pharmaceutical composition for preventing or treating nervous system diseases comprising the highly efficient stem cells.

[0002] The present invention claims priority based on Korean Patent Application No. 10-2022-0107959 filed on August 26, 2022, and all contents disclosed in the specification and drawings of this application are incorporated herein by reference. Background Art

[0003] It is known that neurological diseases occur due to the loss of neurons in the central nervous system or peripheral nervous system, and are currently considered to be diseases for which no specific treatment has been developed. A representative example of a neurological disease is a stroke, which is defined as an interruption of blood flow to brain tissue. This occurs when a specific area of ​​the brain is damaged or the pumping function of the heart is completely impaired. Specifically, a stroke occurs when blood flow to a specific area of ​​the brain is blocked or interrupted due to blockage or rupture of a blood vessel leading to the brain. Specifically, this occurs when an embolism or thrombus forms in an artery supplying blood to the brain, resulting in a blood vessel obstruction, a condition known as an ischemic stroke. Stroke is the third leading cause of death in developed countries. In the United States, the cost of treatment and productivity loss caused by stroke-related deaths is estimated to be approximately $40 trillion. When an ischemic stroke occurs, brain tissue necrosis and cerebral infarction occur. Within 3 to 6 hours after onset, treatment methods such as thrombolysis (including drug administration and rehabilitation) are used to promote the preservation and recovery of brain function. However, thrombolysis carries the risk of increased cerebral hemorrhage, and because it must be performed within hours of a stroke, many patients are unable to receive thrombolysis. These stroke treatments are limited to emergency care or simply stopping the disease from progressing, with no fundamental treatment available to regenerate already damaged brain tissue.

[0004] To overcome these limitations of modern medicine, recent efforts have focused on the use of stem cells that have a regenerative effect on the nervous system for treatment. Stem cells are undifferentiated cells that can differentiate into various types of body tissues. Under appropriate conditions, they have the ability to differentiate into different tissue cells, making them suitable for treatments such as regenerating damaged tissues. This stem cell therapy is expected to regenerate damaged neurons. In addition to direct regeneration, these stem cells are also known to secrete various substances that improve the damaged environment and contribute to the regeneration process. Although stem cell-related technologies are developing rapidly and people have high hopes for their therapeutic effects on nervous system diseases, clinical results using mesenchymal stem cells to treat stroke and other degenerative nervous system diseases have reported limited efficacy in practice. In addition, although stem cell-based therapies are being tried in various disease areas, they have not shown significant efficacy in practice. Therefore, extensive research is being conducted to identify highly effective stem cells. In addition, clinical studies of fetal-derived neural stem cells for patients with stroke and spinal cord injury are underway. However, serious technical problems (such as tumor formation due to incomplete differentiation) and ethical issues have arisen. Summary of the invention [Technical issues]

[0005] Therefore, the inventors conducted research to find a more effective and efficient strategy for using stem cells to treat nervous system diseases. In the process, they first determined the key role of TIMP-1, MCP-1, GROα and IL-6 in the therapeutic activity of stem cells on nervous system diseases, thereby completing the present invention.

[0006] Therefore, the object of the present invention is to provide a method for selecting highly efficient stem cells for treating nervous system diseases, which comprises the step of measuring the expression levels of TIMP-1 (TIMP metallopeptidase inhibitor 1), MCP-1 (monocyte chemoattractant protein-1), GROα (growth regulated oncogene α) and IL-6 (interleukin 6) proteins or their genes.

[0007] Another object of the present invention is to provide a method for in vitro determination of the activity of stem cells for treating nervous system diseases, which comprises measuring and comparing the expression levels of TIMP-1, MCP-1, GROα, IL-6 proteins or genes in stem cells.

[0008] Another object of the present invention is to provide a marker composition for selecting efficient stem cells for treating nervous system diseases, which comprises TIMP-1, MCP-1, GROα, IL-6 protein or its gene.

[0009] Another object of the present invention is to provide a composition for selecting efficient stem cells for treating nervous system diseases, which includes a substance for determining the expression level of TIMP-1, MCP-1, GROα, IL-6 protein or its gene. In addition, the present invention also provides a kit for selecting efficient stem cells for treating nervous system diseases, which includes the above composition.

[0010] Another object of the present invention is to provide a method for screening a preventive or therapeutic agent for a nervous system disease, comprising: (a) contacting the isolated stem cells with a test substance; (b) measuring the expression levels of TIMP-1, MCP-1, GROα and IL-6 proteins or their genes in the stem cells contacted with the test substance and a control group of stem cells not contacted with the test substance; and (c) Selecting a test substance that increases the expression or activity level of TIMP-1, MCP-1, GROα and IL-6 protein or its gene as compared to cells in a control group.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating nervous system diseases, which comprises stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof; or a culture thereof as an active ingredient.

[0012] Another object of the present invention is to provide a composition for promoting the activity of stem cells for preventing or treating nervous system diseases, which comprises a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as an active ingredient.

[0013] Another object of the present invention is to provide a method for promoting the activity of stem cells for preventing or treating nervous system diseases, which comprises the step of treating isolated stem cells with an agent that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins.

[0014] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating nervous system diseases, which comprises isolated stem cells; and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as active ingredients, as well as a pharmaceutical composition for preventing or treating nervous system diseases comprising the above composition.

[0015] Another object of the present invention is to provide a pharmaceutical composition for inducing angiogenesis, which comprises stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof; or a culture thereof as active ingredients.

[0016] Another object of the present invention is to provide a pharmaceutical composition for inducing angiogenesis, which comprises isolated stem cells; and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as active ingredients, and a pharmaceutical composition for inducing angiogenesis comprising the above composition.

[0017] However, the technical problems to be solved by the present disclosure are not limited to the above problems, and other problems not mentioned can also be clearly understood by technicians in the field to which the present disclosure belongs from the following description. [Technical solution]

[0018] To achieve the above objectives, the present invention provides a method for selecting highly efficient stem cells for treating nervous system diseases, which comprises the steps of measuring the expression levels of TIMP-1 (TIMP metallopeptidase inhibitor 1), MCP-1 (monocyte chemoattractant protein-1), GROα (growth regulated oncogene α) and IL-6 (interleukin 6) proteins or their genes.

[0019] In one embodiment of the present invention, TIMP-1, MCP-1, GROα and IL-6 proteins or genes thereof may be highly expressed in highly efficient stem cells for treating nervous system diseases, but is not limited thereto.

[0020] In one embodiment of the present invention, the stem cells can be selected from the group consisting of mesenchymal stem cells, adult stem cells, dedifferentiated stem cells, embryonic stem cells, induced pluripotent stem cells, neural stem cells, tissue-derived stem cells, and any combination of two or more thereof, but are not limited thereto.

[0021] In one embodiment of the present invention, the nervous system disease may be one or more selected from the group consisting of ischemic brain disease, degenerative nervous system disease, nerve injury disease, peripheral nerve disease and traumatic brain injury, but is not limited thereto.

[0022] In one embodiment of the present invention, the ischemic brain disease can be one or more selected from the group consisting of stroke, cerebral infarction, cerebral ischemia, cerebral embolism, cerebral thrombosis, transient ischemic attack (TIA), head trauma, cerebral circulation and metabolic disorders, cerebral coma, cerebral hemorrhage, neonatal intraventricular hemorrhage (IVH), cerebral arteriosclerosis, hypoxic brain damage and neonatal hypoxic-ischemic brain damage, but is not limited thereto.

[0023] In one embodiment of the present invention, the degenerative neurological disease can be one or more selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, striatonigral degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, cortical basal ganglia degeneration, diffuse Lewy body disease, Guam Parkinson-ALS-dementia syndrome and Pick's disease, but is not limited thereto.

[0024] In one embodiment of the present invention, the neurological injury disease may be one or more selected from the group consisting of epilepsy, spinal cord injury disease, behavioral disorder, developmental disorder, intellectual disability, Down syndrome and schizophrenia, but is not limited thereto.

[0025] In one embodiment of the present invention, a method for selecting highly efficient stem cells for treating nervous system diseases may include the following steps, but is not limited thereto: (i) culturing stem cells; (ii) measuring the concentrations of TIMP-1, MCP-1, GROα and IL-6 proteins in the culture of stem cells of step (i); and (iii) If TIMP-1, MCP-1, GROα and IL-6 proteins are highly expressed in the culture, the stem cells are identified and classified as highly potent stem cells for the treatment of nervous system diseases.

[0026] In one embodiment of the present invention, step (iii) is: wherein step (ii) includes measuring the culture, if the concentration of TIMP-1 protein is 6,000 pg / mL or higher, the concentration of MCP-1 protein is 6,000 pg / mL or higher, the concentration of GROα protein is 500 pg / mL or higher, and the concentration of IL-6 protein is 50 pg / mL or higher, then the stem cells are determined to be high-efficiency stem cells, but are not limited thereto.

[0027] To achieve another object of the present invention, the present invention provides a method for determining the activity of stem cells for treating nervous system diseases in vitro, wherein the method includes the steps of measuring and comparing the expression levels of TIMP-1, MCP-1, GROα and IL-6 proteins or their genes in stem cells.

[0028] To achieve another object of the present invention, the present invention provides a marker composition for selecting efficient stem cells for treating nervous system diseases, which comprises TIMP-1, MCP-1, GROα and IL-6 proteins or genes thereof.

[0029] In order to achieve another object of the present invention, the present invention provides a composition for selecting efficient stem cells for treating nervous system diseases, which comprises substances for measuring the expression levels of TIMP-1, MCP-1, GROα and IL-6 proteins or their genes; and a kit for selecting efficient stem cells for treating nervous system diseases, which comprises the composition.

[0030] In addition, the present invention also provides a substance for measuring the expression level of TIMP-1, MCP-1, GROα, IL-6 protein or its gene, or a composition containing the substance as an active ingredient in selecting efficient stem cells for treating nervous system diseases.

[0031] In addition, the present invention also provides the use of a substance for measuring the expression level of TIMP-1, MCP-1, GROα, IL-6 protein or its gene, or a composition containing the substance as an active ingredient in the preparation of a preparation for selecting efficient stem cells for treating nervous system diseases.

[0032] In one embodiment of the present invention, the substance for measuring the expression level of a protein may be a specific antibody against the protein, but is not limited thereto.

[0033] In one embodiment of the present invention, the measurement of the expression level of the gene can be performed by a substance for measuring the mRNA expression level of the gene, but is not limited thereto. At this time, the substance for measuring the mRNA expression level is a probe or primer set that specifically binds to the mRNA, but is not limited thereto.

[0034] In one embodiment of the present invention, the kit for selecting efficient stem cells for treating nervous system diseases according to the present invention may include instructions describing the above method for selecting efficient stem cells for treating nervous system diseases, but is not limited thereto.

[0035] In order to achieve another object of the present invention, the present invention provides a method for screening a preventive or therapeutic agent for a nervous system disease, comprising: (a) contacting the isolated stem cells with a test substance; (b) measuring the expression levels of TIMP-1, MCP-1, GROα and IL-6 proteins or their genes in the stem cells contacted with the test substance and a control group of stem cells not contacted with the test substance; and (c) Selecting a test substance that increases the expression or activity level of TIMP-1, MCP-1, GROα and IL-6 protein or its gene as compared to cells in a control group.

[0036] In one embodiment of the present invention, the stem cells can be selected from the group consisting of mesenchymal stem cells, adult stem cells, dedifferentiated stem cells, embryonic stem cells, induced pluripotent stem cells, neural stem cells, tissue-derived stem cells, and any combination of two or more thereof, but are not limited thereto.

[0037] In one embodiment of the present invention, the measurement of the protein expression or activity level can be carried out using any one of the methods selected from the group consisting of Western blotting, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation, complement fixation assay, flow cytometry and protein chip method, but is not limited thereto.

[0038] In one embodiment of the present invention, the measurement of the gene expression or activity level can be carried out using a method for measuring mRNA expression or activity level, but is not limited thereto. At this time, the measurement of the mRNA expression or activity level can be carried out using any of the following methods: reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative real-time RT-PCR, in situ hybridization, RNase protection assay (RPA), Northern blotting, Southern blotting, RNA sequencing, DNA chip method and RNA chip method, but is not limited thereto.

[0039] To achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating nervous system diseases, which comprises stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or their culture as active ingredients.

[0040] In addition, the present invention provides a method for preventing, improving and / or treating nervous system diseases, comprising the step of administering stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof; or a culture thereof to a subject in need thereof.

[0041] In addition, the present invention provides a method for preventing, improving and / or treating nervous system diseases, comprising the step of administering to a subject in need thereof a composition containing stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or a culture thereof as an active ingredient.

[0042] In addition, the present invention provides stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or their cultures; or compositions containing these as active ingredients for preventing, improving and / or treating nervous system diseases.

[0043] In addition, the present invention provides the use of stem cells that overexpress TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or their cultures; or compositions containing these as active ingredients for preparing (producing) preparations (formulas) for preventing, improving and / or treating nervous system diseases.

[0044] In one embodiment of the present invention, the stem cells may be selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, neural stem cells, tissue-derived stem cells, and any combination of two or more thereof, but are not limited thereto.

[0045] In one embodiment of the present invention, the culture medium (medium) may be a conditioned medium containing TIMP-1, MCP-1, GROα and IL-6 proteins secreted by stem cells, but is not limited thereto.

[0046] In order to achieve another object of the present disclosure, the present invention provides a substance that increases protein expression or activity, which is one or more expression vectors, the expression vectors comprising a polynucleotide encoding TIMP-1 protein; a polynucleotide encoding MCP-1 protein; a polynucleotide encoding GROα protein; and a polynucleotide encoding IL-6 protein.

[0047] In order to achieve another object of the present disclosure, the present invention provides a method for promoting the activity of stem cells for preventing or treating nervous system diseases, which comprises the step of treating isolated stem cells with a substance (agent) that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins.

[0048] In addition, the present invention also provides an agent for increasing the expression or activity of TIMP-1, MCP-1, GROα, IL-6 genes or their proteins, or a composition containing the agent as an active ingredient for use in promoting the activity of stem cells for preventing or treating nervous system diseases.

[0049] In addition, the present invention also provides a substance that increases the expression or activity of TIMP-1, MCP-1, GROα, and IL-6 genes or their proteins, or a composition containing the substance as an active ingredient for use in preparing a preparation for promoting the activity of stem cells for preventing or treating nervous system diseases.

[0050] In one embodiment of the present invention, the substance that increases the expression or activity of a protein may be one or more expression vectors, the expression vectors comprising a polynucleotide encoding a TIMP-1 protein; a polynucleotide encoding an MCP-1 protein; a polynucleotide encoding a GROα protein; and a polynucleotide encoding an IL-6 protein, but are not limited thereto.

[0051] In one embodiment of the present invention, the activity of the stem cells may be one or more selected from the group consisting of self-renewal, neuroprotective ability (neuroprotection), inhibition of neuronal apoptosis, endothelial cell migration, angiogenesis, anti-inflammatory activity and inhibition of reactive gliosis, but is not limited thereto.

[0052] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating nervous system diseases, which comprises isolated stem cells; and substances that increase the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as active ingredients, as well as a pharmaceutical combination preparation for preventing or treating nervous system diseases comprising the composition.

[0053] In addition, the present invention provides a method for preventing, improving and / or treating nervous system diseases, which comprises the steps of administering (particularly co-administering or combined administering) isolated stem cells to a subject in need; and a substance for increasing the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins.

[0054] In addition, the present invention provides a method for preventing, improving or treating nervous system diseases, which comprises the step of administering a composition to a subject in need thereof, the composition comprising isolated stem cells; and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as active ingredients.

[0055] In addition, the present invention provides isolated stem cells; and substances for increasing the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or compositions containing them as active ingredients for preventing, improving and / or treating nervous system diseases.

[0056] In addition, the present invention provides isolated stem cells; and substances for increasing the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or compositions containing these as active ingredients, for use in the preparation (production) of preparations (particularly combination preparations or combination drugs) for preventing, improving and / or treating nervous system diseases.

[0057] In one embodiment of the present invention, the substance that increases the expression or activity of a protein may include one or more expression vectors, wherein the expression vector contains a polynucleotide encoding a TIMP-1 protein; a polynucleotide encoding an MCP-1 protein; a polynucleotide encoding a GROα protein; and a polynucleotide encoding an IL-6 protein, but is not limited thereto.

[0058] To achieve another object of the present invention, the present invention provides a pharmaceutical composition for inducing angiogenesis, which comprises stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof; or their culture as active ingredients.

[0059] In one embodiment of the present invention, the culture medium (medium) may be a conditioned medium comprising TIMP-1, MCP-1, GROα and IL-6 proteins secreted by stem cells, but is not limited thereto.

[0060] In one embodiment of the present invention, the pharmaceutical composition for inducing angiogenesis of the present invention can be used to prevent or treat cerebrovascular diseases, but is not limited thereto.

[0061] In one embodiment of the present invention, the cerebrovascular disease may be one or more selected from the group consisting of cerebral infarction, cerebral ischemia, stroke, vascular dementia, cerebral hemorrhage, subarachnoid hemorrhage and leukodystrophy, but is not limited thereto.

[0062] Therefore, the present invention provides a method for preventing, improving and / or treating cerebrovascular disease (a method for inducing angiogenesis), which comprises administering stem cells or a culture thereof that overexpress TIMP-1, MCP-1, GROα, and IL-6 genes or proteins thereof to a subject in need thereof.

[0063] In addition, the present invention provides a method for preventing, improving and / or treating cerebrovascular diseases (method for inducing angiogenesis), which comprises the step of administering to a subject in need thereof a composition comprising stem cells or a culture thereof overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof as an active ingredient.

[0064] In addition, the present invention provides stem cells that overexpress TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or their cultures; or compositions containing them as active ingredients for preventing, improving and / or treating cerebrovascular diseases (use of inducing angiogenesis).

[0065] In addition, the present invention provides stem cells or cultures thereof that overexpress TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof; or compositions comprising them as active ingredients in the preparation (production) of preparations for preventing, improving and / or treating cerebrovascular diseases (for the manufacture of preparations for inducing angiogenesis).

[0066] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for inducing angiogenesis, which includes isolated stem cells; and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as active ingredients, and a pharmaceutical composition for inducing angiogenesis containing the composition.

[0067] In one embodiment of the present invention, the substance that increases protein expression or activity may include one or more expression vectors, including a polynucleotide encoding TIMP-1 protein; a polynucleotide encoding MCP-1 protein; a polynucleotide encoding GROα protein; and a polynucleotide encoding IL-6 protein, but is not limited thereto.

[0068] In one embodiment of the present invention, the angiogenesis-inducing pharmaceutical composition of the present invention and a pharmaceutical combination (pharmaceutical combination preparation) containing the composition can be used to prevent or treat cerebrovascular diseases, but are not limited thereto.

[0069] Therefore, the present invention provides a method for preventing, improving and / or treating cerebrovascular diseases (angiogenesis induction method), which comprises the steps of administering (particularly co-administering or combined administration) isolated stem cells to a subject in need thereof; and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins.

[0070] In addition, the present invention provides a method for preventing, improving and / or treating cerebrovascular diseases (a method for inducing angiogenesis), which comprises the step of administering (particularly co-administering or combined administration) to a subject in need thereof a composition containing isolated stem cells; and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as active ingredients.

[0071] In addition, the present invention provides isolated stem cells and substances that increase the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or compositions containing these as active ingredients for use in preventing, improving and / or treating cerebrovascular diseases (use of inducing angiogenesis).

[0072] In addition, the present invention provides isolated stem cells; and substances that increase the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or compositions containing these as active ingredients for the preparation (production) of preparations for preventing, improving and / or treating cerebrovascular diseases (particularly, for the preparation of compositions for inducing angiogenesis). [Beneficial Effects]

[0073] According to the present invention, not only is it suggested to use a combination of specific markers including TIMP-1, MCP-1, GROα and IL-6 as a biomarker for selecting stem cells with the highest therapeutic (improving) efficacy for nervous system diseases, but it is also demonstrated that stem cells with increased expression or activity levels of these factors, or the use of substances that regulate (particularly upregulate) these factors in stem cells, can lead to significantly improved efficacy (potency) and efficiency of the nervous system therapeutic effect. In addition, the present invention demonstrates the higher therapeutic potential of allogeneic stem cell transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 Schematic representation of the process for analyzing the composition of substances secreted by neural stem cells by cytokine profiling and LC-MS / MS using conditioned medium obtained from adult human brain tissue (temporal lobe tissue from non-lesional donors, focal cortical dysplasia type IIIa).

[0075] Figure 2 Results are shown evaluating the neuroprotective effects of conditioned medium from neural stem cells isolated from adult human brain tissue in an in vitro stroke model induced by OGD (oxygen glucose deprivation) in primary rat cortical neurons. (Three batches of conditioned medium prepared using the same method were used for repeated experiments (*, p<0.05; **, p<0.01; ***, p<0.001 vs NC).)

[0076] Figure 3a The results of the effects of neutralizing antibodies for MCP-1 or TIMP-1 on neuroprotective activity of the combination of four efficacy factors of the present invention are shown. After pre-incubation of neutralizing antibodies in the conditioned medium of neural stem cells, the medium is applied to an in vitro stroke model induced by OGD (oxygen glucose deprivation). The results show that treatment with neutralizing antibodies of each factor significantly reduced neuroprotective ability (*, p<0.05; **, p<0.01; ***, p<0.001vs NC; #, p<0.05; ##, p<0.01; ###, p<0.001vs CM; $, p<0.05; $$, p<0.01; $$$, p<0.001vs CM+ (each antibody treatment)).

[0077] Figure 3b The results of evaluating the effects of neutralizing antibodies against GROα or IL-6 on neuroprotective activity from four efficacy factor combinations of the present invention are shown. After pre-incubation of neutralizing antibodies in the conditioned medium of neural stem cells, the medium was applied to an in vitro stroke model induced by OGD (oxygen glucose deprivation) in primary rat cortical neurons. The results showed that treatment with neutralizing antibodies of each factor significantly reduced neuroprotective ability (*, p<0.05; **, p<0.01; ***, p<0.001vs NC; #, p<0.05; ##, p<0.01; ###, p<0.001vs CM; $, p<0.05; $$, p<0.01; $$$, p<0.001vs CM+ (each antibody treatment)).

[0078] Figure 4 Shown are the results of evaluating the neuroprotective capacity of conditioned medium from neural stem cells in an in vitro spinal cord injury model created by treating primary rat spinal cord neuronal cells with H2O2. When conditioned medium from neural stem cells was applied under reactive oxygen species (ROS)-induced injury conditions, the survival rate of neurons increased. (Three batches of conditioned medium prepared using the same method were used for repeated experiments (*, p<0.05; **, p<0.01; ***, p<0.001 vs NC).)

[0079] Figure 5a The experimental results of pre-incubation of neutralizing antibodies or IgG for MCP-1 or TIMP-1 in neural stem cell conditioned medium in four kinds of efficacy factor combinations of the present invention are shown. After pre-incubation, the medium is applied to an in vitro spinal cord injury model, which is created by inducing ROS production through H2O2 treatment. The effects of each factor on the neuroprotective ability of neural stem cell conditioned medium are then compared and analyzed. As a result, after treatment with neutralizing antibodies for each factor, the spinal cord neuroprotective ability is significantly reduced (*, p<0.05; **, p<0.01; ***, p<0.001vs IM; #, p<0.05; ##, p<0.01; ###, p<0.001vs CM; $, p<0.05; $$, p<0.01; $$$, p<0.001vs CM+IgG (10 μg / ml)).

[0080] Figure 5bThe experimental results of pre-incubation of neutralizing antibodies or IgG for GROα or IL-6 in neural stem cell conditioned medium in four efficacy factor combinations of the present invention are shown. After pre-incubation, the medium was applied to an in vitro spinal cord injury model, which was created by inducing ROS production through H2O2 treatment. The effects of each factor on the neuroprotective ability of neural stem cell conditioned medium were then compared and analyzed. As a result, after treatment with neutralizing antibodies for each factor, the spinal cord neuroprotective ability was significantly reduced (*, p<0.05; **, p<0.01; ***, p<0.001vs IM; #, p<0.05; ##, p<0.01; ###, p<0.001vs CM; $, p<0.05; $$, p<0.01; $$$, p<0.001vs CM+IgG (10 μg / ml)).

[0081] Figure 6 It is an image showing the results of a wound healing test performed to evaluate changes in the ability of vascular endothelial cells to migrate after treatment with neural stem cell conditioned medium. The test was performed using HUVEC (human umbilical vein endothelial cells). The results showed that the ability of vascular endothelial cells to migrate significantly increased after treatment with neural stem cell conditioned medium (3 batches of conditioned medium prepared using the same method were used for repeated experiments).

[0082] Figure 7 It is a figure that quantitatively shows the results of a wound healing test performed to evaluate the changes in the migration ability of vascular endothelial cells after treatment with neural stem cell conditioned medium, and the test was performed using HUVEC (human umbilical vein endothelial cells). The results showed that the migration ability of vascular endothelial cells was significantly increased after treatment with neural stem cell conditioned medium (3 batches of conditioned medium prepared using the same method were used for repeated experiments) (*, p<0.05; **, p<0.01; ***, p<0.001 vs NC).

[0083] Figure 8a The experimental results of pre-incubation of neutralizing antibodies against MCP-1 or TIMP-1 in neural stem cell conditioned medium in four efficacy factor combinations of the present invention are shown. After pre-incubation, the effect of each factor on the migration ability of vascular endothelial cells (HUVEC) was confirmed by wound healing assay. As a result, the treatment of each factor with neutralizing antibodies significantly reduced the migration ability of vascular endothelial cells (*, p<0.05; **, p<0.01; ***, p<0.001vs NC; #, p<0.05; ##, p<0.01; ###, p<0.001vs CM).

[0084] Figure 8bThe experimental results of pre-incubating neutralizing antibodies against GROα or IL-6 in neural stem cell conditioned medium in the four efficacy factor combinations of the present invention are shown. After pre-incubation, the effect of each factor on the migration ability of vascular endothelial cells (HUVEC) was confirmed by wound healing assay. As a result, the treatment of each factor with neutralizing antibodies significantly reduced the migration ability of vascular endothelial cells (*, p<0.05; **, p<0.01; ***, p<0.001vs NC; #, p<0.05; ##, p<0.01; ###, p<0.001vs CM).

[0085] Figure 9a The results of an experiment using vascular endothelial cells (HUVEC) to evaluate the angiogenic ability induced by neural stem cell conditioned medium are shown, which show tube formation results. Treatment with the medium enhances the tube formation ability of endothelial cells (the experiment was repeated using 3 batches of conditioned medium prepared by the same method).

[0086] Figure 9b It is a figure showing the quantitative analysis of tube length in an experiment using vascular endothelial cells (HUVEC) to evaluate the angiogenesis ability induced by neural stem cell conditioned medium. Treatment with conditioned medium leads to an improvement in the tube forming ability of vascular endothelial cells (the experiment was repeated using 3 batches of conditioned medium prepared by the same method (*, p<0.05; **, p<0.01; ***, p<0.001 vs NC)).

[0087] Fig.10a The experimental results of pre-incubation of neutralizing antibodies against MCP-1 or TIMP-1 in neural stem cell conditioned medium in four efficacy factor combinations of the present invention are shown. After pre-culture, the effect of each factor on angiogenesis ability was confirmed by treating vascular endothelial cells (HUVEC). After treatment with neutralizing antibodies against TIMP-1, the tube formation ability was significantly reduced (*, p<0.05; **, p<0.01; ***, p<0.001vs NC; #, p<0.05; ##, p<0.01; ###, p<0.001vs CM).

[0088] Fig.10b The experimental results of pre-incubation of neutralizing antibodies against GROα or IL-6 in neural stem cell conditioned medium in the four efficacy factor combinations of the present invention are shown. After pre-culture, the effect of each factor on angiogenesis ability was confirmed by treating vascular endothelial cells (HUVEC). After treatment with neutralizing antibodies for each factor, the tube formation ability was significantly reduced (*, p<0.05; **, p<0.01; ***, p<0.001vs NC; #, p<0.05; ##, p<0.01; ###, p<0.001vs CM).

[0089] Fig.11a : is a graph showing the results of the behavioral test BBB score test (Basso, Beattie and Bresnahan score test) performed on an in vivo spinal cord injury animal model. The test was performed at intervals of one week to evaluate the improvement of behavioral defects after injection (transplantation) of the highly efficient neural stem cells selected according to the present invention (G1, vehicle (DMEM F12-phenol-free); G2, 3×10 6 Cells / animal: G3, 1×10 6 cells / animal; *, G1 (n=42) vs G2 (n=41); #, G1 vs G3 (n=42).

[0090] Fig.11b : is a graph showing the results of the rota-rod test performed on an in vivo spinal cord injury animal model. The test was performed at one-week intervals to evaluate the improvement of behavioral deficits after injection (transplantation) of the highly efficient neural stem cells selected according to the present invention (*, G1 (n=42) vs G2 (n=41); #, G1 vs G3 (n=42)).

[0091] Fig.11c is a graph showing the results of von Frey test performed on an in vivo spinal cord injury animal model. The test was performed at one-week intervals to evaluate the improvement of neuropathic pain after injection (transplantation) of highly efficient neural stem cells selected according to the present invention (*, G1 (n=42) vs G2 (n=41); #, G1 vs G3 (n=42)).

[0092] Fig.12a is a schematic timeline showing a series of experiments conducted using an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO) to confirm the therapeutic effect of the highly efficient neural stem cells selected according to the present invention.

[0093] Figure 12b The images are images showing the results of TCC staining performed on the brains of experimental animals to improve cerebral infarction by injecting (transplanting) the highly efficient neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO).

[0094] Fig.12cThe figure is a diagram showing the quantitative measurement of the size (volume) of cerebral infarction in experimental animals after TCC staining performed on the brain of experimental animals to improve cerebral infarction by injecting (transplanting) the highly efficient neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO). In this experiment, the animal groups were divided into a control group (no injury), a control group after induced stroke (HBSS), a neural stem cell transplantation group (NSC, 5×10 5 The results of the cerebral infarction improvement test showed that cerebral infarction was significantly reduced in animals that received intraventricular injection of stem cells (NSC) compared with animals that did not receive stem cells (HBSS) (*, p<0.05; **, p<0.01).

[0095] Fig.13a The figure shows the results of the rotarod test performed once a week, which is used to evaluate the improvement of movement disorders after the administration (transplantation) of the high-efficiency neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO) (*, compared with the control, p<0.05; #, compared with HBSS, p<0.05; ##, compared with HBSS, p<0.01; ###, compared with HBSS, p<0.001).

[0096] Fig.13b is a graph showing the results of a passive avoidance test performed once a week, which was used to evaluate the improvement of cognitive and memory functions after administration (transplantation) of high-efficiency neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO) (*, compared with control, p<0.05; #, compared with HBSS, p<0.05; ##, compared with HBSS, p<0.01; ###, compared with HBSS, p<0.001).

[0097] Fig.13c is a graph showing the results of an adhesive removal test performed once a week, which was used to evaluate the improvement of sensory and motor functions after administration (transplantation) of high-efficiency neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO) (*, compared with control, p<0.05; #, compared with HBSS, p<0.05; ##, compared with HBSS, p<0.01; ###, compared with HBSS, p<0.001).

[0098] Fig.13dis a graph showing the results of an adhesive removal test performed once a week, which was used to evaluate the improvement of sensory and motor functions after administration (transplantation) of high-efficiency neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO) (*, compared with control, p<0.05; #, compared with HBSS, p<0.05; ##, compared with HBSS, p<0.01; ###, compared with HBSS, p<0.001).

[0099] Fig.14a The selected tissue regions for observing the stroke therapeutic effect of injecting (transplanting) the highly efficient neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO) are shown. The damaged ipsilateral and undamaged contralateral regions were observed on average in three sections. The ipsilateral and contralateral regions mentioned in this specification and the drawings correspond to Fig.14a The area shown in .

[0100] Fig.14b The results of NeuN staining to compare the number of neurons in the damaged area after injection (transplantation) of selected high-efficiency neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO) are shown (control, control group; HBSS, injury control group; NSC, test group with neural stem cell transplantation).

[0101] Fig.14c A graph showing the number of neurons in each experimental group quantified after NeuN staining to compare the number of neurons in the damaged area after injection (transplantation) of the highly efficient neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO). It was confirmed that after administration of the highly efficient neural stem cells according to the present invention, the survival rate of nerve cells in damaged brain tissue increased (control, control group; HBSS, injury control group; NSC, neural stem cell transplantation experimental group, *, p<0.05; **, p<0.01; ***, p<0.001).

[0102] Fig.15a The images show the results of immunohistochemical staining with GFAP antibody to compare the extent of reactive gliosis in the damaged area after administration (transplantation) of the highly efficient neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO).

[0103] Fig.15bA graph showing the quantitative measurement of astrocytes in each experimental group after administration (transplantation) of the highly efficient neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO) and immunohistochemical staining with GFAP antibody to compare the extent of reactive gliosis in the damaged area is shown (*, p<0.05; **, p<0.01; ***, p<0.001).

[0104] Fig.15c Shown are images obtained after administration (transplantation) of highly efficient neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO) and immunohistochemical staining with Iba1 antibody to compare the levels of reactive microglia in the damaged area.

[0105] Fig.15d A graph quantifying the microglia in each experimental group after administration (transplantation) of the highly efficient neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO) and immunohistochemical staining with Iba1 antibody to compare the levels of reactive microglia in the damaged area is shown (*, p<0.05; **, p<0.01; ***, p<0.001).

[0106] Fig.16a The image shows the results of Western blotting performed on brain tissue to confirm the expression levels of proteins related to cell survival and apoptosis (such as BAX, Bcl2 and cleaved caspase-3) after administration (transplantation) of the efficient neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO).

[0107] Fig.16b The figure shows the quantitative results of Western blot analysis performed on brain tissue after administration (transplantation) of the efficient neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO) to confirm the expression levels of proteins related to cell survival and apoptosis (such as BAX, Bcl2 and cleaved caspase-3) (*, p<0.05; **, p<0.01, ***, p<0.001).

[0108] Fig.17a The images show the results of CD31 immunofluorescence staining (vascular staining) of the cortical area of ​​the brain to evaluate the recovery of cerebral blood vessels after administration (transplantation) of the highly efficient neural stem cells selected according to the present invention in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO).

[0109] Fig.17b A graph showing the quantitative measurement results of the angiogenesis (regeneration) level in each experimental group after CD31 immunofluorescence staining (vascular staining) of the cortical area of ​​the injured site was performed to evaluate the recovery of cerebral blood vessels after the efficient neural stem cells selected according to the present invention were administered (transplanted) in an in vivo ischemic stroke animal model (middle cerebral artery occlusion model, MCAO) (*, p<0.05; **, p<0.01, ***, p<0.001).

[0110] Fig.18 The present invention summarizes from various aspects the specific combination of the four factors MCP-1, TIMP-1, GROα, and IL-6 when using neural stem cells to treat nervous system diseases, as well as a schematic diagram of the excellent effects of the highly efficient neural stem cells selected based on these factors. DETAILED DESCRIPTION definition

[0111] Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as commonly understood by those skilled in the art.

[0112] In the present invention, the term "neurological disease" refers to a disease caused by a problem of the nervous system (including brain, spinal cord, nerves, etc.), and may also include certain mental illnesses. Nervous system diseases may include diseases of cranial nerves, central nervous system or peripheral nervous system. In the present invention, the types of nervous system diseases are not specifically limited, as long as they are diseases requiring nerve (nerve cell) regeneration. However, they may include any one or more selected from the group consisting of ischemic brain disease, degenerative nervous system disease, nerve injury disease, peripheral nerve disease and traumatic brain injury.

[0113] In the present invention, term "ischemia" refers to a state in which blood supply is obstructed, which results in an ischemic state, thereby causing necrosis of the affected tissue region. Ischemic brain diseases include but are not limited to diseases caused by cerebral ischemia as known in the art. For example, these can include apoplexy, cerebral infarction, cerebral ischemia, cerebral embolism, cerebral thrombosis, transient ischemic attack (TIA), head trauma, cerebrovascular metabolic disorder, cerebral coma, intracerebral hemorrhage, neonatal intraventricular hemorrhage (IVH), cerebral arteriosclerosis, anoxic brain injury and neonatal hypoxic-ischemic brain injury, wherein any one or more can be selected from the group.

[0114] Neurodegenerative diseases include, but are not limited to, diseases known in the art as degenerative neurological diseases. For example, these may include dementia, Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olive pontocerebellar atrophy (OPCA), Shy-Drager syndrome, striatonigral degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, corticobasal degeneration, diffuse Lewy body disease, Guam Parkinson-ALS-dementia syndrome and Pick's disease, any one or more of which may be selected from the group.

[0115] Neurological impairment diseases include, but are not limited to, diseases which can be selected from the group consisting of epilepsy, spinal cord injury diseases, behavioral disorders, developmental disorders, intellectual disabilities, Down syndrome and schizophrenia, any one or more of which can be selected.

[0116] The term "neurogenesis" used throughout this specification refers to the process of nerve regeneration or regrowth. In this specification, nerve regeneration and neurogenesis can be used interchangeably. In the present invention, the term "neurogenesis" refers to the process of generating new neurons, as is generally known in the art.

[0117] In the present invention, the term "protein" is used interchangeably with "polypeptide" to refer to a polymer of amino acid residues such as typically found in naturally occurring proteins.

[0118] In the present invention, TIMP-1 (TIMP Metallopeptidase Inhibitor 1) protein refers to a protein or polypeptide known in the art as TIMP-1, and its specific source and sequence (amino acid sequence composition) are not particularly limited. For example, in the present invention, TIMP-1 is derived from humans, including those known in the art, such as NCBI (GenBank) Accession No. NP_003245.1 (sequence number 1) or UniProtKB / Swiss-Prot Accession No. Q6FGX5. In the present invention, TIMP-1 includes its functional equivalents.

[0119] In the present invention, MCP-1 (Monocyte Chemoattractant Protein-1) protein, if known in the art as MCP-1 protein or polypeptide, its source and sequence (amino acid sequence composition) are not particularly limited. For example, MCP-1 in the present invention is derived from humans (Homo sapiens), including those disclosed in NCBI (Genbank) Accession Nos. AAB20651.1, NP_002973.1 (Sequence No. 2) or UniProtKB / Swiss-Prot Accession No. P13500.1. In the present invention, MCP-1 includes its functional equivalents.

[0120] In the present invention, GROα (growth-regulated oncogene α; chemokine (CXC motif) ligand 1 (CXCL1)) protein, if known in the art as GROα protein or polypeptide, is not specifically limited by its origin and sequence (amino acid sequence composition). For example, GROα in the present invention is derived from humans (Homo sapiens), including those disclosed in NCBI (Genbank) Accession No. NP_001502.1 (Sequence No. 3) or UniProtKB / Swiss-Prot Accession No. P09341. In the present invention, GROα includes its functional equivalents.

[0121] In the present invention, IL-6 (interleukin 6) protein, if known in the art as IL-6 protein or polypeptide, is not specifically limited by its origin and sequence (amino acid sequence composition). For example, IL-6 in the present invention is derived from humans (homo sapiens), including those disclosed in NCBI (Genbank) Accession No. NP_000591.1 (Sequence No. 4) or UniProtKB / Swiss-Prot Accession No. P05231. In the present invention, IL-6 includes its functional equivalents.

[0122] The functional equivalent referred to in this article refers to a polypeptide having a sequence homology (i.e., identity) of at least 70%, preferably more than 80%, and more preferably more than 90% with the amino acid sequence constituting the known TIMP-1, MCP-1, GROα or IL-6 protein. The amino acid sequence constituting the known TIMP-1, MCP-1, GROα or IL-6 protein is preferably exemplified by the amino acid sequence represented by Sequence Number 1, Sequence Number 2, Sequence Number 3 or Sequence Number 4, respectively. For example, polypeptides having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with the parent known protein and exhibiting substantially the same physiological activity as the parent known protein. The term "substantially the same physiological activity" herein refers to the activity of regulating stem cells for preventing, improving, and / or treating diseases of the nervous system. Preferably, in the present invention, the functional equivalents of TIMP-1, MCP-1, GROα or IL-6 can be produced by adding, replacing or deleting a portion of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. The above amino acid substitutions are preferably conservative substitutions. Examples of conservative substitutions of naturally occurring amino acids are as follows: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn) and sulfur-containing amino acids (Cys, Met). In addition, functional equivalents include variants in which some amino acids are deleted in the amino acid sequence of TIMP-1, MCP-1, GROα or IL-6 protein. The deletion or replacement of amino acids is preferably located in a region that is not directly related to the physiological activity of TIMP-1, MCP-1, GROα or IL-6. In addition, functional equivalents include variants in which several amino acids are added to the N-terminus or C-terminus or amino acid sequence of TIMP-1, MCP-1, GROα or IL-6. In addition, the scope of the functional equivalents disclosed herein includes polypeptide derivatives in which certain chemical structures of the polypeptide are modified while maintaining the basic framework and physiological activity of TIMP-1, MCP-1, GROα or IL-6. For example, structural modifications intended to change the stability, storage capacity, volatility or solubility of the protein are included in this scope.

[0123] In this specification, sequence homology and identity are defined as the percentage of identical matching residues (amino acid residues or nucleotides) between the original sequence (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 of an amino acid sequence; or SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 of a nucleotide sequence) and the candidate sequence after aligning the sequences and introducing gaps. If desired, conservative substitutions are not considered part of the sequence identity to achieve the maximum sequence homology percentage. In addition, for determining protein sequence homology or identity, extensions, deletions or insertions within the N-terminal, C-terminal or internal sequences are not considered to affect sequence homology or identity. In addition, sequence identity can be determined using common standard methods for comparing similar regions of the amino acid sequences of two polypeptides. Computer programs (e.g., BLAST or FASTA) align two polypeptides so that each amino acid is optimally matched (along the full length of one or both sequences, or along the predicted portion of one or both sequences). These programs provide default open penalties and default gap penalties and can be used in conjunction with scoring matrices such as PAM250 (a standard scoring matrix; Dayhoff et al., in Atlas of Protein Sequence and Structure, vol 5, supp 3, 1978) that can be linked to computer programs. For example, percent identity can be calculated as follows. The total number of identical matches is multiplied by 100 and then divided by the sum of the length of the longer sequence within the match span and the number of gaps introduced into the longer sequence to align the two sequences.

[0124] In the present invention, the term "nucleic acid", "DNA sequence", "RNA sequence" or "polynucleotide" refers to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form. Unless otherwise limited, the term "nucleic acid" also includes analogs of known naturally occurring nucleotides that hybridize with nucleic acids in a manner similar to naturally occurring nucleotides.

[0125] In the present invention, the term "polynucleotide encoding TIMP-1" may refer to a nucleotide (nucleic acid) sequence encoding an amino acid sequence designated as SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity therewith. Nucleic acids may include DNA, cDNA and RNA sequences. In other words, a nucleic acid encoding TIMP-1 may have a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity therewith, or a complementary nucleotide sequence thereof, including DNA, cDNA and RNA (e.g., TIMP-1 mRNA). Preferably, it may include a nucleotide sequence represented by NCBI (Genbank) Accession No. NM_003254.3 (SEQ ID NO: 5), and more preferably, it may consist of a nucleotide sequence represented by SEQ ID NO: 5.

[0126] In the present invention, the term "polynucleotide encoding MCP-1" may refer to a nucleotide (nucleic acid) sequence encoding an amino acid sequence designated as SEQ ID NO: 2 or an amino acid sequence having at least 70% sequence identity therewith. Nucleic acids may include DNA, cDNA and RNA sequences. In other words, the nucleic acid encoding MCP-1 may have a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 70% sequence identity therewith, or a complementary nucleotide sequence thereof, including DNA, cDNA and RNA (e.g., MCP-1 mRNA). Preferably, it may include a nucleotide sequence represented by NCBI (GenBank) Accession No. NM_002982.3 (SEQ ID NO: 6), and more preferably, it may consist of a nucleotide sequence represented by SEQ ID NO: 6.

[0127] In the present invention, the term "polynucleotide encoding GROα" may refer to a nucleotide (nucleic acid) sequence encoding an amino acid sequence designated as SEQ ID NO: 3 or an amino acid sequence having at least 70% sequence identity therewith. Nucleic acids may include DNA, cDNA, and RNA sequences. In other words, a nucleic acid encoding GROα may have a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 70% sequence identity therewith, or a complementary nucleotide sequence thereof, including DNA, cDNA, and RNA (e.g., GROα mRNA). Preferably, it may include a nucleotide sequence represented by NCBI (Genbank) Accession No. NM_001511.4 (SEQ ID NO: 7), and more preferably, it may consist of a nucleotide sequence represented by SEQ ID NO: 7.

[0128] In the present invention, the term "polynucleotide encoding IL-6" may refer to a nucleotide (nucleic acid) sequence encoding an amino acid sequence designated as SEQ ID NO: 4 or an amino acid sequence having at least 70% sequence identity therewith. Nucleic acids may include DNA, cDNA, and RNA sequences. In other words, the nucleic acid encoding IL-6 may have a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 70% sequence identity therewith, or a complementary nucleotide sequence thereof, including DNA, cDNA, and RNA (e.g., IL-6 mRNA). Preferably, it may include a nucleotide sequence represented by NCBI (Genbank) Accession No. NM_000600.5 (SEQ ID NO: 8), and more preferably, it may consist of a nucleotide sequence represented by SEQ ID NO: 8.

[0129] In the present invention, the polynucleotide encoding the target protein for expression may undergo various modifications within the coding region without changing the amino acid sequence of the protein expressed from the coding region due to the degeneracy of the codons or considering the codons preferred by the organism to express the protein. In addition, modifications may occur in regions outside the coding region as long as they do not affect the expression of the gene. Those skilled in the art will also understand that such modified genes belong to the scope of the present invention. In other words, the polynucleotide of the present invention may be modified by substitution, deletion, insertion or a combination thereof of one or more nucleotides as long as it encodes a protein with equivalent activity, and such modifications are also included within the scope of the present invention.

[0130] In the present invention, the term "analog" refers to a substance that is structurally similar to a reference molecule, but in which a specific substituent of the reference molecule is replaced by a substituent, thereby modifying the target or regulatory mechanism of the substance. Compared with the reference molecule, the analog has the same, similar or enhanced utility, as expected by those skilled in the art. Synthesis and screening to identify analogs of compounds with improved properties (e.g., having a higher binding affinity to a target molecule) is a well-known method in the field of medicinal chemistry science.

[0131] In the present invention, the term "homologue" refers to a protein and / or protein sequence that is naturally or artificially derived from a common ancestral protein or protein sequence. Similarly, nucleic acids and / or nucleic acid sequences are considered to be homologous when they are naturally or artificially derived from a common ancestral nucleic acid or nucleic acid sequence.

[0132] In the present invention, the term "contacting" refers to its general meaning, which involves combining two or more agents (e.g., two polypeptides) or combining agents with cells (e.g., proteins and cells). Contact can occur in vitro. For example, it refers to combining two or more compositions in a test tube or other container, or combining a test composition with a cell or cell lysate. In addition, contact can occur in a cell or in situ. For example, contact can involve co-expressing a recombinant polynucleotide encoding two polypeptides in a cell, thereby contacting the two polypeptides in a cell or cell lysate.

[0133] In the present invention, the term "test substance" or "test agent" refers to any substance, molecule, element, compound, entity or combination thereof. For example, but not limited to, it includes proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, etc. In addition, it can be a natural product, a synthetic compound, a chemical compound or a combination of two or more substances. Unless otherwise specified, preparations, substances and compounds can be used interchangeably.

[0134] More specifically, the test agents that can be screened using the screening method of the present invention include polypeptides, antibodies, β-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycine, oligocarbamates, carbohydrates, fatty acids, purines, pyrimidines or their derivatives, structural analogs or combinations. Some test agents can be synthetic substances (synthetic compounds), while other test agents can be natural substances (natural compounds, plant extracts, etc.). Test agents can be obtained from a wide and diverse source, including obtaining from a synthetic or natural compound library. Combinatorial libraries can be generated using various types of compounds that can be synthesized by a stepwise process. Many compounds in the combinatorial library can be prepared using the ESL (encoded synthetic library) method (WO 95 / 12608, WO 93 / 06121, WO 94 / 08051, WO 95 / 395503 and WO 95 / 30642). Peptide libraries can be prepared using phage display methods (WO 91 / 18980). Libraries of natural compounds can be obtained from commercial sources or collected from the field in the form of extracts from bacteria, fungi, plants and animals. Known agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification or amidation reactions, to produce structural analogs.

[0135] The test agent can be a naturally occurring protein or a fragment thereof. Such test agents can be obtained from natural sources, such as from cells or tissue lysates. A polypeptide agent library can be obtained, for example, by conventional methods or from commercially available cDNA libraries. The test agent can be a peptide, for example, having about 5 to 30 amino acids, preferably about 5 to 20 amino acids, more preferably about 7 to 15 amino acids. The peptide can be a fragment of a naturally occurring protein, a random peptide, or a "biased" random peptide.

[0136] In addition, the test agent can be a nucleic acid. In addition, the nucleic acid test agent can be a naturally occurring nucleic acid, a random nucleic acid, or a "biased" random nucleic acid. In addition, fragments of prokaryotic or eukaryotic genomes can be used in a similar manner to that described above.

[0137] In addition, the test agent can be a small molecule (eg, a molecule with a molecular weight of about 1,000 or less). High throughput assays can be applied to screen for small molecule modulators. Many assays are available for screening.

[0138] The test agent library screened in the method of the present invention can be produced based on the structural study of the full-length protein or fragment (fragment polypeptide) of TIMP-1, MCP-1, GROα or IL-6 or its analogs. Such structural studies can identify test agents that may bind or interact with the above-mentioned proteins. Various methods (such as crystal structure and molecular modeling) can be used to study the three-dimensional structure of the above-mentioned proteins. Protein structure research methods using X-ray crystallography are well known in the art. Computer modeling of protein structure provides an alternative method for the design of test agents for screening. Molecular modeling methods are known in the art and are disclosed, for example, in the following references: U.S. Patent No. 5,612,894 and U.S. Patent No. 5,583,973. In addition, the protein structure can be determined by neutron diffraction and nuclear magnetic resonance (NMR).

[0139] In the present invention, the term "primer" refers to a single-stranded oligonucleotide that can be used as a starting point for guiding DNA synthesis as a template under appropriate conditions in an appropriate buffer (e.g., four different nucleoside triphosphates and polymerases, such as DNA, RNA polymerases or reverse transcriptases) and at an appropriate temperature. The appropriate length of the primer may vary depending on the intended use, but is generally between 15 and 30 nucleotides. The term "substantially complementary" used in conjunction with a primer refers to a primer that is sufficiently complementary to a template nucleic acid sequence to selectively hybridize under certain annealing conditions, and the annealed primer is extended by a polymerase to produce a complementary strand of the template. Therefore, the term "substantially complementary" has a different meaning from the term "fully complementary" or related terms. Short primer molecules generally require lower temperatures to form a stable hybrid with the template. The primer sequence does not need to be fully complementary to the template, but it must be sufficiently complementary (substantially complementary) to hybridize with the template. The primer hybridizes with the target DNA containing the polymorphic region and initiates the amplification of the allele form showing perfect homology. The primer is used in combination with a second primer that hybridizes to the opposite strand. Amplification results in a product amplified from two primers, indicating the presence of a specific allele. The primers of the present invention include polynucleotide fragments for ligase chain reaction (LCR).

[0140] In the present invention, the term "probe" refers to a generally labeled oligonucleotide having a sequence complementary to the target nucleic acid sequence and forming a double-stranded structure by complementary base pairing. The probe generally includes a "hybridization region" corresponding to the target sequence region, which is preferably composed of at least 30 nucleotides, and in some cases is composed of more than 50 nucleotides. The term "correspondence" refers to a substance identical or complementary to a specified nucleic acid. The probe may preferably not include a sequence complementary to a sequence for preparing PCR. Typically, the 3' end of the probe will be "blocked" to prevent it from being integrated into the primer extension product. "Blocking" can be achieved by adding nucleotides to the 3' hydroxyl of the base, which can achieve a dual purpose: preventing integration into the primer extension product, and acting as a marker for subsequent detection or capture of labeled nucleic acids, which uses non-complementary bases or chemical moieties, such as biotin, phosphate groups or fluorophores (depending on the selected part) to achieve. Blocking can be achieved by removing the 3'-OH group or by using nucleotides (such as dideoxynucleotides) without a 3'-OH group.

[0141] In the present invention, the term "TIMP-1 antibody" or "antibody against TIMP-1" refers to a specific protein molecule against the antigenic site of TIMP-1. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to the TIMP-1 protein, and it includes polyclonal antibodies, monoclonal antibodies and recombinant antibodies. For the purpose of the present invention, the antibody may preferably be a monoclonal antibody, which is a group of antibodies having substantially the same amino acid sequence in the heavy chain and the light chain. In the present invention, when referring to or involving "MCP-1 antibody", "GROα antibody" or "IL-6 antibody", those skilled in the art will clearly understand its meaning based on the description of "TIMP-1 antibody".

[0142] As described above, the production of antibodies against TIMP-1, MCP-1, GROα or IL-6 can be easily achieved using techniques known in the art. Polyclonal antibodies can be produced using methods known in the art, wherein TIMP-1, MCP-1, GROα or IL-6 protein antigens are injected into animals and serum containing antibodies is obtained by collecting blood from the animals. Such polyclonal antibodies can be produced from host animals of any species, including goats, rabbits, sheep, monkeys, horses, pigs, cattle and dogs.

[0143] Monoclonal antibodies can be produced using the hybridoma method, which is well known in the art, or by using phage antibody library technology.

[0144] In addition, in the present invention, the term antibody includes not only the complete form with two full-length light chains and two full-length heavy chains, but also the functional fragments of antibody molecules. The functional fragment of an antibody molecule refers to a fragment that retains at least the antigen binding function, preferably, the fragment retains at least 50%, 60%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to TIMP-1, MCP-1, GROα or IL-6. Specifically, the functional fragment can be Fab, F(ab)2, Fab', F(ab')2, Fv, diabody, scFv or other similar forms. Fab (antigen binding fragment) is an antigen binding fragment of an antibody, consisting of a variable region and a constant region of both the heavy chain and the light chain. F(ab')2 is a fragment produced by pepsin digestion of an antibody, which is formed by two Fab fragments connected by a disulfide bond in the hinge region of the heavy chain. F(ab') is a monomeric antibody fragment obtained by reducing the disulfide bonds of the F(ab')2 fragment, wherein the hinge region of the heavy chain is added to the Fab fragment. Fv (variable fragment) is an antibody fragment consisting only of the variable regions of both the heavy chain and the light chain. scFv (single-chain variable fragment) is a recombinant antibody fragment in which the variable region of the heavy chain (VH) and the variable region of the light chain (VL) are connected by a flexible peptide linker. Diabodies are fragments in which the VH and VL of the scFv are connected by very short linkers to prevent them from binding to each other. Instead, they pair with the VH and VL of another scFv of the same form to form a dimer.

[0145] The antibody applicable to the present invention is not limited thereto, but may be selected from the group consisting of IgG, IgA, IgM, IgE and IgD, and preferably, may be an IgG antibody.

[0146] In the present invention, the term "expression" refers to the production of a protein or nucleic acid in a cell.

[0147] In the present invention, the term "host cell" refers to a prokaryotic or eukaryotic cell containing heterologous DNA introduced into the cell by any means (eg, electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, etc.).

[0148] The present invention will be described in detail below.

[0149] The inventors of the present invention, by studying other functions of neural stem cells besides differentiation into neurons, which is the basic function of neural stem cells, discovered for the first time that in neural stem cells derived from human brain tissue, TIMP-1, MCP-1, GRO-α and IL-6 play a key role in the treatment of nervous system diseases and directly contribute to the therapeutic activity.

[0150] Therefore, the inventors were able to select stem cells with the best therapeutic (improving) effect on nervous system diseases by using a combination of specific markers, TIMP-1, MCP-1, GROα and IL-6. Using these selected stem cells, they have demonstrated significant therapeutic effects in in vivo nervous system disease animal models.

[0151] In addition, by directly utilizing stem cells with increased expression or activity levels of TIMP-1, MCP-1, GROα, and IL-6 factors, or by utilizing substances that regulate (especially upregulate) the expression or activity of these factors in stem cells, nervous system diseases can be treated more significantly and effectively with high efficiency (high performance) and high efficiency.

[0152] Therefore, the present invention provides a method for selecting highly efficient stem cells for treating nervous system diseases, which comprises the step of measuring the protein or gene expression levels of TIMP-1 (TIMP metallopeptidase inhibitor 1), MCP-1 (monocyte chemoattractant protein-1), GROα (growth regulated oncogene α) and IL-6 (interleukin 6).

[0153] In the above method for selecting highly efficient stem cells for treating nervous system diseases, the TIMP-1, MCP-1, GROα, IL-6 protein or gene is characterized in that it is highly expressed in highly efficient stem cells for treating nervous system diseases.

[0154] In the present invention, the specific source or type of the 'stem cells' or 'isolated stem cells' before selection is not particularly limited, as long as they are known in the art for the treatment of nervous system diseases. However, preferably, they refer to stem cells having the ability to differentiate into neural cells. In the present invention, the stem cells are preferably, but not limited to, selected from mesenchymal stem cells, adult stem cells, dedifferentiated stem cells, embryonic stem cells, induced pluripotent stem cells, neural stem cells, tissue-derived stem cells, and any combination of two or more thereof.

[0155] In the present invention, the 'stem cells' or 'isolated stem cells' before selection can be isolated and obtained according to methods known in the art. Examples of such known methods include, but are not limited to, density gradient separation (especially density gradient centrifugation), immunoselection, layer separation culture method, differential adhesion separation, and fluorescence activated cell sorting (FACS).

[0156] In a preferred embodiment of the present invention, the stem cells (especially neural stem cells) of the present invention can be derived from the human brain or human neural tissue (central or peripheral neural tissue), but are not limited thereto. As described in the embodiments of the present application, the inventors have solved the ethical issues associated with the use of embryonic stem cells by isolating and culturing neural stem cells isolated from the temporal lobe tissue of a donor with localized cortical dysplasia type IIIa (a non-lesioned area removed during brain surgery from a donor with localized cortical dysplasia IIIa), while easily providing transplantable human adult neural stem cells. In the present invention, human adult neural stem cells isolated from brain tissue of a donor with localized cortical dysplasia type IIIa are capable of autologous and allogeneic transplantation.

[0157] In the present invention, the neural stem cells may include neural progenitor cells.

[0158] In the present invention, the term "stem cell culture" refers to a product obtained by culturing stem cells according to a known stem cell culture method, and its specific form, such as solid or liquid, is not particularly limited. In one embodiment of the present invention, the culture can be used in the form of a liquid culture medium.

[0159] The composition of the culture is not particularly limited if the culture contains the proteins TIMP-1, MCP-1, GROα and IL-6 secreted by stem cells. In the present invention, the culture encompasses a composition containing stem cells in the culture and a composition not containing stem cells in the culture.

[0160] In one embodiment of the present invention, the culture may not include stem cells that overexpress TIMP-1, MCP-1, GROα, IL-6 genes or proteins. That is, in the present invention, the culture may be a conditioned medium for culturing stem cells that overexpress TIMP-1, MCP-1, GROα, IL-6 genes or proteins. In the present invention, conditioned medium refers to a secretome produced by stem cells that overexpress TIMP-1, MCP-1, GROα, IL-6 genes or proteins, which is obtained by culturing stem cells in a conventional basal medium known in the art (preferably, a medium for mammalian cell culture, more preferably, a commonly used stem cell conditioned medium). The stem cells themselves that overexpress TIMP-1, MCP-1, GROα and IL-6 genes or proteins are removed, and only the culture medium is collected.

[0161] In the present invention, 'basal medium' refers to a mixture containing basic components such as sugars, amino acids, water and other substances necessary for cell survival. The basal medium of the present invention may be a medium synthetically prepared for use or a commercially available medium. Commercially available culture media include, but are not limited to, DMEM (Dulbecco's Modified Eagle's Medium), low glucose DMEM, MEM (minimum essential medium), MEM-α medium, BME (Basal Eagle's Medium), IMEM (Modified Minimum Essential Medium), K-SFM (keratinocyte serum-free medium), EGM-2 (Endothelial cell growth medium 2), RPMI 1640, F-10, F-12, Ham F-12 medium, α-MEM (α-minimum essential medium), G-MEM (Glasgow Minimum Essential Medium), McCoy 5A medium, Eagle's basal medium (BME, Eagle's basal medium), CMRL medium, CMRL 1066 medium, Glasgow's Minimum Essential Medium (GMEM), Leibovitz L-15 medium, KSB-3 basal medium, KSB-3 complete medium, and Iscove's modified Dulbecco's medium. In addition, the culture medium of the present invention may also include a nutrient mixture. Nutrient mixture refers to a mixture of various amino acids, vitamins and inorganic salts commonly used in cell culture. The mixture can be prepared by combining amino acids, vitamins and inorganic salts, or commercially available nutrient mixtures can be used. Commercially available nutrient mixtures include, for example, F12, M199, MCDB110, MCDB131, MCDB202 and MCDB302. Preferably, F12 nutrient mixture (F12 nutrient mixture), M199 or MCDB culture medium can be used.

[0162] In the present invention, the type of culture medium is not particularly limited, as long as it is a culture medium known in the art for culturing mammalian cells, more preferably, a conventional stem cell conditioned medium known in the art. For example, the culture medium can be selected from the group consisting of DMEM culture medium, M199 / F12 culture medium, MEM-α culture medium, low-glucose DMEM, MCDB131 culture medium, IMEM culture medium, K-SFM culture medium, DMEM / F12 culture medium, and EGM-2 culture medium.

[0163] In one embodiment of the present invention, the medium used to obtain the conditioned medium may not include additional serum components, and specifically, may be used under low serum medium conditions or serum-free medium conditions (conditions considered in the art to be substantially serum-free).

[0164] In one embodiment of the present invention, it relates to a method for selecting highly efficient stem cells for treating nervous system diseases, which method may include the following steps: (I) culturing stem cells; (II) measuring the expression levels of TIMP-1, MCP-1, GRO-α and IL-6 in stem cells; and (III) When the expression levels of TIMP-1, MCP-1, GRO-α, and IL-6 are high, the stem cells are identified and classified as highly effective stem cells (candidates) for treating nervous system diseases.

[0165] Step (I) is a step of culturing each stem cell bank, and the stem cell bank is in a state before the present disclosure is selected. If the method is commonly used in the art, the type of culture medium used (specifically referring to the description of one or more basic culture media above), culture temperature, culture time and culture conditions are not particularly limited.

[0166] In step (II), measuring the expression levels of TIMP-1, MCP-1, GROα, and IL-6 specifically refers to measuring the protein expression levels of TIMP-1, MCP-1, GROα, and IL-6, or the expression levels of their respective genes.

[0167] The measurement of TIMP-1, MCP-1, GROα, IL-6 expression levels specifically refers to the level of the measured protein or its corresponding gene. The method for measuring the protein expression level is not limited in the present invention, as long as it is a method known in the art. For example, methods such as Western blotting, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation, complement fixation assay, flow cytometry and protein chip method can be used, but are not limited thereto.

[0168] The measurement of gene expression level is not limited to any particular method, as long as it is a technology known in the art. In a specific embodiment, the measurement of gene expression level may include measuring the mRNA expression level of the gene, but is not limited thereto. The measurement of mRNA expression level can be carried out using any method known in the art without limitation. For example, the measurement of mRNA expression level can be carried out using but is not limited to any of the following methods: reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative real-time RT-PCR, in situ hybridization, RNase protection assay (RPA), Northern blotting, Southern blotting, RNA sequencing, DNA chip method or RNA chip method.

[0169] In step (II), the measurement may be performed on the culture supernatant of the stem cells cultured in step (I), the culture medium obtained after the culture in step (I), or the lysate of the stem cells cultured in step (I), but is not limited thereto.

[0170] In the present invention, the term "high efficiency" means that the activity of stem cells is enhanced compared to unselected stem cells, and the therapeutic activity for nervous system diseases is significantly superior. It is not limited thereto, but preferably, it may refer to having significantly superior ability in neuroprotection, inhibition of reactive gliosis, inhibition of inflammatory response or angiogenesis of neural tissue.

[0171] Step (III) may further include the step of comparing the expression levels of TIMP1, MCP1, GRO-α and IL-6 measured in step (II) with those of a reference group.

[0172] The reference group may preferably refer to unselected stem cells.

[0173] In a specific embodiment of the present invention, step (III) may include comparing the expression levels of TIMP1, MCP1, GRO-α and IL-6 measured in step (II) with the expression levels of a reference group, wherein the reference group is an unselected stem cell. If the levels of TIMP1, MCP1, GRO-α and IL-6 increase compared to the reference group, the stem cells are determined to be "overexpressed" and classified as high-performance stem cells (candidates) for treating nervous system diseases. In this case, comparison with the reference group may include comparing the expression levels of TIMP1, MCP1, GRO-α and IL-6 with the cutoff values ​​of the expression levels in the reference group, but is not limited thereto. Methods for deriving cutoff values ​​according to various statistical analysis methods are well known in the art.

[0174] In one embodiment of the present invention, the cutoff value is determined by any method selected from the group consisting of: receiver operating characteristic curve analysis (ROC curve analysis); linear or nonlinear regression method; linear or nonlinear classification analysis method; analysis of variance (ANOVA) method; hierarchical analysis or cluster analysis method; hierarchical algorithm using decision tree; kernel principal component analysis method; Markov Blanket analysis method; recursive feature elimination (RFE) analysis method; forward or backward floating search (FS) analysis method; and a combination of two or more of these methods, but not limited to this.

[0175] In another embodiment of the present invention related to the efficient stem cell screening method, the method of the present invention may also include the following steps: In another embodiment of the method of the present invention for selecting efficient stem cells for treating nervous system diseases, the method may also include step (IV), i.e. measuring whether the stem cells classified in step (III) have one or more of the following abilities: neuronal differentiation ability; neuroprotective ability; anti-inflammatory ability; and angiogenesis ability.

[0176] In another embodiment of the present invention relating to a highly efficient stem cell screening method, the method of the present invention may further comprise the following steps:

[0177] (IV) a step of confirming the preventive or therapeutic effect of the stem cells classified in step (III) on nervous system diseases in a non-human animal model of nervous system diseases.

[0178] In a specific embodiment of the method for selecting high-performance stem cells for treating nervous system diseases according to the present invention, the method may include the following steps: (i) culturing stem cells; (ii) measuring the protein concentrations of TIMP-1, MCP-1, GROα and IL-6 in the stem cell conditioned medium from step (i); and (iii) When one or more proteins selected from the group consisting of TIMP-1, MCP-1, GROα and IL-6 are highly expressed in the culture medium, the stem cells are identified and classified as high-performance stem cells for treating a nervous system disease.

[0179] Step (i) should be understood with reference to the description of step (I) mentioned above. In one embodiment of the present invention, in step (i), 2 The reference number of cultured stem cells is preferably between 8000 cells / cm 2 and 10,000 cells / cm 2 Between (i.e. 8000 cells / cm 2 Up to 10,000 cells / cm 2 ), more preferably 8500 cells / cm 2 and 9500 cells / cm 2 Between (i.e. 8500 cells / cm 2 Up to 9500 cells / cm 2 ).

[0180] In one embodiment of the present invention, step (i) is preferably performed using one medium selected from the group consisting of DMEM medium, low-glucose DMEM medium and DMEM / F12 medium.

[0181] In one embodiment of the present invention, step (i) is preferably carried out at a temperature ranging from 35°C to 39°C, more preferably at a temperature ranging from 36°C to 38°C.

[0182] In one embodiment of the present invention, step (i) is preferably performed for a culturing time of 20 to 30 hours, more preferably for a culturing time of 24 to 26 hours.

[0183] In this method, step (ii) can be understood with reference to the description of step (II) above. In step (ii), the culture medium to be measured is preferably a culture medium not containing stem cells (more preferably a conditioned culture medium).

[0184] In the method, step (iii) may include: if the TIMP-1 protein concentration in the culture medium measured in step (ii) is 6,000 pg / mL or higher, the MCP-1 protein concentration is 6,000 pg / mL or higher, the GROα protein concentration is 500 pg / mL or higher, and the IL-6 protein concentration is 50 pg / mL or higher, then the stem cells are determined to have high efficacy. The present invention is characterized in that these concentrations are used as cutoff values. Those skilled in the art can derive the range of changes in the cutoff value when changing specific culture conditions with reference to the truncation characteristics of the present invention. Therefore, this range of changes (or modified values) should also be understood to be included within the scope of the present invention.

[0185] In another specific embodiment of the present invention related to the method for highly efficient stem cell selection, the method of the present invention may further comprise the following steps: (iv) measuring whether the stem cells classified in step (iii) have one or more abilities selected from the group consisting of neuronal differentiation ability, neuroprotective ability, anti-inflammatory ability, and angiogenic ability.

[0186] In another specific embodiment of the present invention, the method of the present invention may further include the following steps: (iv) verifying the preventive or therapeutic effects of the stem cells classified in step (iii) in an animal (non-human animal) model of a nervous system disease.

[0187] In addition, the present invention provides a method for determining the neurogenic disease therapeutic activity of stem cells in vitro, wherein the method comprises measuring and comparing the expression levels of proteins or genes of TIMP-1, MCP-1, GROα and IL-6 in stem cells.

[0188] In the method for determining the neurogenic disease therapeutic activity of stem cells in vitro, the proteins or genes of TIMP-1, MCP-1, GROα, and IL-6 are highly expressed in stem cells with high neurogenic disease therapeutic activity, and their composition is based on the contents described in the aforementioned method for selecting high-potential stem cells for neurogenic disease treatment, which will be easily understood by those skilled in the art.

[0189] In addition, the present invention provides a marker composition for selecting high-potential stem cells for treating neurogenic diseases, which comprises proteins or genes of TIMP-1, MCP-1, GROα, and IL-6.

[0190] As a specific embodiment, the present invention provides a composition for selecting efficient stem cells for neurogenic disease treatment, which includes substances for measuring the expression levels of proteins or genes of TIMP-1, MCP-1, GROα, and IL-6.

[0191] The present invention also provides a kit for selecting efficient stem cells for the treatment of neurogenic diseases, which includes the composition as described above. In a specific embodiment of the present invention, it provides a kit for selecting efficient stem cells for the treatment of neurogenic diseases, which includes a substance for measuring the protein TIMP-1, MCP-1, GROα, and IL-6 or its gene expression level. In another specific embodiment of the present invention, the kit of the present invention may include instructions for the method for selecting efficient stem cells for the treatment of neurogenic diseases as described above.

[0192] Furthermore, the present invention provides a preparation for measuring the expression levels of proteins TIMP-1, MCP-1, GROα and IL-6, or a composition containing these proteins as active ingredients, which is used for selecting highly efficient stem cells for the treatment of neurogenic diseases.

[0193] In addition, the present invention also provides the use of preparing a preparation for selecting efficient stem cells for treating neurogenic diseases, wherein the preparation comprises a preparation for measuring the expression levels of proteins TIMP-1, MCP-1, GROα and IL-6, or a composition containing these as active ingredients.

[0194] In the composition or kit for selecting efficient stem cells for treating neurogenic diseases of the present invention, the preparation for measuring the expression level of a protein is not particularly limited to a specific type, as long as it is a means known in the art. For example, it can be a specific antibody against a protein.

[0195] In the composition or kit for selecting efficient stem cells for the treatment of neurogenic diseases of the present invention, the measurement of gene expression level is not specifically limited to a specific type, as long as it is a means and / or method known in the art. For example, a preparation for measuring the mRNA expression level of a gene can be used to measure. The preparation for measuring the mRNA expression level is not specifically limited to a specific type, as long as it is a means known in the art. For example, it can be a probe or primer set that specifically binds to mRNA.

[0196] The measuring agent can be labeled with a detectable marker to facilitate identification, detection, measurement and quantification under given conditions. For example, detectable labels can include chemical labels (e.g., biotin), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase, peroxidase, luciferase, α-glucuronidase, α-galactosidase, chloramphenicol acetyltransferase, β-galactosidase, and β-glucosidase), radioactive labels (e.g., 14C, 125I, 32P, and 35S), fluorescent labels (e.g., coumarin, fluorescein, FITC (fluorescein isothiocyanate), rhodamine 6G, rhodamine B, TAMRA (6-carboxytetramethyl-rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2-phenylindole), HEX, TET, Dabsyl, and FAM), luminescent labels, chemiluminescent labels, FRET (fluorescence resonance energy transfer) labels, or metal labels (e.g., gold and silver). Likewise, detectable groups can be substrates, cofactors, inhibitors, or affinity ligands.

[0197] The confirmation, detection, measurement and quantification of the measured substance can be analyzed by detecting the signal emitted from the label. For example, when alkaline phosphatase is used as a label, a colorimetric reaction substrate such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate or enhanced chemiluminescence (ECF) is used to detect the signal. When horseradish peroxidase is used as a label, a substrate such as chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-fluorescein, lucigenin (bis-N-methylacridine nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and catechol), TMB (tetramethylbenzidine), ABTS (2,2'-azine-di[3-ethylbenzothiazoline sulfonate]), o-phenylenediamine (OPD) or naphthol / pyrone can be used to detect the signal.

[0198] In addition, the kit for selecting efficient stem cells for the treatment of neurogenic diseases according to the present invention may include, in addition to preparations for measuring the expression levels of proteins TIMP-1, MCP-1, GROα and IL-6, one or more other compositions suitable for analytical methods, the aforementioned markers, substances for detecting them, and equipment.

[0199] As a specific embodiment, the test kit can be a test kit characterized in that it comprises the basic components required for reverse transcription polymerase chain reaction. The reverse transcription polymerase chain reaction test kit includes a specific primer pair for each marker gene. The primer is a nucleotide with a specific sequence for the nucleotide sequence of each marker gene, and a length of about 7bp to 50bp, more preferably about 10bp to 30bp. In addition, a specific primer for the nucleotide sequence of a control gene can also be included. The reverse transcription polymerase chain reaction test kit can also include a test tube or other suitable containers, a reaction buffer (with different pH and magnesium concentrations), deoxynucleotide triphosphates (dNTPs), enzymes (such as Taq polymerase and reverse transcriptase), DNase, RNase inhibitors, DEPC-treated water (DEPC-water), sterile water, etc.

[0200] Alternatively, the kit is characterized in that it contains the essential components required for the DNA chip assay. The DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, preparations, enzymes and other components for preparing fluorescently labeled probes. In addition, the substrate may include a cDNA or oligonucleotide corresponding to a control gene or a fragment thereof.

[0201] Alternatively, the kit is characterized in that it includes the basic components required for performing ELISA. The ELISA kit includes specific antibodies for marker proteins. Antibodies are antibodies with high specificity and affinity for each marker protein and minimal cross-reactivity with other proteins, and may include monoclonal antibodies, polyclonal antibodies or recombinant antibodies. In addition, the ELISA kit may include specific antibodies for control proteins. In addition, the ELISA kit may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., bound to antibodies), substrates for enzymes, or other substances that may be bound to antibodies.

[0202] Furthermore, the kit of the present invention may include a washing solution or elution buffer, which removes unbound proteins, such as enzymes and substrates for colorimetric reactions, and retains only bound protein markers.

[0203] In addition, the present invention provides a method for screening a preventive or therapeutic agent for a neurogenic disease, comprising the following steps: (a) contacting a test substance with isolated stem cells; (b) measuring the expression levels of TIMP-1, MCP-1, GROα and IL-6 proteins or their genes in the stem cells contacted with the test substance and control stem cells not contacted with the test substance; and (c) selecting a test substance that increases the expression or activity level of TIMP-1, MCP-1, GROα and IL-6 protein or its gene compared to control cells.

[0204] In the screening method of the present invention, any method known in the art can be used to perform the measurement of protein expression or activity level without limitation. For example, any one or more methods selected from the group consisting of Western blotting, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation analysis, complement fixation assay, flow cytometry (FACS) and protein chip method can be used for measurement, but not limited thereto.

[0205] In the screening method of the present invention, the measurement of gene expression or activity level can be carried out using any method known in the art, without limitation. In a specific embodiment, the measurement of gene expression or activity level can include measuring the mRNA expression or activity level of each gene, but is not limited thereto. In this case, the measurement of mRNA expression or activity level can be carried out using any method known in the art, without limitation. For example, the measurement of mRNA expression or activity level can be carried out using one or more methods selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative real-time RT-PCR, in situ hybridization, RNase protection assay (RPA), Northern blotting, Southern blotting, RNA sequencing, DNA chip and RNA chip, but is not limited thereto.

[0206] Those skilled in the art may understand the screening method comprising steps (a), (b) and (c) of the present invention as a method for screening candidate substances for preventing and / or treating neurogenic diseases.

[0207] Therefore, in one embodiment of the present invention, the present invention provides a method for screening candidate substances for preventing and / or treating neurogenic diseases, comprising the following steps: (a) contacting the isolated stem cells with a test substance; (b) measuring the expression levels of proteins TIMP-1, MCP-1, GROα and IL-6 or the expression levels of their genes in stem cells contacted with the test substance and control stem cells not contacted with the test substance; and (c) identifying and / or selecting a test substance as a candidate substance, in which the expression or activity level of the proteins TIMP-1, MCP-1, GROα and IL-6 or their genes is increased compared to control cells.

[0208] The screening method of the present invention comprising steps (a), (b) and (c) can be understood by those skilled in the art as a method for screening stem cell excipients (stem cell activators, stem cell activation promoting combinations or stem cell activators for the prevention or treatment of neurogenic diseases) for preventing or treating neurogenic diseases.

[0209] Therefore, in one embodiment of the present invention, the present invention provides a method for screening stem cell supplements (stem cell activators, neurogenic disease prevention or treatment stem cell activation promoters (activators)) for preventing or treating neurogenic diseases, which comprises the following steps: (a) contacting a test substance with isolated stem cells; (b) measuring the expression or activity levels of proteins TIMP-1, MCP-1, GROα and IL-6 or their genes in stem cells contacted with the test substance and control stem cells not contacted with the test substance; and (c) Screening of test substances based on the increased expression or activity levels of TIMP-1, MCP-1, GROα and IL-6 proteins or their genes compared to control cells.

[0210] In the above screening method of the present invention, the measurement of the expression level of TIMP-1, MCP-1, GROα and IL-6 proteins or their genes in step (b) may include measuring the expression (secretion) level of stem cells contacted with the test substance and cultured in the culture medium and control stem cells not contacted with the test substance in the culture medium.

[0211] In the above screening method of the present invention, the term "selection" in step (c) may refer to selecting the test substance based on the steps or selection criteria (e.g., cutoff values, etc.) of the "efficient stem cell selection method for the treatment of neurogenic diseases" described in the present invention.

[0212] Specifically, in one embodiment of the present invention, the "selection" in step (c) is preferably performed by selecting the test substance based on the following criteria: 2 Up to 10,000 cells / cm 2 (More preferably 8500 cells / cm 2 Up to 9500 cells / cm 2) and select test substances in which the TIMP-1 protein concentration secreted from the stem cells is increased to 6,000 pg / mL or higher, the MCP-1 protein concentration is 6,000 pg / mL or higher, the GROα protein concentration is 500 pg / mL or higher, and the IL-6 protein concentration is 50 pg / mL or higher. The present invention can be characterized by using the above concentrations as cutoff values. It is easy for those skilled in the art to understand that by referring to the cutoff characteristics of the present invention, a range of changes in the cutoff value can be obtained by changing specific culture conditions. Therefore, this range of changes (modified value) is also included in the scope of the present invention.

[0213] The above screening method of the present invention may further include the following steps after step (c) to enable the final selection of the test substance: (d) A step of confirming the neurogenic disease preventive or therapeutic effect of the test substance (preventive or therapeutic candidate) selected in step (c) in an animal (non-human animal) model of neurogenic disease.

[0214] In step (d), the (candidate) test substance may be co-administered (combined administration) with the stem cells from step (a), but is not limited thereto.

[0215] Furthermore, the present invention provides a composition for promoting the activity of stem cells for preventing or treating neurogenic diseases, comprising as an active ingredient a substance that increases the expression or activity of genes or proteins TIMP-1, MCP-1, GROα and IL-6.

[0216] Furthermore, the present invention provides a method for promoting the activity of stem cells for preventing or treating neurogenic diseases, comprising the step of treating isolated stem cells with a substance that increases the expression or activity of genes or proteins TIMP-1, MCP-1, GROα and IL-6.

[0217] In addition, the present invention provides a substance that increases the expression or activity of the gene or protein TIMP-1, MCP-1, GROα and IL-6; or a composition containing these as active ingredients, for use in promoting the activity of stem cells for preventing or treating neurogenic diseases.

[0218] In addition, the present invention provides a substance that increases the expression or activity of the genes or proteins TIMP-1, MCP-1, GROα and IL-6; or a composition containing these as active ingredients for the preparation of a preparation for promoting the activity of stem cells for preventing or treating neurogenic diseases.

[0219] The activity of stem cells is not limited to but preferably may include one or more selected from the group consisting of self-renewal (neurogenesis), neuroprotection ability, inhibition of neuronal apoptosis, endothelial cell migration, angiogenesis, anti-inflammatory effect and inhibition of reactive gliosis.

[0220] In the present specification, the term 'increasing substance' encompasses the meanings of terms commonly used in the art, such as enhancer, promoter, enhancer and activator.

[0221] In the present specification, the term 'increase in protein expression or activity' includes the meaning of increasing the extracellular secretion of the relevant protein.

[0222] The protein expression or activity increasing substances are not limited to the following, but an exemplary preferred embodiment may include one or more expression vectors (recombinant expression vectors) containing a polynucleotide encoding a TIMP-1 protein; a polynucleotide encoding an MCP-1 protein; a polynucleotide encoding a GROα protein; and a polynucleotide encoding an IL-6 protein.

[0223] As a specific embodiment of the present invention, the protein expression or activity enhancing substance provides one or more expression vectors (recombinant expression vectors), wherein the expression vectors include a promoter and one or more polynucleotides operably linked thereto, as follows: Polynucleotide encoding TIMP-1 protein; polynucleotide encoding MCP-1 protein; polynucleotide encoding GROα protein; polynucleotide encoding IL-6 protein.

[0224] The polynucleotides encoding each of TIMP-1, MCP-1, GROα or IL-6 proteins provided by the present invention can be integrated into a single expression vector, or they can also be integrated into two or more expression vectors, for example, into 2 to 4 expression vectors, individually or in combination.

[0225] The term "promoter" refers to a DNA sequence that regulates the expression of a polynucleotide sequence operably connected thereto in a specific host cell, and "operably connected" refers to a polynucleotide fragment being connected to another polynucleotide fragment in such a way that its function or expression is affected by another polynucleotide fragment. In addition, the sequence may also include any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating transcription and translation termination. The promoter may be a constitutive promoter that always induces the expression of the target gene, or an inducible promoter that induces the expression of the target gene at a specific location or time. Examples of such promoters include SV40 promoter, CMV promoter, CAG promoter, CaMV 35S promoter, Rsyn7 promoter (U.S. Patent Application No. 08 / 991,601), rice actin promoter, ubiquitin promoter, and ALS promoter (U.S. Patent Application No. 08 / 409,297), etc. In addition, promoters disclosed in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142, etc. can also be used.

[0226] Meanwhile, the expression vector can be introduced into the target cell (host cell) in an expression form by methods known in the art, such as infection, transfection or transduction.

[0227] The gene delivery method using a plasmid expression vector is a method for delivering plasmid DNA directly to mammalian cells, and is a method approved by the FDA for humans. Plasmid DNA has the advantage of being able to be evenly purified, unlike viral vectors. The plasmid expression vectors that can be used in the present invention include mammalian expression plasmids known to those skilled in the art. For example, but not limited to, pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91 / 08291) and pVL1392 (PharMingen) are representative. Plasmid expression vectors can be introduced into target cells by methods known in the art, including but not limited to transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene guns and other known methods of introducing DNA into cells.

[0228] In addition, as the method applicable to the present invention, the viral expression vector containing nucleic acid includes but is not limited to retrovirus, adenovirus, herpes virus, fowl pox virus and slow virus. Retroviral vector is a vector in which all viral genes have been removed or changed so that non-viral proteins are produced in cells infected by viral vectors. The major advantage of retroviral vectors for gene therapy is that they can deliver a large amount of genes to replicating cells, accurately integrate the delivered genes into the DNA of cells, and prevent persistent infection after gene transfection. The retroviral vector approved by FDA is produced using PA317 dual tropic retroviral packaging cell line. Non-retroviral vectors include adenovirus, as described above. The major advantage of adenovirus is that they can carry larger DNA fragments (36kb genome) and infect non-replicating cells with very high titers. In addition, herpes virus can also be used for human gene therapy. In addition, other known suitable viral vectors can be used in the present invention.

[0229] In addition, the present invention provides a pharmaceutical composition for preventing or treating nervous system diseases, which comprises stem cells (stem cell therapy) overexpressing genes or proteins of TIMP-1, MCP-1, GROα and IL-6 and / or their culture medium as active ingredients.

[0230] In addition, the present invention provides a method for preventing, improving and / or treating nervous system diseases, which comprises the step of administering stem cells that overexpress genes or proteins of TIMP-1, MCP-1, GROα and IL-6, or their culture medium; or a composition containing these as active ingredients to a subject in need thereof.

[0231] In addition, the present invention provides stem cells that overexpress TIMP-1, MCP-1, GROα and IL-6 genes or proteins, or their culture media; or compositions containing these as active ingredients for preventing, improving and / or treating nervous system diseases.

[0232] In addition, the present invention provides stem cells that overexpress genes or proteins of TIMP-1, MCP-1, GROα and IL-6, or their culture media; or compositions containing these as active ingredients, for use in preparing preparations (products) for preventing, improving and / or treating nervous system diseases.

[0233] In addition, the present invention provides a pharmaceutical composition for inducing angiogenesis, which comprises stem cells (stem cell therapeutic agent) overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or proteins and / or their culture medium as active ingredients.

[0234] The culture medium may be a conditioned medium containing proteins of TIMP-1, MCP-1, GROα, and IL-6 secreted by stem cells, but is not limited thereto.

[0235] In addition, the present invention provides a method for inducing angiogenesis, which comprises the step of administering stem cells or a culture thereof overexpressing genes of TIMP-1, MCP-1, GROα and IL-6 or their proteins, or a composition comprising these as active ingredients to a subject in need thereof.

[0236] In addition, the present invention provides a method for preventing, improving and / or treating cerebrovascular diseases, which comprises the step of administering stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or their proteins, or a culture thereof, or a composition comprising these as active ingredients to a subject in need thereof.

[0237] In addition, the present invention provides use of stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or their proteins, or a culture thereof, or a composition comprising these as active ingredients for inducing angiogenesis.

[0238] In addition, the present invention provides the use of stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or their proteins, or cultures thereof, or compositions containing these as active ingredients for preventing, improving and / or treating cerebrovascular diseases.

[0239] In addition, the present invention provides the use of stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or their proteins, or cultures thereof, or compositions containing these as active ingredients for preparing therapeutic agents for inducing angiogenesis.

[0240] In addition, the present invention provides the use of stem cells that overexpress TIMP-1, MCP-1, GROα and IL-6 genes or their proteins, or their cultures, or compositions containing these as active ingredients in the preparation (production) of therapeutic agents for preventing, improving and / or treating cerebrovascular diseases.

[0241] In this specification, the term "stem cell therapeutic agent" is used with its generally accepted meaning in the art, and preferably refers to a pharmaceutical composition comprising stem cells as an active ingredient. The term "administering" stem cells in this specification includes the meaning of "implanting" cells.

[0242] In the present invention, "overexpression of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins" can be achieved by treating (or contacting) with a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or proteins as described above.

[0243] In a preferred embodiment of the present invention, overexpression refers to the process of artificially expressing a gene containing a sequence of a respective polypeptide encoding TIMP-1, MCP-1, GROα or IL-6 in a cell to produce a level of the expressed encoded polypeptide that exceeds the expression level of the same polypeptide in a precursor host cell. Therefore, when the above terms are used together with genes, the term "overexpression" can also be used together with proteins to refer to the increased level of the protein produced by the overexpression of the gene encoding it. In some embodiments, the overexpression of a protein-encoding gene is achieved by increasing the copy number of the protein-encoding gene. In other embodiments, the overexpression of a protein-encoding gene is achieved by increasing the binding affinity of a promoter region and / or a ribosome binding site to enhance the transcription and / or translation of the protein-encoding gene. In other embodiments, overexpression can be achieved by increasing the copy number of a gene and enhancing the binding affinity of a promoter region and / or a ribosome binding site. In some embodiments, the overexpression of a protein-encoding gene is caused by the expression of one or more copies of the corresponding encoding polynucleotide present in multiple copies of a plasmid introduced into the genome of a host cell. In other embodiments, overexpression of a protein-encoding gene results from expression of two or more copies of the corresponding encoding polynucleotide integrated into the genome of the host cell.

[0244] In addition, the present invention also provides a pharmaceutical composition for preventing or treating nervous system diseases, which comprises isolated stem cells and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as active ingredients, as well as a pharmaceutical combination preparation for preventing or treating nervous system diseases comprising the composition.

[0245] In addition, the present invention also provides a method for preventing, improving and / or treating nervous system diseases, which comprises administering to a subject in need thereof isolated stem cells and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins, or a composition comprising the substance as an active ingredient.

[0246] In addition, the present invention provides isolated stem cells and substances that increase the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins, or compositions containing the substances as active ingredients for preventing, improving and / or treating nervous system diseases.

[0247] In addition, the present invention provides the use of isolated stem cells and substances that increase the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins, or compositions containing the substances as active ingredients for the manufacture (production) of preparations (especially combined or combined therapeutic preparations) for preventing, improving and / or treating nervous system diseases.

[0248] In addition, the present invention provides a pharmaceutical composition for inducing angiogenesis, which comprises isolated stem cells and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as active ingredients, as well as a pharmaceutical composition for inducing angiogenesis comprising the pharmaceutical composition.

[0249] In addition, the present invention provides a method for inducing angiogenesis, which comprises the step of administering (particularly by co-administration or combined administration) isolated stem cells and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as active ingredients, or a composition containing them to a subject in need thereof.

[0250] In addition, the present invention provides a method for preventing, improving and / or treating cerebrovascular diseases, which comprises administering (isolated stem cells) and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as active ingredients, or a composition containing them, to a subject in need, in particular by (co-administration) or (combined administration).

[0251] In addition, the present invention provides the use of isolated stem cells and substances that increase the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or compositions containing them as active ingredients for inducing angiogenesis.

[0252] In addition, the present invention provides isolated stem cells and substances that increase the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or compositions containing them as active ingredients, for use in preventing, improving and / or treating cerebrovascular diseases.

[0253] In addition, the present invention provides isolated stem cells and substances that increase the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or compositions containing them as active ingredients, for use in the preparation (production) of angiogenesis inducing preparations (particularly combined preparations or co-administration preparations).

[0254] In addition, the present invention provides isolated stem cells and substances that increase the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or compositions containing them as active ingredients, for use in preparing (producing) preparations (particularly combined preparations or co-administered preparations) for preventing, improving and / or treating cerebrovascular diseases.

[0255] In the present invention, the term "for inducing angiogenesis" may preferably refer to the use for preventing or treating cerebrovascular diseases. The above-mentioned cerebrovascular diseases refer to neurological dysfunction caused by damage to the normal blood supply to the brain. The types of cerebrovascular diseases are not particularly limited as long as they are recognized in the art. Examples include ischemic stroke, cerebral ischemia, cerebrovascular accident, vascular dementia, intracerebral hemorrhage, subarachnoid hemorrhage and leukodystrophy, and any one or more can be selected from this group.

[0256] The pharmaceutical composition of the present invention can be formulated into a single composition in which the isolated stem cells and the genes or activators of TIMP-1, MCP-1, GROα and IL-6 are combined, or can be formulated into separate compositions. The method of formulating these components can utilize techniques generally known in the art.

[0257] The genes of TIMP-1, MCP-1, GROα and IL-6, or substances that enhance the expression or activity thereof, can be administered simultaneously, separately or sequentially with the isolated stem cells.

[0258] The active ingredients of the present invention (which include isolated stem cells and genes for TIMP-1, MCP-1, GROα and IL-6, or substances that enhance their expression or activity) can be contained together in a single pharmaceutical preparation and administered simultaneously through the same administration site; or provided as separate preparations and administered simultaneously or sequentially through the same administration site; or provided as separate preparations and administered simultaneously or sequentially through different administration sites.

[0259] Specifically, the term "simultaneously" refers to the administration of two active ingredients together by the same route of administration, or by the same or different routes of administration at substantially the same time (e.g., within 15 minutes or shorter administration time intervals). The term "separately" refers to the administration of two active ingredients at certain time intervals by the same or different routes of administration. The term "sequentially" refers to the administration of two active ingredients by the same or different routes of administration in a specific order according to the patient's condition. The order of administration of the pharmaceutical composition according to the present invention, including which component is administered at what time, whether it is administered simultaneously, separately or sequentially, can be determined by a doctor or expert. This order of administration may depend on many factors.

[0260] The route of administration can be oral or parenteral. It can be administered by various routes. All methods of administration can be foreseen by those skilled in the art, including but not limited to oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, epidural injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, by oral or nasal spray, skin administration and transdermal administration.

[0261] The pharmaceutical composition of the present invention may include only the above-mentioned active ingredients in a pharmaceutically effective amount, or may additionally include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. The term "pharmaceutically effective amount" refers to an amount that produces a greater reaction than that of a negative control, and preferably refers to an amount sufficient to treat or prevent a nervous system disease.

[0262] Those skilled in the art can, within the scope of the present invention, consider factors such as the patient's pain level, sensitivity to drugs, side effects, etc., to select and administer the combination ratio, dosage and administration time of the active ingredients of the present invention, wherein the active ingredients are selected from the group consisting of isolated stem cells and one or more gene or protein expression or activity enhancers.

[0263] On the other hand, when the active ingredients of the present invention are co-administered as a single formulation, the composition (formulation) can be prepared by appropriately selecting the ratio of each ingredient according to the amount of each ingredient in the daily dose. Then, the combined preparation can be administered once or more per day.

[0264] However, the pharmaceutically effective amount can be appropriately varied according to a variety of factors, such as the disease and its severity, the patient's age, weight, health, sex, route of administration, and duration of treatment. For example, when administered to human patients, the total daily dose of the composition according to the present invention can be determined by medical judgment within the therapeutic range. The specific therapeutically effective amount for a particular patient should be adjusted based on various factors, including the desired type and degree of response, whether a different formulation is used in certain circumstances, the patient's age, weight, general health, sex, diet, time of administration, route of administration, secretion rate of the composition, duration of treatment, and other similar factors well known in the pharmaceutical field.

[0265] The pharmaceutical composition according to the present invention may also include suitable carriers, excipients and diluents commonly used in the preparation of the pharmaceutical composition. Carriers include all types of solvents, dispersion media, water-in-oil or oil-in-water emulsions, aqueous compositions, liposomes, microbeads and microsponges.

[0266] The auxiliary material may include one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a humectant, a film coating material, and a controlled release additive.

[0267] The pharmaceutical composition according to the present invention can be formulated into various forms, such as powder, granules, slow-release granules, enteric granules, liquids, eye drops, elixirs, emulsions, suspensions, tinctures, syrups, lozenges, aerosols, tablets, slow-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, slow-release capsules, enteric-coated capsules, powders, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusions, lotions, pastes, sprays, inhalants, patches, sterile injections or aerosols. External preparations can have forms such as creams, gels, patches, sprays, ointments, lotions, liniments, pastes or cataplasms.

[0268] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, glucose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0269] The composition of the present invention can also be formulated into a non-oral preparation. Preparations for non-oral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. For sterile aqueous solutions, suitable buffer solutions can be used, such as Hank's solution, Ringer's solution or physiologically buffered saline. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable esters such as ethyl oleate. If necessary, preservatives, stabilizers, wetting agents or emulsifiers, salts and / or buffers for regulating osmotic pressure can be used. For suppositories, conventional substrates can be used, such as Witepsol, Macrogol, Tween 61, cocoa butter, lauric acid and glycerin gelatin.

[0270] When formulating, a diluent or excipient (such as commonly used fillers, expanders, binders, wetting agents, disintegrants and surfactants) is usually used to prepare the composition.

[0271] The additives of the tablets, powders, granules, capsules, lozenges and sugar tablets according to the present invention may include corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, monocalcium phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate and primogel as excipients; gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, carboxymethyl cellulose calcium, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin, hydroxy cellulose, hydroxypropyl starch, hydroxypropyl methyl cellulose, purified shellac, starch powder, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone as binders; hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, carboxymethyl cellulose Vitamin calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicon dioxide, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginate, amylose, guar gum, sodium bicarbonate, polyvinyl pyrrolidone, calcium phosphate, gelling starch, gum arabic, pullulan, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium silicate, D-sorbitol solution and anhydrous silicon dioxide as disintegrants; calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, kaolin, vaseline, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG6000, liquid paraffin, hydrogenated soybean oil (Lubri Wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, polyethylene glycol, synthetic aluminum silicate, anhydrous silica, higher fatty acids, polymer alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid esters, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, anhydrous silica as lubricants.

[0272] The additives of the liquid preparation according to the present invention may include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose stearate, polyoxyethylene sorbitan fatty acid esters (Tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamin, polyvinyl pyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc.

[0273] The syrup preparation according to the present invention may include sucrose solution, other sugars or sweeteners, and, if necessary, may also include flavoring agents, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers and thickening agents.

[0274] The emulsion according to the present invention may include purified water, and if necessary, an emulsifier, a preservative, a stabilizer and a flavoring agent may be used.

[0275] The suspending agent according to the present invention may include suspending agents such as gum arabic, gum tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose, HPMC 1828, HPMC 2906, HPMC 2910, and if necessary, surfactants, preservatives, stabilizers, colorants and flavoring agents may be used.

[0276] The injection according to the present invention may include a solvent, such as sterile water for injection, 0.9% sodium chloride injection, Ringer's injection, glucose injection, glucose + sodium chloride injection, PEG (polyethylene glycol), lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils, such as perilla oil, cottonseed oil, safflower oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate and benzyl benzoate; solubilizers, such as sodium benzoate, sodium salicylate, sodium acetate, urea, carbamate, monoethylacetamide, butazolidine, propylene glycol, Tweens, nicotinamide, hexamine, dimethylacetamide; buffers, such as weak acids and their salts (acetic acid, and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones and gums; denaturants such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen (N2) and ethylenediaminetetraacetic acid; antioxidants such as 0.1% sodium bisulfite, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid and sodium bisulfite acetone; anesthetics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose and calcium gluconate; suspending agents such as sodium CMC, sodium alginate, Tween 80 and aluminum monostearate.

[0277] Suppositories according to the invention may include excipients such as cocoa butter, lanolin, Witepsol, polyethylene glycol, glycerinated gelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, safflower oil, palm oil, cocoa butter + cholesterol, lecithin, lanette wax, glyceryl monostearate, Tween or Span, Imhausen, Monolen (propylene glycol monostearate), glycerol, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, Hexaride Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrocote25, Hydrocote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosufostal-N, Paramount-B, Suposhiro (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs) and Tejester triglyceride base (TG-95, MA, 57).

[0278] The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route or administration method as long as it exhibits the effects of the present invention.

[0279] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined based on factors such as the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, time of administration, route of administration, excretion rate, duration of treatment, and other factors well-known in the medical field (including any concurrently used drugs).

[0280] The pharmaceutical combination preparation of the present invention can be formulated so that the active ingredient is included in a single dosage form as a component according to the method of administration and the route of administration. Alternatively, each component can be prepared separately and included in a single package according to the dosage unit (e.g., daily or single dose administration). The formula of the active ingredient prepared separately can be the same or different. The specific type, preparation method and pharmaceutically acceptable carrier that can be included in the pharmaceutical combination preparation of the present invention are as described in the pharmaceutical composition above.

[0281] In the present invention, the term "pharmaceutically acceptable" refers to a physiologically acceptable, non-toxic substance or composition that does not inhibit the action of the active ingredient when administered to the human body and generally does not cause allergic reactions or similar reactions such as gastrointestinal disturbances or dizziness.

[0282] The active ingredients as the components of the pharmaceutical combination preparation according to the present invention can be used simultaneously, individually or in a predetermined order (sequentially). The combination preparation can be formulated so that the daily dose is included in a single administration, but can also be formulated into divided doses for administration, for example 2, 3 or 4 times a day.

[0283] The preferred dosage of the pharmaceutical combination preparation of the present invention can be appropriately varied according to several factors, such as the disease and its severity, the patient's age, weight, health status, sex, route of administration and treatment period. Since the bioavailability (bioavailability) of the active pharmaceutical ingredient may vary between individuals, it may be desirable to monitor the blood concentration of each drug using a monoclonal antibody-based assay, etc., in the initial stage of administration of the pharmaceutical preparation of the present invention, as known in the art.

[0284] In the present invention, the term "prevention" refers to any action to inhibit or delay the onset of the disease targeted by the present invention (neurological diseases in the present disclosure) by administering the pharmaceutical composition of the present invention before the onset of the target disease.

[0285] In the present invention, the term "treatment" refers to any behavior that improves or beneficially changes the symptoms of the disease (neurological disease in the present invention) targeted by the present invention by administering the pharmaceutical composition of the present invention after the onset of the target disease. In the present invention, the term "improvement" refers to any behavior that reduces parameters associated with the disease targeted by the present invention (e.g., the degree of symptoms).

[0286] In the present invention, the term "subject" refers to an entity in need of treatment of a disease (not limited thereto, but in the present invention, as an example, it may refer to a subject in need of inhibition of neuronal cell damage and induction of angiogenesis in the brain). More specifically, it may be a mammal, including a human or a non-human mammal, such as a mouse, a rat, a dog, a cat, a horse, and a cow, but not limited thereto.

[0287] The following preferred embodiments and experimental examples are provided to assist in understanding the present invention. However, the following embodiments and experimental examples are only used to help better understand the present invention, and the scope of the present invention is not limited to the contents of the following embodiments and experimental examples. Example 1. Obtaining Conditioned Medium of Selectively Isolated Neural Stem Cells of the Present Invention, Cytokine Analysis and Liquid chromatography-mass spectrometry (LC / MS / MS) analysis. 1-1. Cultivating neural stem cells and obtaining conditioned medium.

[0288] Neural stem cells were isolated and cultured from adult brain tissue (temporal lobe tissue from non-lesional donors, classified as focal cortical dysplasia type IIIa). First, the surgical specimens were mechanically minced and then treated with an enzyme mixture containing papain (10 units / ml, Sigma), DNase I (0.1 mg / ml, Roche), and D,L-cysteine ​​(4 mg / ml, Sigma) at 37°C for 15 to 30 minutes. Subsequently, after a gentle grinding process, the mixture was filtered using a cell strainer (40 μm, BD Biosciences). After Percoll purification, the cells were cultured adherently in poly-L-ornithine (PLO)-coated culture dishes using DMEM / F12 medium supplemented with 1% B27 (Invitrogen), 1% penicillin / streptomycin mixture (Invitrogen), EGF (50 ng / ml, R&D Systems), bFGF (50 ng / ml, R&D Systems), and 0.5% FBS (Invitrogen). Subsequently, the cells were passaged using Accutase (PAA Laboratories, Austria) and cultured to 80% confluence. Using the above methods, various human neural stem cell lines were obtained, thereby creating different neural stem cell line banks.

[0289] After thawing, the frozen neural stem cells were seeded at a density of 6,000 cells per square centimeter in a T75 flask containing 20 ml of proliferation medium, which included 0.1 ml of FBS, 0.4 ml of B-27 TM Supplement (Xenofree; Gibco, A14867-01), 0.2 ml N-2 supplement (Gibco, 17502-048), 1 μL hEGF, 1 μL hbFGF2, 10 μL gentamicin in DMEM / F12 (Gibco, 11330-032). Neural stem cells were cultured in T75 flasks to 80% to 90% confluence and then washed three times with PBS. Subsequently, cells were detached using trypsin / EDTA, the cell number was measured, and then 9,000 cells / cm 2Inoculate into a T175 flask containing proliferation medium for subculture (CO2 incubator [Thermofisher (HERACELL VIOS160i)], 37.0°C, 5% CO2). After 48 hours of cell subculture, the obtained medium was replaced with a basic medium DMEM / F12 [Gibco (11330-032)] containing gentamicin, and then cultured in an incubator for 24 hours. After 24 hours of culture, all the medium was collected, transferred to a 50 mL tube, and centrifuged at 300 RCF for 3 minutes. The supernatant was carefully transferred to a new tube and used as a conditioned medium. 1-2. Cytokine analysis and liquid chromatography analysis

[0290] Cytokine analysis was performed using the conditioned medium obtained from stem cells in Example 1-1 to verify secreted proteins (ie, protein expression). The analysis was performed using the R&D Proteome Profiler Human XL Cytokine Array Kit and Raybio's Human Cytokine Array Kit according to the manufacturer's protocol.

[0291] In addition, in order to cross-validate the potency factors in the conditioned medium, the liquid chromatography analysis based on protein quantification was outsourced to eBiogen Inc. (Korea), where the analysis was performed. The analysis process can be briefly described as follows. First, for the proteomic analysis of the conditioned medium samples, the UPLC-Exactive device was used and three samples were used for a total of three runs. For data analysis, the LC-MS / MS data of each sample was analyzed using Proteome Discoverer, and the database search was performed using the Uniprot human database. The modifications analyzed included oxidation (M), carbamylation (N-terminus), and carbamoylmethylation (C). Among the identified proteins, sequences marked as Con (contaminant) and Rev (reverse) were filtered out before analysis.

[0292] like Figure 1 As briefly shown, cytokine analysis confirmed the expression levels of about 120 cytokines in the conditioned medium, while LC-MS / MS analysis revealed the presence of about 496 proteins. After many trials and errors, the inventors of the present invention successfully identified MCP-1, TIMP-1, GROα and IL-6 as efficacy factors among many substances commonly found in the conditioned medium.

[0293] The following examples specifically describe the use of neural stem cells in the present invention, stem cells selected based on a specific combination of four factors: MCP-1, TIMP-1, GROα, and IL-6, and the superior therapeutic effects (prevention or treatment of nervous system diseases) of stem cells selected based on these factors. 1-3. Selection of efficient neural stem cells for treating nervous system diseases according to the present invention

[0294] According to the present invention, the protein concentrations of TIMP-1 (SEQ ID NO: 1), MCP-1 (SEQ ID NO: 2), GROα (SEQ ID NO: 3) and IL-6 (SEQ ID NO: 4) were measured in the stem cell conditioned medium obtained from various human neural stem cell lines derived from the stem cell bank in Example 1-1. Neural stem cells containing high concentrations of these four factors were selected.

[0295] The specific method is as follows. First, 9,000 cells / cm 2 Neural stem cells (various human neural stem cell lines from a stem cell bank) were seeded into a T175 flask containing 35 mL of a basic DMEM / F12 medium [Gibco (11330-032)] containing 0.05% gentamicin and cultured for 24 hours in a CO2 incubator [Thermofisher (HERACELLVIOS160i)] at 37.0°C and 5% CO2. After 24 hours of culture, all the culture medium was collected, transferred to a 50 mL tube, and centrifuged at 300 RCF for 3 minutes. The supernatant was carefully transferred to a new tube and used as a conditioned medium.

[0296] The concentration (protein amount) of TIMP-1 (SEQ ID NO: 1), MCP-1 (SEQ ID NO: 2), GROα (SEQ ID NO: 3) and IL-6 (SEQ ID NO: 4) in the conditioned medium was quantitatively determined using an enzyme-linked immunosorbent assay (ELISA) method. ELISA (enzyme-linked immunosorbent assay) is a method for measuring protein amount by labeling antibodies or antigens with enzymes as markers and quantitatively measuring enzyme activity. This allows the intensity and amount of antigen-antibody reactions to be quantified. ELISA tests were performed using the following kits: Human MCP-1 Quantikine ELISA (R&D Systems, Cat no. DCP00), Human TIMP-1 Quantikine ELISA (R&D Systems, Cat no. DTM100), Human CXCL1 / GROα DuoSet ELISA (R&D Systems, Cat no. DY275-05) and Human IL-6 DuoSet ELISA (R&D Systems, Cat no. DY970-05). Testing was performed according to the manufacturer's instructions for each kit.

[0297] In the conditioned medium, the concentrations of the four proteins are measured, and when the following conditions are met at the same time, the stem cells (i.e., stem cells expressing the four protein factors) are identified and selected as high-efficiency neural stem cells: TIMP-1 concentration is 6,000pg / mL or higher, MCP-1 concentration is 6,000pg / mL or higher, GROα concentration is 500pg / mL or higher, and IL-6 concentration is 50pg / mL or higher.

[0298] Using the above method, NSC#1, NSC#2 and NSC#3 cell lines were successfully selected as efficient neural stem cells. Each of NSC#1, NSC#2 or NSC#3 neural stem cells was used, and three different batches of conditioned medium were prepared using the same method. These were used in the following experiments. Example 2. Creation of an in vitro stroke model 2-1. Isolation and culture of primary rat cortical neurons

[0299] Pregnant rats (E18, OriGenebio) were euthanized using CO2 gas, and then the embryos were removed by cesarean section using surgical scissors. After removing the embryos from the placental sac, the embryo skull was removed using forceps, and the brainstem and cerebellum tissues were carefully dissected under a microscope. After removing the meninges covering each hemisphere and removing the hippocampus, the cortical tissue was obtained. Then, 10mL 1XHBSS was added and the tissue was minced by pipetting with a 10mL serological pipette. After centrifuging the tissue to precipitate, the supernatant was discarded. Next, 3mL 1X HBSS was added and then pipetted. Next, trypsin-EDTA was added, the mixture was pipetted again, and then incubated in a 37°C water bath for 2 minutes. After centrifugation, the supernatant was discarded and the precipitate was resuspended in DMEM (containing 10% FBS and 1% P / S). The suspension was then filtered through a filter to obtain a single cell suspension, which was collected in a 50mL conical tube. After counting the number of detached cells, 1×105 cells / well were seeded onto PDL-coated plates containing DMEM (containing 10% FBS and 1% P / S). After 4 hours, half of the medium was replaced with complete cortical neuron medium (Neurobasal medium [Gibco (21103-049)] containing 2% B27 supplement [50X, serum-free, Gibco (17504-044)], L-glutamine and 1% penicillin / streptomycin [P / S]). After 12 hours, another half medium replacement was performed using the same medium. Subsequently, the cells were cultured for 3 days with a complete medium replacement once a day. 2-2. Creation of in vitro stroke model (OGD injury conditions)

[0300] The brain neurons isolated in Example 2-1 were seeded onto a 96-well plate and cultured with a growth medium for 7 days to allow the cells to mature into neurons. A medium without glucose was then added and the cells were exposed to 1% oxygen glucose deprivation (OGD) for 1 hour to establish an in vitro stroke model. Example 3. Analysis of the neuroprotective effect of neural stem cell conditioned medium in an in vitro stroke model

[0301] The inhibitory effect of in vitro neuronal cell death treated with the conditioned medium of the selected efficient neural stem cells according to the present invention was analyzed. Specifically, after the in vitro stroke model established in Example 2-2 was treated with the conditioned medium of the neural stem cells obtained in Example 1-3, the neuronal cell survival rate was measured by MTT assay. As a control group, growth medium (GM, 100 μl) not treated with OGD was used as a positive control. In addition, the incomplete medium (IM, 100 μl) described in Example 1-1 and the conditioned medium (CM, 100 μl) obtained from three batches of selected neural stem cells in Example 1-3 were used for analysis.

[0302] First, after treating the in vitro stroke model (OGD-induced cells) established in Example 2-2 with the corresponding test substances of each control group and experimental group, MTT solution (Sigma, M5655) was applied at a concentration of 0.5 mg / ml. The plate was incubated for 1 hour, during which the formation of formazan was observed under a microscope. The culture medium was removed and 100 μl of DMSO was added to each well. The formazan was then dissolved in DMSO in an incubator at 37°C and 5% CO2 for 30 minutes. The plate was placed in a microplate spectrophotometer (X-MARK microplate absorbance spectrophotometer [Bio-rad (168-1150)]) and the optical density (OD) value was measured at a wavelength of 570 nm.

[0303] like Figure 2 As shown, compared with normal neuronal cells (PC, positive control), the in vitro stroke model (NC, negative control) showed a significant increase in neuronal cell death induced by OGD. On the other hand, in the group treated with conditioned medium from three batches of neural stem cells under OGD injury conditions, a significant inhibition of neuronal cell death was observed.

[0304] This confirms that the proteins secreted by neural stem cells exhibit excellent neuroprotective effects on neuronal cells. Example 4: Effect of neural stem cell conditioned medium on efficacy factors after treatment with neutralizing antibodies in an in vitro stroke model Analysis of changes in neuroprotective effects

[0305] In order to confirm whether the neuroprotective effect of the highly effective neural stem cell conditioned medium observed in the in vitro stroke model (OGD-induced conditions) in Example 3 is regulated by the four combined efficacy factors of the present invention, the control group and the experimental group were treated with IM (incomplete medium, 100 μl), CM (conditioned medium, 100 μl) and CM+neutralizing antibodies (10 μg / mL, 100 μl) of each factor 1 hour after OGD-induced injury in the in vitro stroke model. After incubation for 24 hours, the neuroprotective effect was analyzed using the MTT assay (as described in Example 3).

[0306] The neutralizing antibody information of each factor used in this experiment is as follows: MCP-1Ab [Santa Cruz (sc-32771)], TIMP-1Ab [Santa Cruz (sc-21734)], GROαAb [Santa Cruz (sc-514065)], IL-6Ab [SantaCruz (sc-57315)].

[0307] The experimental results showed that cell death was increased in the in vitro stroke model induced by the negative control OGD (OGD+IM) compared with normal cells (control). However, in the group treated with conditioned medium (OGD+CM), cell death was significantly inhibited. When the conditioned medium was pre-incubated with 10 μg / well of neutralizing antibodies against MCP-1, TIMP-1, GROα or IL-6 and then applied to nerve cells, the neuroprotective effect of the conditioned medium (i.e., its ability to inhibit nerve cell death) was observed to be significantly reduced ( Figure 3a and 3b ).

[0308] Notably, even when only one of the factors, MCP-1, TIMP-1, GROα, or IL-6, was inhibited using neutralizing antibodies, the extent of cell death was either greater than that observed in the negative control (OGD+IM) or similar to that observed in the negative control, indicating that cell death was significant ( Figure 3a and 3b ). In other words, if even one of the four factors is absent, the neuroprotective capacity (neuroprotective effect) is largely lost. This confirms that the combination of the four factors (MCP-1, TIMP-1, GROα and IL-6) of the present invention must be present simultaneously. Example 5. Neuroprotective effect of neural stem cell conditioned medium in an in vitro spinal cord injury model (neuron Analysis of cell death inhibition

[0309] The neuroprotective effect of the conditioned medium was evaluated on spinal cord neurons subjected to cell death induced by reactive oxygen species (ROS), a secondary damaging factor associated with spinal cord injury. This analysis was performed in comparison to the results obtained in Example 3, in which the neural stem cell conditioned medium was applied to an in vitro stroke model. 5-1. Isolation and culture of primary rat spinal cord neurons (motor neurons)

[0310] After euthanizing pregnant rats (E15, purchased from Orient Bio) using CO2 gas, cesarean sections were performed with surgical scissors to remove the embryos. The embryos were removed from the placental sac and their bodies were fixed with forceps. The outer membrane of the spinal cord was carefully removed with forceps. After removing the outer membrane, the spinal cord was carefully separated from the rat body. The meninges and dorsal root ganglia surrounding the spinal cord were then removed to separate the pure spinal cord, which was transferred to a conical tube containing 15ml 1X HBS. The tissue was pelleted using a centrifuge and the supernatant was removed. 10ml 1X HBSS was added to the tube, then gently pipetted and centrifuged. After removing the supernatant, 3ml 1X HBSS was added and then pipetted. Trypsin-EDTA was then added, the mixture was pipetted again, and then placed in a 37°C water bath for 2 minutes. After centrifugation, the supernatant was removed and the pellet was resuspended in DMEM (containing 10% FBS and 1% P / S). The suspension was then passed through a 70 μm filter connected to a 50 mL conical tube to separate it into single cells. The separated cells were counted and then plated into plates. After at least 2 hours, it was confirmed that the cells had adhered to the plate. Subsequently, a half medium change was performed using complete motor neuron medium, and the process was repeated once a day for three days. After that, a full medium change was performed every three days. 5-2. Establishment of an in vitro spinal cord injury model induced by H2O2 and analysis of the neuroprotective effect of neural stem cell conditioned medium on spinal cord neurons

[0311] The spinal cord neurons isolated in Example 5-1 were exposed to ROS (H2O2, hydrogen peroxide 0.1mM), a secondary factor of spinal cord injury, for 1 hour to establish an in vitro spinal cord injury model. Specifically, the spinal cord neurons isolated in Example 5-1 were cultured in GM (growth medium), and then switched to IM (basal medium) containing 0.1mM H2O2 (hydrogen peroxide) and cultured for 1 hour to establish an in vitro spinal cord injury model.

[0312] Fresh IM (basal medium) was used as a control, and CM (conditional medium of neural stem cells obtained in Example 1-3) was used as an experimental group. After 6 hours, the spinal cord neuron survival effect (neuroprotective ability) was analyzed by MTT assay. The MTT assay was performed using the same method described in Example 3.

[0313] like Figure 4 As shown, compared with normal spinal cord neurons (PC, positive control), neuronal cell death was significantly increased under spinal cord injury conditions induced by H2O2 treatment (NC, negative control). However, under the same injury conditions, neuronal cell death was significantly inhibited in the group treated with the neural stem cell conditioned medium according to the present invention.

[0314] This confirms that the protein secreted by neural stem cells not only protects brain neurons, but also shows good protective effects on spinal cord neurons. Example 6. Neutralizing antibodies against effective factors in neural stem cell conditioned medium in an in vitro spinal cord injury model Analysis of changes in neuroprotective effects after treatment

[0315] In order to verify whether the neuroprotective effect of the conditioned medium observed in Example 5-2 is regulated by the four candidate effective factors of the present invention, neutralizing antibodies of each factor were added to the conditioned medium in the same manner as described in Example 4. Then, the changes in spinal cord neuronal cell death were observed using the same method as in Example 5-2.

[0316] At this time, the results were also compared with those obtained by treatment with general IgG (10 μg / ml, IgG [Santa Cruz (sc-2025)]).

[0317] like Figure 5a and 5b As shown, in an in vitro model of spinal cord injury induced by ROS (IM+H2O2), spinal cord neuron death was significantly increased compared with the normal cell group (PC). In contrast, in the treatment group with the neural stem cell conditioned medium (CM, containing all four factors) of the present invention, cell death was significantly reduced. When the conditioned medium was pre-incubated with neutralizing antibodies against MCP-1, TIMP-1, GROα or IL-6 and then treated with spinal cord neurons, the spinal cord neuron death inhibitory effect of the conditioned medium was significantly reduced ( Figure 5a and 5b Specifically, even when only one of MCP-1, TIMP-1, GROα, or IL-6 was neutralized with an antibody, a cell death level similar to that of the negative control group (IM) was observed ( Figure 5a and 5b ). This confirms that the combination of the four factors (MCP-1, TIMP-1, GROα and IL-6) of the present invention plays a key role in inhibiting the death of spinal cord neurons in the conditioned medium. Example 7. Effect of neural stem cell conditioned medium on the migration ability of endothelial cells and vascular cells

[0318] In angiogenesis, the migration of endothelial cells is an essential step in the process of angiogenesis, which occurs simultaneously with the degradation of the extracellular matrix components around the blood vessels. Using HUVEC (human umbilical vein endothelial cells), the changes in the endothelial cell migration ability induced by the neural stem cell conditioned medium obtained in Example 1-3 were analyzed by wound healing assay. HUVEC was seeded into 12-well plates using endothelial cell growth medium 2 [PromoCell (C-22211)] supplemented with growth medium 2 supplement mixture [PromoCell (C-39216), 2.5%] and penicillin-streptomycin (P / S, 1%). The cells were then cultured in an incubator at 5% CO2 and 37°C for 16 hours. After the cells were cultured to a confluence of 90%, the supernatant was removed using an aspiration system. Subsequently, 2 mL of IM (incomplete medium, endothelial cell growth medium 2 [PromoCell (C-22211)] supplemented with penicillin-streptomycin (P / S, 1%)) was added, and the cells were starved (cell nutrient deprivation) for 7 hours. A cross-shaped scratch was made in each well using a 200 μL pipette tip. The supernatant was then removed using an aspiration system, and 1.5 mL of CM (conditioned medium) was added to each well. Images of the cross-shaped area were captured under a microscope, focusing on the center of the scratch along the vertical and horizontal axes. After 17 hours, images were captured at the same position as the initial photograph. The distance between the scratch edges was measured, and the distance difference between 0 hours and 17 hours was calculated and converted into a percentage. These data were then organized into graphs.

[0319] like Figure 6 and 7 As shown, the group treated with neural stem cell conditioned medium (repeated in three separate batches) exhibited a significant enhancement in endothelial cell migration compared with IM (negative control, NC). Example 8. Neutralizing antibodies against functional factors in neural stem cell conditioned medium inhibit endothelial cell migration Analysis of changes in the system

[0320] In order to determine whether the increase in endothelial cell (HUVEC) migration observed by the conditioned medium in Example 7 is regulated by the four functional factors of the present invention, neutralizing antibodies against each factor were added to the conditioned medium in the same manner as described in Example 4. The changes in endothelial cell migration ability were analyzed using the same method as in Example 7.

[0321] like Figure 8a and 8bAs shown, compared with the group treated with conditioned medium (CM), the treatment of conditioned medium with neutralizing antibodies against MCP-1, TIMP-1, GROα or IL-6 significantly reduced endothelial cell migration. In other words, when any one of the four factors is absent, the migration ability of endothelial cells is substantially lost. Thus, it is verified that the combination of the four factors (MCP-1, TIMP-1, GROα and IL-6) in the present invention must exist simultaneously. Example 9. Evaluation of the tube-forming ability of endothelial cells induced by treatment with neural stem cell conditioned medium

[0322] In a broad sense, angiogenesis refers to a series of processes that allow new blood vessels to form. This involves the proliferation and migration of cells that make up existing blood vessels, ultimately producing new tubular structures that play a vital role in nutrition and blood supply. This process is closely related to the regeneration of damaged tissues and can serve as an important indicator for evaluating the overall therapeutic effect. In order to verify the tube-forming ability of vascular cells induced by proteins secreted by neural stem cells, neural stem cell conditioned medium was applied to vascular endothelial cells to evaluate whether the tube-forming ability could be enhanced.

[0323] Matrigel was used to evaluate the tube-forming ability of vascular endothelial cells induced by neural stem cell conditioned medium. After coating a 96-well plate with Matrigel, 1×104HUVEC cells were mixed with the culture medium and distributed into the wells. After 10 minutes, the attachment of the cells was confirmed and the culture medium was replaced with IM (incomplete culture medium) to induce cell starvation for 2 hours. The culture medium was then replaced with CM (conditioned culture medium) and microscopic images were captured (0 hours). After 20 hours, microscopic images were taken for each sample and the total length of the tube formed was measured using the ImageJ program. The mean and standard deviation were calculated and the results were summarized in a chart.

[0324] like Figure 9a and 9b As shown, it was confirmed that when the neural stem cell conditioned medium according to the present invention was applied, the tube-forming ability of endothelial cells was significantly enhanced. Example 10. Tubular formation of endothelial cells after treatment with neutralizing antibodies against efficacy factors in neural stem cell conditioned medium Analysis of changes in ability

[0325] In order to verify whether the increase in the tube-forming ability of endothelial cells induced by the conditioned medium confirmed in Example 9 can be regulated by the four combined efficacy factors of the present invention, each neutralizing antibody against each factor was treated in the conditioned medium according to the same method as described in Example 4. Subsequently, the changes in the tube-forming ability of endothelial cells were observed using the same method as described in Example 9.

[0326] like Fig.10a and 10bAs shown, in the group to which the neutralizing antibody against TIMP-1, GROα or IL-6 was added to the medium, the tube-forming ability of endothelial cells was significantly reduced compared with the group treated with conditioned medium (CM).

[0327] In summary, the combination of the four factors of the present invention (MCP-1, TIMP-1, GROα and IL-6) plays a vital role in regulating the migration ability of endothelial cells, and it has been confirmed and verified that these factors can enhance the tube-forming ability of endothelial cells (especially TIMP-1, GROα and IL-6). In other words, if any one of the four factors is missing, the migration ability of endothelial cells will be significantly reduced. This verifies that the combination of the four factors of the present invention (MCP-1, TIMP-1, GROα and IL-6) must exist simultaneously. These results confirm the efficacy of these factors as agents that play a key role in the regeneration and protection of damaged neural tissue. Example 11. Evaluation of the effect of injection of selected neural stem cells according to the present invention in an in vivo spinal cord injury animal model. Behavioral improvements after cell death.

[0328] In order to conduct behavioral assessments after injection of neural stem cells in an in vivo spinal cord injury animal model, a spinal cord injury model was created and tested using the following method. Ten-week-old rats were anesthetized by inhalation using approximately 50 ml of isoflurane mixed with oxygen (O2). After exposing the thoracic vertebrae (T8-T10), the muscles attached to the spine were separated, and a laminectomy was performed to remove the lamina of the T9 vertebra. Subsequently, an impactor was used to apply pressure to the dorsal dura mater of the spinal cord to create a spinal cord injury model. Seven days later, in the spinal cord injury model, a vehicle was administered to the control group (G1), and 1×10 2 (G3) or 3×10 2 (G2) Neural stem cells (highly effective neural stem cells selected according to the present invention). After neural stem cell transplantation, neurobehavioral assessment and neuropathic pain assessment were performed weekly. For neurobehavioral assessment, BBB scoring test (Basso, Beattie and Bresnahan scoring test) and rotarod test were performed. For neuropathic pain assessment, von Frey test was performed.

[0329] Specifically, the BBB scoring test was performed 7 times in total, with evaluation performed before administration of the test substance and once a week thereafter. The scoring of the test was based on the method evaluated by DM Basso et al. (J Neurotrauma. 1995 Feb; 12(1): 1-21).

[0330] The rotarod test was performed 7 times in total, evaluated before applying the test substance, and then performed once a week, using the rotarod latency test. The animal was carefully placed on a rotarod treadmill (JD-A-07RA5, BS Technolab Inc., South Korea), and the rotation speed was set to increase from 4rpm to 40rpm at stable intervals within 300 seconds. The time when the animal loses balance and falls to the ground is measured in seconds. If the animal falls to the ground, the time is automatically measured using a sensor on the floor of the rotarod treadmill. For each animal, after measuring once, repeat the measurement once again, a total of three times, and use the mean value to test.

[0331] The von Frey test was performed before the test substance was applied, and then once a week for a total of seven times. The animals were stabilized in a plastic box with a fine metal mesh on the floor, and after they were adapted to the environment, the pain level was assessed by observing the withdrawal reaction to the mechanical stimulation using the von Frey filament. The von Frey filament was used to apply mechanical stimulation to the animal's paw through the hole on the wire mesh floor of the box. A positive reaction was defined as a sharp retraction of the foot, scratching or shrinking of the animal in response to the filament stimulation. The filament strength during the positive reaction was then measured. In order to ensure that the measurement was accurate, pressure was applied in about 3-5 seconds until the von Frey filament was bent. The filament strength increased in the following order: 0.6, 1, 1.4, 2, 4, 6, 8, 10, 15, 26, 60, 100, 180 and 300g until a positive reaction was observed. For each animal, a measurement was initially performed once, and then additional measurements were performed every other week, for a total of three measurements, and the average value was used for testing.

[0332] like Fig.11a and Fig.11b As shown, the results of the BBB behavior score test and the rotarod test for evaluating motor ability confirmed that the G2 and G3 groups receiving the highly efficient neural stem cells selected according to the present invention showed significant improvement in behavior compared to the vehicle control group (G1). In addition, it was observed that this effect difference became more obvious 5 weeks after the neural stem cell transplantation.

[0333] In addition, if Fig.11c As shown, the results of neuropathic pain assessment (von Frey test) showed that starting from one week after transplantation, both the G2 and G3 groups receiving the highly efficient neural stem cells selected according to the present invention showed significant improvement, showing a clear pain relief effect.

[0334] Thus, it was confirmed and verified that the neural stem cells selected according to the present invention can improve behavioral disorders caused by spinal cord injury.However, no significant difference was observed between the G2 and G3 groups in which the number of neural stem cell injections (transplantations) increased. Example 12. Validation in an in vivo ischemic stroke animal model (middle cerebral artery occlusion rat model, MCAO) The neural stem cells selected according to the present invention have the effect of reducing brain tissue shrinkage (damage) after injection.

[0335] After the neural stem cells selected according to the present invention were injected into an in vivo ischemic stroke animal model, the following experiments were performed to evaluate the therapeutic effect of the disease and the improvement degree of brain tissue damage. 12-1. Establishment of MCAO ischemic stroke animal model

[0336] After anesthetizing 8-week-old Sprague-Dawley (SD) rats with a mixture of isoflurane and oxygen, the neck skin was shaved to expose the area. After disinfection with an alcohol swab, a 3 cm incision was made along the midline of the neck with surgical scissors. After fixing the salivary glands toward the trachea, the muscle tissue was clamped with a curved Crile clamp to expose the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The common carotid artery (CCA) was exposed using a curved iris forceps and a silk suture was looped around it. Similarly, the external carotid artery (ECA) and internal carotid artery (ICA) were exposed and sutures were looped around them. The ligatures around the CCA and ECA (excluding the ICA) were tightened to secure them in place. A syringe was used to create a small hole at the junction of the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The silicon-coated MCAO catheter was then inserted into the ICA from the bifurcation of the CCA. The ICA was fixed by tying the ICA to the MCAO catheter. The salivary glands were repositioned and the incised skin was sutured. One hour after occlusion, the same approach was performed: under anesthesia, the CCA, ECA, and ICA were exposed, the previously fixed ligatures were loosened, and the inserted MCAO sutures were removed to start reperfusion. After hemostasis was achieved, the skin was sutured. One day after reperfusion, neurological symptoms were evaluated to confirm the presence of cerebral infarction. 12-2. Experimental method for confirming the therapeutic effect of MCAO ischemic stroke using stem cells selected according to the present invention

[0337] Seven days after the MCAO ischemic stroke animal model described in Example 12-1 was established, 5×105 high-efficiency neural stem cells selected according to the present invention were transplanted into the cerebral ventricle. The experimental protocol for neurobehavioral assessment and tissue analysis of the above animal model is as follows. Fig.12aThe timeline shown in the figure is briefly summarized. For three groups - control group (wild-type normal rats), MCAO+HBSS (Hanks balanced salt solution, 20 μL 1×HBSS) and MCAO+NSC (highly efficient neural stem cells selected according to the present invention, 5×105 cells suspended in 20 μL 1×HBSS) - neurobehavioral assessments were performed one week before MCAO surgery, one week before HBSS or NSC transplantation, and then 1, 2, 3 and 4 weeks after transplantation. The three groups of rats were killed on days 1, 14 and 28 after transplantation. Tissue samples were prepared for immunohistochemical staining and immunofluorescence (IF). Triphenyltetrazolium chloride (TTC) staining was also performed on day 28, and the specific details of the method are provided in each of the corresponding examples. 12-3. Confirmation of therapeutic effect on brain damage in MCAO ischemic stroke model after administration of neural stem cells selected according to the present invention

[0338] The brain tissues obtained from the control group and experimental group rats were sliced ​​using brain matrix, and tissue sections were prepared at intervals of 2 mm along the frontal pole to the parietal lobe surface. The brain tissue sections were stained by placing them in a 2% (in phosphate buffered saline, PBS) 2,3,5-triphenyltetrazolium chloride (TTC) solution and incubating them at 37°C for 1 hour. The stained tissue sections were placed in a 4% (in PBS) paraformaldehyde solution and fixed at 4°C for 1 day. Subsequently, the brain tissues were placed on OHP films in turn and photographed. The infarct volume of the brain tissue sections was calculated using the following percentage (%) calculation formula. The infarct volume was measured using the ImageJ program (National Institutes of Health, USA).

[0339] Infarct volume (% of infarct volume) = {(contralateral hemisphere volume - non-ischemic ipsilateral hemisphere volume) / contralateral hemisphere volume} × 100

[0340] The results of TTC staining performed 28 days after the injection of the stem cells selected according to the present invention showed that the infarct volume of the group injected with high-efficiency neural stem cells was significantly and significantly reduced compared with the tissue damage area of ​​the HBSS-treated group ( Figure 12b and Fig.12c ). The results confirmed and verified that transplantation of the highly efficient neural stem cells selected according to the present invention reduced the infarct area of ​​ischemic stroke. Example 13. Administration of the drug according to the present invention in an in vivo ischemic stroke animal model (midbrain occlusion rat model, MCAO) Evaluation of behavioral improvements using neural stem cells selected by the invention

[0341] In the animal experiment conducted according to the protocol of Example 12-2, after administration of the neural stem cells selected according to the present invention, neurobehavioral assessment was performed every other week to evaluate the effect of the neural stem cells on the improvement of brain damage.

[0342] The rotarod test is a method for evaluating the balance and motor coordination of experimental animals. The experimental animals began training three times a day three days before the MCAO operation, and animals showing good learning performance were selected to create the MCAO model. The animal experiment was then carried out according to the scheme outlined in Example 12-2. The experimental animals were then placed on a rotarod treadmill accelerated from 4rpm to 40rpm, and the time they fell was measured. The maximum observation time for each experiment was set to 3 minutes. The measurement was terminated under the following conditions: 1) when the experimental animal fell completely off the treadmill, and 2) when the experimental animal rotated continuously for more than three laps while grabbing the treadmill. The measurement was re-performed under the following conditions: 1) when the experimental animal ran in the opposite direction, and 2) when the experimental animal jumped off the treadmill or behaved absent-mindedly and did not walk correctly. If the same result is obtained when re-measured, the animal is excluded from the group.

[0343] The passive avoidance test is a method used to assess short-term and long-term memory in experimental animals. The passive avoidance test is performed by conducting a training trial and then a memory test trial the next day. In the training trial, the animal is allowed to adapt to the dark room without lighting for 30 seconds, and then the light is turned on. After a 15-second adaptation period, the door that allows the test animal to enter the dark room is opened for 300 seconds. When the test animal enters the dark room, the door is closed and a 15-second adaptation period is given, followed by a 15-second electric shock at 0.3mA. If the test animal does not move, it is observed until it eventually moves. The next day, a memory test is performed to assess memory and cognition. During this test, electric shocks are not applied to avoid stressing the animal. The memory test is performed under the same conditions as the learning test to ensure consistency. In the learning test, the time required for the animal to move to the dark room after the light is turned on is measured.

[0344] The adhesive removal test is a method used to assess tactile responses and asymmetry in experimental animals. Paper dot stickers (9 mm in diameter) are attached to the front paws of experimental animals, and the time required for the animals to recognize and remove the stickers is measured. Measurements are performed separately for the right and left front paws, and the maximum duration of each experiment is set to 1 minute. The procedure is repeated three times, and the average value is calculated (excluding values ​​outside the median ± 50% of the raw data).

[0345] like Fig.13a As shown, the results of the rotarod test performed to evaluate motor function showed that the motor ability of the high-efficiency neural stem cell injection group according to the present invention was significantly improved compared with the HBSS injection group.

[0346] like Fig.13b As shown, the results of the passive avoidance test performed to evaluate cognitive and memory abilities confirmed that the group injected with the high-efficiency neural stem cells according to the present invention showed improved cognitive memory abilities compared with the group injected with HBSS.

[0347] like Fig.13c and 13d As shown, the results of the adhesive removal test conducted to evaluate sensory and motor abilities confirmed that in the highly efficient neural stem cell transplantation group according to the present invention, the sensory and motor functions of the injured forelimbs were significantly improved, and particularly significant improvements were observed at 2 weeks. In other words, it was confirmed that the neurobehavioral deficits were significantly alleviated in the highly efficient neural stem cell transplantation group according to the present invention. Example 14. In vivo ischemic stroke animal model (midbrain occlusion rat model, MCAO) by injection of root Analysis of the histological improvement effect (neuronal expression) of the selected neural stem cells according to the present invention

[0348] In the animal experiment performed according to the protocol of Example 12-2, brain tissue was collected, fixed with 4% PFA using conventional methods, and paraffin blocks were prepared. For various immunofluorescence staining, tissue sections were made into 5 μm thickness, placed on slides, and dewaxed before NeuN staining to evaluate the increase or decrease of neuronal cells.

[0349] The specific method is as follows. First, the frozen rat brain tissue sections were washed three times with 1X PBS for 5 minutes each time. The tissue samples were incubated with 50% ethanol at room temperature for 30 minutes and then treated with 2.5% normal goat serum (VECTOR, S-1012) for 1 hour. The samples were treated with the primary antibody NeuN Ab (dilution: 1:100, [abcam (ab177487)]) and incubated overnight at 4°C. Before applying the secondary antibody, the samples were incubated with 1% normal goat serum for 15 minutes. Afterwards, the samples were incubated with the secondary antibody at room temperature for 2 hours. The secondary antibody (goat anti-rabbit IgG (H+L) Alexa Fluor 594) was diluted in 1X PBS at a ratio of 1:500 and used for incubation. 4',6-diamidino-2-phenylindole (DAPI) solution (SIGMA, D9564) was diluted in PBS at a ratio of 1:100,000 and incubated for 5 minutes. Finally, the immunofluorescence-stained brain tissue slides were mounted with VECTASHIELD mounting solution (VECTOR Laboratories, H-1400) and covered with coverslips (Deckglδser, 24x32 mm). Then, three areas were observed at a magnification of 400 times using a confocal laser microscope (Leica): the ipsilateral area of ​​the cortical infarct area and the non-infarct area of ​​the contralateral area, which were randomly selected. The tissue areas observed in the damaged ipsilateral area and the undamaged contralateral area were as follows: Fig.14a shown.

[0350] like Fig.14b and Fig.14cAs shown, the results confirmed that the group injected with HBSS had a reduced number of neurons compared to the control group. Compared to the HBSS group, the group implanted with the efficient neural stem cells according to the present invention showed a significant increase in neuronal marker expression, and it was found that the number of neurons increased to the level observed in the control group. Therefore, it was confirmed and verified that the transplantation of the efficient NSC according to the present invention has an excellent promoting effect on the recovery and improvement of damaged neuronal cells. Example 15. In vivo ischemic stroke animal model (midbrain occlusion rat model, MCAO), injection according to the present invention Analysis of the amelioration of reactive gliosis by selected stem cells (astrocytes, microglia).

[0351] In the animal experiment conducted according to the protocol of Example 12-2, brain tissues were collected, and in order to evaluate the changes in reactive gliosis caused by the injection of the highly efficient stem cells of the present invention, the damaged cerebral cortex area was immunohistochemically stained with GFAP antibody and Iba1 antibody, respectively (see Fig.14a Increased reactive gliosis by astrocytes was assessed by measuring GFAP (glial fibrillary acidic protein) levels by immunohistochemical staining ( Fig.15a and Fig.15b ), and the increase in reactive gliosis by microglia was assessed by measuring Iba1 (ionized calcium binding adaptor molecule 1) levels ( Fig.15c and Fig.15d Immunofluorescence tissue staining was performed using primary antibody GFAP Ab ([NSJ Bioreagents (V2129)], dilution 1:100) or Iba1 Ab ([abcam (ab178846)], dilution 1:500), and except for using these specific antibodies, the rest of the operation was the same as described in Example 16.

[0352] like Fig.15a , Fig.15b , Fig.15c and Fig.15d As shown, the number of reactive astrocytes in the HBSS group increased compared with the control group. However, in the group transplanted with the efficient neural stem cells according to the present invention, the expression of reactive astrocyte markers decreased compared with the HBSS group. In addition, the number of reactive microglia in the HBSS group increased compared with the control group. However, in the group transplanted with the efficient neural stem cells of the present invention, the expression of reactive microglia markers decreased compared with the HBSS group. In other words, it was confirmed that the transplantation of the efficient neural stem cells selected according to the present invention resulted in a significant reduction in reactive gliosis in the tissue, indicating that tissue damage was significantly improved. Example 16. In vivo ischemic stroke animal model (midbrain occlusion rat model, MCAO) was induced by injection of root Analysis of the cell death-improving effect of the selected neural stem cells according to the present invention

[0353] In the animal experiment conducted according to the protocol of Example 12-2, brain tissues were collected, and in order to evaluate the extent of improvement of cell death in damaged tissues by injecting high-efficiency neural stem cells according to the present invention, the tissues were homogenized, and the expressions of proteins BAX, Bcl2, and cleaved caspase-3, which are associated with cell survival and death, were evaluated by Western blotting.

[0354] Brain tissue was dissolved in RIPA buffer (ChemCruz, sc-24948). The protein concentration of the sample was quantified using Bradford protein assay dye (BIO-RAD, #5000006). Equal amounts of protein were loaded onto OneGel PAGE Kit Plus (BIOSOLUTION) and separated using 1X Tris-Glycine-SDS buffer (DYNE BIO, CBT3141) according to the manufacturer's protocol. The separated protein was transferred to a nitrocellulose membrane in 1X Tris-glycine buffer (DYNE BIO, CBT3291) containing methanol (DAEJUNG, 5558-4410). Protein separation and transfer were performed using Tetra vertical electrophoresis and blotting system (BIORAD). The membrane was blocked with 5% skim milk (CellNest, CNS109-0500) in Tris-buffered saline (DYNE BIO, CBT3051) containing Tween-20 (VWR LIFE SCIENCE, 0777-1L) for 1 hour at room temperature. The membrane was then incubated with primary antibodies in 5% bovine serum albumin (BSA) (SIGMA, A-2153) at 4°C overnight. The primary antibodies used were: Bax Ab ([Santa Cruz (sc-7480)], dilution 1:1,000), Bcl-2 Ab ([Santa Cruz (sc-7382)], dilution 1:100), β Ab ([Cell signaling (9664)], dilution 1:1,000) and Cleaved-caspase3 Ab ([Santa Cruz (sc-47778)], dilution 1:1,000). Then, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:10,000) at room temperature for 1 hour. The detection kit solution (WESTSAVEup, LF-QC0101) was added to the membrane, and the signal was detected using X-ray film (AGFA) or a detection system (JPI Healthcare Co., JP-33). The bands were quantified using ImageJ software and normalized by the signal of β-actin.

[0355] like Fig.16aand Fig.16b As shown, the Western blot results confirmed that the expression of cell death-related proteins Bax and Cleaved-caspase3 in the HBSS group increased, while the expression of survival-related protein Bcl2 decreased compared with the control group. In contrast, the group transplanted with the efficient stem cells selected according to the present invention showed a decrease in the expression of Bax and Cleaved-caspase3, while an increase in the expression of Bcl2 compared with the HBSS group. In particular, in the group transplanted with the efficient stem cells according to the present invention, it was found that the levels of decrease in the expression of Bax and Cleaved-caspase3 and the level of increase in the expression of Bcl2 were similar to those observed in the control group.

[0356] Therefore, based on the above results, it was confirmed and verified that the injection of the high-efficiency stem cells according to the present invention significantly reduced tissue damage caused by ischemia, thereby showing a good effect of alleviating tissue damage. Example 17. In vivo ischemic stroke animal model (midbrain occlusion rat model, MCAO) by injection of root Analysis of the effect of stem cells selected according to the present invention on cerebral vascular recovery.

[0357] In the animal experiment performed according to the protocol of Example 12-2, brain tissue was collected, and in order to evaluate the extent of brain vascular restoration by injection of the highly efficient stem cells according to the present invention, CD31 immunofluorescence staining (for vascular staining) was performed in the cerebral cortex. Immunofluorescence staining was performed using CD31Ab ([abcam (ab28364)], dilution 1:50) as the primary antibody, and except for this change, it was performed in the same manner as Example 14.

[0358] like Fig.17a and Fig.17b As shown, CD31 immunohistochemical staining results showed that the vascular expression in the stroke injury tissue of the HBSS group was significantly reduced compared with the control group. However, in the group receiving the efficient stem cells selected according to the present invention, the vascular expression was significantly increased compared with the HBSS group. Based on these results, it was confirmed and verified that the transplantation of the efficient stem cells selected according to the present invention induced angiogenesis, showing highly effective results. On the contrary, in the tissue (contralateral tissue) where cerebral infarction did not occur, there was no significant difference in the degree of vascular expression between the three groups.

[0359] In summary (see Fig.18), the inventors have confirmed that by selecting and separating stem cells through the efficacy factors (MCP-1, TIMP-1, GROα and IL-6) of the present invention and transplanting the selected stem cells, the prevention of cranial nerve damage can be effectively enhanced, and stroke (a typical nervous system disease) and angiogenesis that may be secondary to brain damage can be promoted. In addition, the present invention also confirms that not only the brain histology and biochemical recovery effects of stroke damage are significantly improved, but also behavioral sensation and motor ability are significantly improved. In other words, the specific neuroprotective ability of factors in neurons and endothelial cells, as well as their ability to promote angiogenesis, have been confirmed. In addition, it has been confirmed that neural stem cells have a low possibility of inducing immune rejection and have a strong immunosuppressive ability.

[0360] In other words, the inventors have confirmed that transplantation of neural stem cells selected according to the present invention has a therapeutic effect on neurological diseases including spinal cord injury and stroke. This is verified by an increase in the survival rate of nerve and vascular cells in damaged tissues and by improving behavior by reducing reactive gliosis, immune cells and cell death caused by neuronal loss. Therefore, not only by isolating and culturing neural stem cells, but also by identifying specific efficacy factors secreted by isolated and cultured neural stem cells, the inventors have proposed a new treatment strategy that can treat neurological diseases more significantly more efficiently and with high performance.

[0361] Based on the above results (see Fig.18 ), those skilled in the art can clearly understand that the specific combination of four factors, MCP-1, TIMP-1, GROα and IL-6, using neural stem cells in the present invention significantly enhances the therapeutic effect of neural stem cells in the treatment of various nervous system diseases and symptom improvement.

[0362] The description of the present invention provided above is for illustrative purposes only, and those skilled in the art to which the present invention belongs will readily appreciate that the present invention can be modified into other specific forms without changing the technical spirit or basic features of the present invention. Therefore, the above embodiments should be understood to be exemplary in all aspects, rather than restrictive. [Industrial Applicability]

[0363] According to the present invention, the use of stem cells showing increased expression or activity of TIMP-1, MCP-1, GROα and IL-6 factors or the use of substances that regulate (especially upregulate) these factors in stem cells can effectively (effectively) and efficiently treat nervous system diseases. Therefore, the present invention has industrial applicability.

Claims

1. A method for selecting highly efficient stem cells for treating nervous system diseases, comprising the step of measuring the expression levels of TIMP-1 (TIMP metallopeptidase inhibitor 1), MCP-1 (monocyte chemoattractant protein-1), GROα (growth regulated oncogene α) and IL-6 (interleukin 6) proteins or their genes. 2 . The method according to claim 1 , wherein the TIMP-1, MCP-1, GROα and IL-6 proteins or genes thereof are highly expressed in the high-efficiency stem cells for treating the nervous system diseases.

3. The method according to claim 1, wherein the stem cell is selected from the group consisting of mesenchymal stem cells, adult stem cells, dedifferentiated stem cells, embryonic stem cells, induced pluripotent stem cells, neural stem cells, tissue-derived stem cells, and any combination of two or more thereof.

4. The method according to claim 1, wherein the nervous system disease is one or more selected from the group consisting of ischemic brain disease, degenerative nervous system disease, nerve injury disease, peripheral nerve disease and traumatic brain injury.

5. The method according to claim 4, wherein the ischemic brain disease is one or more selected from the group consisting of stroke, cerebral infarction, cerebral ischemia, cerebral embolism, cerebral thrombosis, transient ischemic attack, head trauma, cerebral circulation and metabolic disorders, cerebral coma, cerebral hemorrhage, neonatal intraventricular hemorrhage (IVH), cerebral arteriosclerosis, hypoxic brain damage and neonatal hypoxic-ischemic brain damage.

6. The method of claim 4, wherein the degenerative neurological disease is one or more selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, striatonigral degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, cortical basal degeneration, diffuse Lewy body disease, Guam Parkinson-ALS-dementia syndrome, and Pick's disease.

7. The method according to claim 4, wherein the neurological injury disease is one or more selected from the group consisting of epilepsy, spinal cord injury disease, behavioral disorder, developmental disorder, intellectual disability, Down syndrome and schizophrenia.

8. The method according to claim 1, wherein the method comprises the following steps: (i) culturing stem cells; (ii) measuring the concentrations of TIMP-1, MCP-1, GROα and IL-6 proteins in the culture of the stem cells of step (i); and (iii) If TIMP-1, MCP-1, GROα and IL-6 proteins are highly expressed in the culture, the stem cells are identified and classified as highly efficient stem cells for the treatment of nervous system diseases.

9. The method of claim 8, wherein step (iii) comprises: The culture obtained in step (ii) is measured, and if the concentration of the TIMP-1 protein is 6,000 pg / mL or higher, the concentration of the MCP-1 protein is 6,000 pg / mL or higher, the concentration of the GROα protein is 500 pg / mL or higher, and the concentration of the IL-6 protein is 50 pg / mL or higher, the stem cells are determined to be high-efficiency stem cells.

10. A method for determining in vitro the activity of stem cells for treating nervous system diseases, wherein the method comprises the steps of measuring and comparing the expression levels of TIMP-1, MCP-1, GROα and IL-6 proteins or genes thereof in the stem cells.

11. A marker composition for selecting highly efficient stem cells for treating nervous system diseases, comprising TIMP-1, MCP-1, GROα and IL-6 proteins or genes thereof.

12. A composition for selecting highly efficient stem cells for treating nervous system diseases, comprising a substance for measuring the expression levels of TIMP-1, MCP-1, GROα and IL-6 proteins or their genes.

13. The composition according to claim 12, wherein the substance used to measure the expression level of the protein is a specific antibody against the protein. 14 . The composition according to claim 12 , wherein the measurement of the expression level of the gene is performed by a substance for measuring the mRNA expression level of the gene. 15 . The composition according to claim 14 , wherein the substance for measuring the expression level of the mRNA is a probe or a primer set that specifically binds to the mRNA.

16. A kit for selecting highly efficient stem cells for treating nervous system diseases, comprising substances for measuring the expression levels of TIMP-1, MCP-1, GROα and IL-6 proteins or their genes.

17. The kit according to claim 16, wherein the kit comprises instructions describing the method according to claim 1.

18. A method for screening a preventive or therapeutic agent for a nervous system disease, comprising: (a) contacting the isolated stem cells with a test substance; (b) measuring the expression levels of TIMP-1, MCP-1, GROα and IL-6 proteins or genes thereof in the stem cells contacted with the test substance and a control group of stem cells not contacted with the test substance; and (c) selecting a test substance that increases the expression or activity level of TIMP-1, MCP-1, GROα and IL-6 protein or its gene as compared to the cells of the control group.

19. The method of claim 18, wherein the stem cells are selected from the group consisting of mesenchymal stem cells, adult stem cells, dedifferentiated stem cells, embryonic stem cells, induced pluripotent stem cells, neural stem cells, tissue-derived stem cells, and any combination of two or more thereof.

20. The method according to claim 18, wherein: The measurement of the protein expression or activity level is performed using one of the methods selected from the group consisting of Western blotting, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation, complement fixation assay, flow cytometry and protein chip method.

21. The method according to claim 18, wherein: The measurement of the gene expression or activity level is performed using a method for measuring the mRNA expression or activity level.

22. The method of claim 21, wherein the measurement of the mRNA expression or activity level is performed using one of the methods selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative real-time RT-PCR, in situ hybridization, RNase protection assay (RPA), Northern blotting, Southern blotting, RNA sequencing, DNA chip method, and RNA chip method.

23. A pharmaceutical composition for preventing or treating nervous system diseases, comprising stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof; or a culture thereof as active ingredients.

24. The composition according to claim 23, wherein the culture is a conditioned medium comprising TIMP-1, MCP-1, GROα and IL-6 proteins secreted by the stem cells.

25. The composition of claim 23, wherein the stem cell is selected from the group consisting of mesenchymal stem cells, adult stem cells, dedifferentiated stem cells, embryonic stem cells, induced pluripotent stem cells, neural stem cells, tissue-derived stem cells, and any combination of two or more thereof.

26. A composition for promoting the activity of stem cells for preventing or treating nervous system diseases, comprising as an active ingredient a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof.

27. The composition according to claim 26, wherein the substance that increases the expression or activity of the protein is one or more expression vectors, the expression vectors comprising a polynucleotide encoding the TIMP-1 protein; a polynucleotide encoding the MCP-1 protein; a polynucleotide encoding the GROα protein; and a polynucleotide encoding the IL-6 protein.

28. The composition of claim 26, wherein the activity of the stem cell is one or more selected from the group consisting of self-renewal, neuroprotective ability, inhibition of neuronal apoptosis, endothelial cell migration, angiogenesis, anti-inflammatory activity, and inhibition of reactive gliosis.

29. A method for promoting the activity of stem cells for preventing or treating nervous system diseases, comprising the step of treating isolated stem cells with a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof.

30. The method according to claim 29, wherein the substance that increases the expression or activity of the protein is one or more expression vectors, the expression vectors comprising a polynucleotide encoding the TIMP-1 protein; a polynucleotide encoding the MCP-1 protein; a polynucleotide encoding the GROα protein; and a polynucleotide encoding the IL-6 protein.

31. The method of claim 29, wherein the activity of the stem cells is one or more selected from the group consisting of self-renewal, cytoprotective ability, inhibition of apoptosis, endothelial cell migration, angiogenesis, anti-inflammatory activity, and inhibition of reactive gliosis.

32. A pharmaceutical composition for preventing or treating a nervous system disease, comprising isolated stem cells; and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as active ingredients.

33. The composition according to claim 32, wherein the substance that increases the expression or activity of the protein is one or more expression vectors, the expression vectors comprising a polynucleotide encoding the TIMP-1 protein; a polynucleotide encoding the MCP-1 protein; a polynucleotide encoding the GROα protein; and a polynucleotide encoding the IL-6 protein.

34. A pharmaceutical composition for preventing or treating a nervous system disease, comprising the composition according to claim 32.

35. A pharmaceutical composition for inducing angiogenesis, comprising stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof; or a culture thereof as active ingredients.

36. The composition of claim 35, wherein the culture is a conditioned medium comprising TIMP-1, MCP-1, GROα and IL-6 proteins secreted by the stem cells.

37. The composition according to claim 35, wherein the composition is used for preventing or treating cerebrovascular disease.

38. The composition of claim 37, wherein the cerebrovascular disease is one or more selected from the group consisting of cerebral infarction, cerebral ischemia, stroke, vascular dementia, cerebral hemorrhage, subarachnoid hemorrhage, and leukodystrophy.

39. A pharmaceutical composition for inducing angiogenesis, comprising isolated stem cells; and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof as active ingredients.

40. The composition according to claim 39, wherein the substance that increases the expression or activity of the protein is one or more expression vectors, the expression vectors comprising a polynucleotide encoding the TIMP-1 protein; a polynucleotide encoding the MCP-1 protein; a polynucleotide encoding the GROα protein; and a polynucleotide encoding the IL-6 protein.

41. The composition of claim 39, wherein the composition is used to prevent or treat cerebrovascular disease.

42. A pharmaceutical composition for inducing angiogenesis, comprising the composition according to claim 39.

43. Use of a substance for measuring the expression levels of TIMP-1, MCP-1, GROα and IL-6 proteins or their genes, wherein the preparation is used to prepare a preparation for selecting highly efficient stem cells for treating nervous system diseases.

44. A method for preventing, treating or ameliorating a nervous system disease, comprising the step of administering a composition to a subject in need thereof, wherein the composition comprises stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof; or a culture thereof as an active ingredient.

45. A stem cell overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or their proteins; or a use of a culture thereof in preparing a preparation for preventing, treating or ameliorating a nervous system disease.

46. ​​Use of a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof in the preparation of a preparation for promoting the activity of stem cells for preventing or treating nervous system diseases.

47. A method for preventing, treating or ameliorating a nervous system disease, comprising the step of administering a composition to a subject in need thereof, wherein the composition comprises isolated stem cells; and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins as active ingredients.

48. Use of isolated stem cells; and substances that increase the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins in the manufacture of preparations for preventing, treating or ameliorating nervous system diseases.

49. A method for inducing angiogenesis, comprising administering to a subject in need thereof a composition comprising stem cells overexpressing TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof; or a culture thereof as an active ingredient.

50. Use of stem cells or cultures thereof that overexpress TIMP-1, MCP-1, GROα and IL-6 genes or proteins thereof in the preparation of a preparation for inducing angiogenesis.

51. A method for inducing angiogenesis, comprising administering to a subject in need thereof a composition comprising isolated stem cells; and a substance that increases the expression or activity of TIMP-1, MCP-1, GROα, and IL-6 genes or proteins thereof as active ingredients.

52. Use of isolated stem cells; and substances that increase the expression or activity of TIMP-1, MCP-1, GROα and IL-6 genes or their proteins in the preparation of a composition for inducing angiogenesis.

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