Composition for inducing differentiation of stem cells into astrocytes, containing CPI-455

Abstract

The present invention relates to a composition for inducing the differentiation of stem cells into astrocytes, which contains CPI-455. It is expected that using the composition of the present invention will not only induce differentiation into astrocytes without using existing stem cell differentiation promoters, but will also enable the effective treatment of neurodegenerative diseases using the differentiated astrocytes.

Description

Technical Field

[0001] The present invention relates to a composition for inducing the differentiation of stem cells containing CPI-455 into astrocytes, etc.

[0002] This application claims priority based on Korean Patent Application No. 10-2023-0044632 filed on April 5, 2023, and all the contents disclosed in the specification and drawings of the said application are incorporated herein by reference.

Background Art

[0003] Neurodegenerative disorders refer to diseases that occur in the brain or spinal cord among degenerative diseases that occur with aging, and are diseases that appear due to the progressive loss of the structure and function of specific brain cell groups in the brain and spinal cord caused by unknown causes, genetic defects, or environmental factors. The death or damage of nerve cells caused by degenerative nervous system diseases is known to cause problems in the formation and function of synapses that transmit information between the most important brain nerve cells and brain nerve cells for information transmission in the brain nervous system, as well as abnormal increases or decreases in the electrical activity of brain nerves. Nerve cells in the brain and spinal cord perform very diverse functions depending on their location, and damage to nerve cells in a specific site induces characteristic functional disorders, and also shows very diverse clinical manifestations depending on how such functional disorders progress. Such degenerative nervous system diseases include amyotrophic lateral sclerosis (ALS) known as Lou Gehrig's disease, Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease (HD), multiple sclerosis (MS), etc.

[0004] Meanwhile, for research into Alzheimer's disease, researchers have been differentiating cells primarily into nerve cells abundant in the forebrain, and then conducting studies on the etiology. Differentiation into forebrain nerve cells is performed using nerve cell differentiation methods that proceed without the addition of special external substances. Recently, methods such as monolayer culture via "dual-SMAD inhibition," methods via embryoid body formation, and methods via cerebral organoid formation have been developed, and both "glutamatergic neurons" and "GABAergic neurons" are frequently used in research to uncover the etiology of Alzheimer's disease. According to a recently published paper, it was confirmed that when patient-derived induced pluripotent stem cells were differentiated into "GABAergic neurons," they showed greater resistance to amyloid-beta-induced cell death. In other words, this demonstrates that diverse phenotypes can emerge through differentiation into specific nerve cells.

[0005] Astrocytes, along with nerve cells, have been reported to play a crucial role in the pathogenesis of Alzheimer's disease. Astrocytes have been reported to play a critical role in synapse formation and maturation, and thus may be able to treat neurodegenerative diseases. Generally, astrocytes differentiate from the same progenitor cells as nerve cells, and are a mixed cell population of the two types, allowing for cell fractionation and purification using astrocyte-specific expression factors.

[0006] On the other hand, it is unknown whether CPI-455 is effective in inducing neural stem cells into astrocytes. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The inventors of this invention have made diligent efforts to find a method for inducing stem cells into astrocytes, and have completed this invention by confirming that CPI-455 of the present invention can be used to treat neural stem cells into astrocytes.

[0008] The object of the present invention is to provide a composition for inducing the differentiation of stem cells into astrocytes, comprising CPI-455 as an active ingredient.

[0009] Another object of the present invention is to provide a method for differentiating stem cells into astrocytes, comprising the step of treating stem cells with a composition for inducing differentiation of stem cells into astrocytes according to the present invention and culturing the stem cells.

[0010] Another object of the present invention is to provide a kit for inducing differentiation of stem cells into astrocytes, comprising CPI-455 or a composition for inducing differentiation of stem cells into astrocytes according to the present invention.

[0011] Another object of the present invention is to provide CPI-455 or the stem cell differentiation induction composition according to the present invention for use in inducing the differentiation of stem cells into astrocytes.

[0012] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, comprising CPI-455 as an active ingredient.

[0013] Another object of the present invention is to provide a method for preventing or treating neurodegenerative diseases, comprising the step of administering CPI-455, or a composition containing it as an active ingredient, to an individual in need.

[0014] Another object of the present invention is to provide CPI-455, or compositions containing it as an active ingredient, for use in the prevention or treatment of neurodegenerative diseases.

[0015] Another object of the present invention is to provide applications for the manufacture of therapeutic agents for neurodegenerative diseases using CPI-455, or compositions containing it as an active ingredient.

[0016] Another object of the present invention is to provide a cell therapy agent for the prevention or treatment of neurodegenerative diseases, comprising astrocytes produced by a method for differentiating stem cells into astrocytes according to the present invention.

[0017] Another object of the present invention is to provide a method for preventing or treating neurodegenerative diseases, which includes the step of administering a cell therapy agent according to the present invention to an individual in need thereof.

[0018] Another object of the present invention is to provide a cell therapy agent according to the present invention for the prevention or treatment of neurodegenerative diseases.

[0019] Another object of the present invention is to provide a method for producing astrocytes, which are cell-based therapeutic agents produced by the method of differentiating stem cells into astrocytes according to the present invention.

[0020] However, the technical problems that this invention aims to solve are not limited to those mentioned above, and other problems not mentioned should be clearly understood by a person with ordinary skill in the art to which this invention pertains from the description below. [Means for solving the problem]

[0021] To achieve the above objective, the present invention provides a composition for inducing the differentiation of stem cells into astrocytes, comprising CPI-455 as an active ingredient.

[0022] As one embodiment of the present invention, the stem cells may be one or more selected from the group consisting of embryonic stem cells, adult stem cells, embryonic germ stem cells, and embryonic tumor stem cells, but are not limited thereto.

[0023] As another embodiment of the present invention, the CPI-455 may, but is not limited to, be included in the overall composition at a concentration of 0.1 to 100 μM.

[0024] As yet another embodiment of the invention, the CPI-455 may satisfy one or more of the following features, but is not limited to these:

[0025] (a) Promote the phosphorylation of STAT3 (Signal transducer and activator of transcription 3); and

[0026] (b) Increase the secretion of BMP2 (Bone morphogenic protein 2).

[0027] The present invention also provides a method for differentiating stem cells into astrocytes, including the step of culturing the stem cells by treating them with the composition for inducing the differentiation of stem cells into astrocytes according to the present invention.

[0028] As an embodiment of the present invention, the astrocytes can express GFAP (Glial fibrillary acidic protein), but are not limited thereto.

[0029] The present invention also provides a kit for inducing the differentiation of stem cells into astrocytes, including CPI-455 or the composition for inducing the differentiation of stem cells into astrocytes according to the present invention.

[0030] The present invention also provides the use of CPI-455 or the composition for inducing the differentiation of stem cells into astrocytes according to the present invention for inducing the differentiation of stem cells into astrocytes.

[0031] The present invention also provides a pharmaceutical composition for preventing or treating neurodegenerative diseases, containing CPI-455 as an active ingredient.

[0032] The present invention also provides a cell therapy agent for preventing or treating neurodegenerative diseases, including astrocytes produced by the method for differentiating stem cells into astrocytes according to the present invention.

[0033] As an embodiment of the present invention, the neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis, adrenoleukodystrophy, Alexander disease, Alpas disease, vasodilatory ataxia, Batten disease, bovine spongiform encephalopathy (BSE), Canavan disease, corticobasal degeneration, Creutzfeldt-Jakob disease, Lewy body dementia, fatal familial insomnia, frontotemporal lobar degeneration, Huntington's disease, Kennedy disease, Krabbe disease, Lyme disease, and Machad Joseph disease. One or more of the following may be selected from the group consisting of disease, multiple sclerosis, multiple system atrophy, neuroacanthocytosis, Niemann-Pick disease, Parkinson's disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelin-disintegrating sclerosis, spinocerebellar ataxia, subacute combined degeneration of the spinal cord, spinal syphilis, Tay-Sachs disease, toxic encephalopathy, transmissible spongiform encephalopathy, and unstable Hedgehog syndrome.

[0034] Furthermore, the present invention provides a method for preventing or treating neurodegenerative diseases, comprising the step of administering CPI-455, or a composition containing it as an active ingredient, to an individual in need.

[0035] Furthermore, the present invention provides CPI-455, or a composition containing it as an active ingredient, for use in the prevention or treatment of neurodegenerative diseases.

[0036] Furthermore, the present invention provides applications for the manufacture of neurodegenerative disease treatment agents using CPI-455, or compositions containing it as an active ingredient.

[0037] Furthermore, the present invention provides a method for preventing or treating neurodegenerative diseases, which includes the step of administering the cell therapy agent according to the present invention to an individual in need of it.

[0038] Furthermore, the present invention provides a cell therapy agent according to the present invention for the prevention or treatment of neurodegenerative diseases.

[0039] Furthermore, the present invention provides a method for producing astrocytes, which are cell-based therapeutic agents produced by differentiating stem cells into astrocytes according to the present invention. [Effects of the Invention]

[0040] The inventors have demonstrated a technique for inducing stem cells into astrocytes using CPI-455. This technique not only allows for differentiation into astrocytes without the use of conventional stem cell differentiation promoters, but also enables the effective treatment of neurodegenerative diseases using the differentiated astrocytes. [Brief explanation of the drawing]

[0041] Figure 1(a) shows the results of Western blotting to confirm the expression levels of TUBB3 (Tubulin beta 3 class III), GFAP, and OLIG2 (Oligodendrocyte transcription factor 2) after treating neural stem cells with 0.1% DMSO or CPI-455 for 4 days without EGF and FGF2 (D: DMSO, same applies below).

[0042] Figure 1(b) shows the results of measuring the expression of tubeb3 and gfap mRNA by RT-qPCR after treating neural stem cells with 0.1% DMSO or CPI-455 for 3 days without EGF and FGF2.

[0043] Figure 2(a) shows the results of immunostaining with TUBB3 (green) or anti-GFAP (red) after treating neural stem cells with 0.1% DMSO or CPI-455 for 4 days without EGF and FGF2. (Top panel) 0.1% DMSO treatment, (Bottom panel) CPI-455 treatment. Nuclei are stained with DAPI (blue). Scale bar = 100 μm.

[0044] Figure 2(b) shows the ratio of TUBB3-positive cells and GFAP-positive cells to DAPI-positive cells after neural stem cells were treated with 0.1% DMSO or CPI-455 for 4 days without EGF and FGF2.

[0045] Figure 3(a) shows the results of Western blotting to confirm the degree of STAT3 phosphorylation in neural stem cells treated with CPI-455.

[0046] Figure 3(b) shows the results of measuring the expression of il-6, lif, cntf, and bmp2 mRNA in neural stem cells treated with CPI-455 using RT-qPCR. [Best Mode for Carrying Out the Invention]

[0047] Unless otherwise specified, the terms, techniques, and other terms used in this specification are used in the sense commonly used in the art to which the present invention pertains. Furthermore, all documents referenced herein are incorporated herein as references to illustrate the present invention.

[0048] In this specification, "active ingredient" means an ingredient that exhibits the desired activity on its own, or an ingredient that can exhibit the desired activity in combination with a carrier or the like that is inactive on its own.

[0049] The present invention will be described in detail below.

[0050] The present invention provides a composition for inducing the differentiation of stem cells into astrocytes, comprising CPI-455 as an active ingredient.

[0051] In this invention, "CPI-455" is named 4,7-dihydro-6-(1-methylethyl)-7-oxo-5-phenyl-pyrazolo[1,5-a]pyrimidine-3-carbonitrile (Cas No. 1628208-23-0) and is represented by the following chemical formula 1. Furthermore, CPI-455 can be obtained commercially or manufactured by known methods (Benjamin R. Leadem. Novel Histone Demethylase Inhibitors Synergistically).

[0052] [ka]

[0053] In the present invention, the CPI-455 is expressed in the following concentrations relative to the overall composition: 0.1-100 μM, 1-90 μM, 1-80 μM, 1-70 μM, 1-60 μM, 1-55 μM, 1-50 μM, 1-45 μM, 1-40 μM, 10-90 μM, 10-80 μM, 10-70 μM, 10-60 μM, 10-50 μM, 20-80 μM, It may be present in concentrations of 30-70 μM, 40-60 μM, 43-60 μM, 45-60 μM, 47-60 μM, 50-58 μM, 50-55 μM, 50-54 μM, 50-52 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, 51 μM, 52 μM, 53 μM, or 54 μM, but is not limited to these.

[0054] According to one embodiment of the present invention, CPI-455 can induce differentiation of neural stem cells into astrocytes by increasing the secretion of BMP2 and promoting the phosphorylation of STAT3, and is characterized by satisfying one or more of the following features: (a) Promote the phosphorylation of STAT3 (Signal transducer and activator of transcription 3); and (b) Increases BMP2 (Bone morphogenic protein 2) secretion.

[0055] In this invention, "BMP2 (Bone morphogenic protein 2)" is one of the TGF-beta series cytokines, a polypeptide dimer composed of 114 amino acids, and is known to interact with fibroblast growth factors to induce not only the formation of chondrocytes and osteoblast precursors, but also the development of epidermal tissue and the differentiation of nerve cells.

[0056] In this invention, "STAT3 (Signal transducer and activator of transcription 3)" is a member of the STAT family, existing as an inactive form in the cytoplasm. After phosphorylation occurs due to stimulation by various types of cytokines, growth factors, and hormones, it forms a dimer and then moves into the nucleus to transmit signals. STAT3 induces cell differentiation, survival, proliferation, and death by regulating the expression of various types of genes, and is known to be particularly increased in many tumor cells. The activity of STAT3 is regulated by the phosphorylation of Janus kinase 2 (JAK2), an upstream kinase of STAT that promotes STAT3 phosphorylation, the expression level of the suppressor of cytokine signaling 3 (SOCS3) protein that binds to JAK2 and suppresses its activity, and the activity of SH2 (domain-containing protein tyrosine phosphatase (SHP-1)), a tyrosine phosphatase.

[0057] In this invention, "stem cells" are not particularly limited as long as they are cells that, while in an undifferentiated state, have the function of unlimited proliferation and differentiation into astrocytes. Specifically, examples of stem cells include embryonic stem cells, adult stem cells, embryonic germ stem cells, and embryonic tumor stem cells, but are not limited thereto. According to one embodiment of this invention, the stem cells may be neural stem cells.

[0058] In this invention, "astrocyte" may refer to astrocytes with increased GFAP expression levels, which are astrocyte markers, but is not limited to this.

[0059] Furthermore, the present invention provides a method for differentiating stem cells into astrocytes, comprising the step of treating the stem cells with the stem cell differentiation induction composition according to the present invention and culturing the stem cells.

[0060] According to one embodiment of the present invention, astrocytes produced by the method for differentiating stem cells into astrocytes according to the present invention may express GFAP (Glial fibrillary acidic protein), which is an astrocyte marker, and may have an increased GFAP expression level compared to stem cells.

[0061] In this invention, "culturing" can be performed using any one of the cell culture media selected from DMEM (Dulbecco's Modified Eagle Medium), DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), RPMI1640 (Roswell Park Memorial Institute 1640), MEM (Minimum Essential Media), and Ham F10, to which sodium pyruvate, glutamine, etc., may be further added. The culture period may be 1 to 10 days, 1 to 8 days, 1 to 6 days, 2 to 6 days, 3 to 6 days, 3 to 5 days, or 3 to 4 days, but is not limited thereto.

[0062] The aforementioned "culture medium" or "culture culture medium" refers to a mixture for the differentiation and proliferation of cells such as astrocytes in vitro, containing elements essential for cell growth and proliferation, such as sugars, amino acids, various nutrients, serum, growth factors, and minerals. In particular, in this invention, "culture medium" refers to a medium for the differentiation and proliferation of astrocytes. In this invention, the term "culture" includes the differentiation and proliferation of astrocytes.

[0063] In this invention, the "culture medium" can be any basic culture medium known in the industry without limitation. Basic culture media can be manufactured by artificial synthesis, and commercially manufactured culture media include, but are not limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), and Iscove's Modified Dulbecco's Medium.

[0064] Furthermore, the culture medium of the present invention may further contain a nutrient mixture. The nutrient mixture is a mixture containing various amino acids, vitamins, inorganic salts, etc., commonly used in cell culture, and can be produced by mixing the amino acids, vitamins, inorganic salts, etc., or a commercially produced nutrient mixture can be used. Examples of commercially produced nutrient mixtures include, but are not limited to, B27, N2, F-12, M199, MCDB110, MCDB202, MCDB302, etc.

[0065] Furthermore, the culture medium of the present invention may not contain growth factors. The growth factors may be, for example, EGF or FGF2, but are not limited to these.

[0066] The culture medium used to culture stem cells in this invention includes all the culture media commonly used for astrocyte differentiation in the field. The culture medium used for culturing generally contains a carbon source, a nitrogen source, and trace element components.

[0067] More specifically, a method for differentiating stem cells into astrocytes may include the step of culturing stem cells in a differentiation medium treated with a composition containing CPI-455 as an active ingredient to differentiate them into astrocytes.

[0068] Examples of the aforementioned stem cells include, but are not limited to, embryonic stem cells, adult stem cells, embryonic germline stem cells, and embryonic tumor stem cells.

[0069] The differentiation medium may, but is not limited to, contain one or more selected from the group consisting of antibiotics, antifungal agents, and B27.

[0070] The differentiation medium may, but is not limited to, contain one or more selected from the group consisting of 0.1-5% (v / v) of an antibiotic-antifungal agent and 0.5-5% (v / v) of B27.

[0071] The differentiation medium may, but is not limited to, not contain the growth factors EGF and FGF2.

[0072] Furthermore, the present invention provides a kit for inducing the differentiation of stem cells into astrocytes, comprising CPI-455 or a composition for inducing the differentiation of stem cells into astrocytes according to the present invention.

[0073] The kit is not limited to those described above and may include other reagents and equipment. For example, it may include a culture plate for culturing target cells, reagents capable of evaluating the differentiation state into astrocytes, and may also include the stem cells to be cultured.

[0074] The kit according to the present invention may be provided in a single container containing the differentiation induction medium additive, or CPI-455, or the nutrient mixture, growth factor, or basal medium and all other reagents in appropriate doses and / or forms, or each may be provided in separate containers.

[0075] The kit according to the present invention may include instructions describing procedures for carrying out the method according to the present invention as described above.

[0076] Furthermore, the present invention provides CPI-455 or the composition for inducing differentiation of stem cells into astrocytes according to the present invention for use in inducing differentiation of stem cells into astrocytes.

[0077] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, comprising CPI-455 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0078] The CPI-455 according to the present invention may be used on its own or in the form of a pharmaceutically acceptable salt.

[0079] In this invention, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.

[0080] Examples of suitable acids include hydrochloric acid, bromate, sulfuric acid, nitric acid, perchloric acid, propionic acid, maleic acid, tartaric acid, gluconic acid, trifluoroacetic acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydrobromic acid, hydroiodic acid, nitrite, benzoic acid, citric acid, and lactic acid. Acid addition salts can be produced by conventional methods, for example, by dissolving a compound in an excess of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Alternatively, they can be produced by heating the same molar amount of the compound and an acid or alcohol in water, followed by evaporating and drying the mixture, or by suction filtration of the precipitated salt.

[0081] Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium. Alkali metal or alkaline earth metal salts can be obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the insoluble compound salt, and then evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are particularly pharmaceutically appropriate as metal salts, and corresponding silver salts can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0082] The term "neurodegenerative disease" as used in this invention is not particularly limited. Non-limiting examples of the neurodegenerative disease include Alzheimer's disease, amyotrophic lateral sclerosis, adrenoleukodystrophy, Alexander disease, Alpas disease, ataxia vasodilator, Batten disease, bovine spongiform encephalopathy (BSE), Canavan disease, corticobasal degeneration, Creutzfeldt-Jakob disease, Lewy body dementia, fatal familial insomnia, frontotemporal lobar degeneration, Huntington's disease, Kennedy disease, Krabbe disease, Lyme disease, and Machad-Joseph disease. This may include, but is not limited to, one or more selected from the group consisting of multiple sclerosis, multiple system atrophy, neuroacanthocytosis, Niemann-Pick disease, Parkinson's disease, Pick disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelin-disintegrating sclerosis, spinocerebellar ataxia, subacute combined degeneration of the spinal cord, spinal syphilis, Tay-Sachs disease, toxic encephalopathy, transmissible spongiform encephalopathy, and unstable Hedgehog syndrome.

[0083] The content of the compound in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the patient's condition, etc. For example, it may be 0.0001 to 99.9% by weight, 0.001 to 80% by weight, or 0.01 to 50% by weight based on the total weight of the composition, but is not limited thereto. The content ratio is a value based on the dry weight after removing the solvent.

[0084] In this invention, "pharmaceutical composition" means a substance manufactured for the purpose of preventing or treating a disease, and each can be formulated into various forms by conventional methods and used. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can also be formulated into topical preparations, suppositories, and sterile injection solutions for use.

[0085] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be one or more selected from the group consisting of, for example, diluents, binders, disintegrants, lubricants, adsorbents, humectants, film coating substances, and controlled-release additives.

[0086] The pharmaceutical compositions according to the present invention can be formulated by conventional methods into external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, elixirs, emulsions, suspensions, alcoholic preparations, lozenges, aromatic preparations, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, ointment extracts, dried extracts, liquid extracts, injections, capsules, perfusion solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, and these external preparations may have dosage forms such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or catarplasma preparations.

[0087] Examples of carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0088] When formulating, the product is typically prepared using fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients.

[0089] Excipients for tablets, powders, granules, capsules, pills, and lozenges according to the present invention 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 gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, Primogel, etc.; gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium casein Binders such as sodium carboxymethylcellulose, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch paste, hydroxypropylcellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used; disintegrants such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, acacia gum, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ethers, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.

[0090] Possible additives for the liquid formulation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, proamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, and the like.

[0091] The syrup according to the present invention may contain a solution of sucrose, other sugars, or sweeteners, and may, if necessary, contain fragrances, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, and the like.

[0092] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0093] The suspending agent according to the present invention may include suspending agents such as gum arabic, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose (HPMC), HPMC1828, HPMC2906, and HPMC2910, and may also include surfactants, preservatives, stabilizers, colorants, and fragrances as needed.

[0094] The injectable preparations according to the present invention include solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidinedione, propylene glycol, twins, nicotinamide, hexamine, and dimethylacetamide; weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), and organic compounds. It may contain buffering agents such as proteins, albumin, peptone, and rubbers; isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; antioxidants such as 0.1% sodium bisulfide, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and sodium acetone bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cyenate, sodium alginate, Twin 80, and aluminum monostearate.

[0095] The suppositories according to the present invention contain cocoa butter, lanolin, whitepsol, polyethylene glycol, glycerol gelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, twin or span, Imhausen, monolen (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, and Cebes Pharma 16. 16) Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrocote 25, Hydrocote 711, Idropostal, Massaestralium Bases such as estrarium (A, AS, B, C, D, E, I, T), Masa-MF, Maspol, Maspol-15, Neospostal-En, Paramount-B, Sposilo (OSI, OSIX, A, B, C, D, H, L), Suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Spostal (N, Es), Wecobi (W, R, S, M, Fs), and Tezestr triglyceride base (TG-95, MA, 57) may be used.

[0096] Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. Such solid preparations are manufactured by mixing at least one excipient from the aforementioned extracts, such as starch, calcium carbonate, sucrose, or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0097] Liquid formulations for oral administration include suspensions, oral solutions, emulsions, and syrups. Besides commonly used simple diluents such as water and liquid paraffin, various excipients may be included, such as humectants, sweeteners, fragrances, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0098] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined by factors including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and elimination ratio, the duration of treatment, drugs used concurrently, and other factors well known in the medical field.

[0099] The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by an ordinary person skilled in the art to which the present invention pertains.

[0100] The pharmaceutical composition of the present invention can be administered to an individual by a variety of routes. All methods of administration are conceivable, but for example, it can be administered by oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, perispinal space (intradural) injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, etc.

[0101] The pharmaceutical composition of the present invention is determined by the type of drug that is the active ingredient, along with many relevant factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.

[0102] Furthermore, the present invention provides a method for preventing or treating neurodegenerative diseases, comprising the step of administering CPI-455, or a composition containing it as an active ingredient, to an individual in need.

[0103] Furthermore, the present invention provides CPI-455, or a composition containing it as an active ingredient, for use in the prevention or treatment of neurodegenerative diseases.

[0104] Furthermore, the present invention provides applications for the manufacture of neurodegenerative disease treatment agents using CPI-455, or compositions containing it as an active ingredient.

[0105] In this invention, "individual" means an entity that requires treatment for a disease, and more specifically, it means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, or cow.

[0106] In this invention, "administration" means providing a predetermined composition of the present invention to an individual by any suitable method.

[0107] In the present invention, "prevention" means all actions that suppress or delay the onset of the target disease; "treatment" means all actions that improve or beneficially alter the target disease and its associated metabolic abnormalities through the administration of the pharmaceutical composition according to the present invention; and "improvement" means all actions that reduce parameters associated with the target disease, such as the severity of symptoms, through the administration of the composition according to the present invention.

[0108] Furthermore, the pharmaceutical composition of the present invention can be administered in parallel with known compounds that have preventive or therapeutic effects on neurodegenerative diseases.

[0109] In this invention, stem cells were differentiated into astrocytes using CPI-455. The differentiated astrocytes have very high potential for use as cell therapy agents for patients.

[0110] Accordingly, the present invention provides a cell therapy agent for the prevention or treatment of neurodegenerative diseases, comprising astrocytes produced by a method for differentiating stem cells into astrocytes according to the present invention.

[0111] Furthermore, the present invention provides a method for preventing or treating neurodegenerative diseases, comprising the step of administering the cell therapy agent according to the present invention to an individual in need thereof.

[0112] Furthermore, the present invention provides a cell therapy agent according to the present invention for the prevention or treatment of neurodegenerative diseases.

[0113] In this invention, the term "cellular therapeutic agent" means a pharmaceutical product (as defined by the U.S. FDA) that uses cells and tissues isolated from humans, cultured, and manufactured through special manipulations for therapeutic, diagnostic, and preventive purposes. It also means a pharmaceutical product in which living autologous, allogenic, or xenogenic cells are grown and selected outside the body, or their biological properties are altered by other means, in order to restore the function of cells or tissues, and these cells are used for the therapeutic, diagnostic, and preventive purposes of disease.

[0114] The administration route of the cell therapy agent according to the present invention may be any common route as long as it can reach the target tissue. Parenteral administration may be, for example, intraperitoneal, intravenous, intramuscular, subcutaneous, or intradermal.

[0115] The cell therapy agents can be formulated in appropriate forms with pharmaceutically acceptable carriers commonly used in cell therapy. "Pharmacologically acceptable" means a composition that, when administered to humans, is physiologically acceptable and does not typically cause allergic reactions or similar reactions such as gastrointestinal disturbances or dizziness. Examples of pharmaceutically acceptable carriers include parenteral carriers such as water, suitable oils, saline solutions, aqueous glucose, and glycols, and may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. For other pharmaceutically acceptable carriers, please refer to the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0116] Furthermore, the composition may be administered by any device capable of delivering the cell therapy agent to target cells.

[0117] The cell therapy agent according to the present invention may contain a therapeutically effective amount of astrocytes for the treatment of a disease. "Therapeutically effective amount" means the amount of active ingredient or therapeutic agent that induces a biological or medical response in a tissue system, animal or human, as can be determined by researchers, veterinarians, physicians or other clinical experts, and this includes the amount that induces relief of the symptoms of the disease or disorder being treated.

[0118] It is obvious to those skilled in the art that the cells contained in the cell therapy agent according to the present invention will change according to the desired effect. Therefore, the optimal content of the cell therapy agent can be easily determined by those skilled in the art and can be adjusted by various factors including the type of disease, the severity of the disease, the content of other components contained in the composition, the type of formulation, and the patient's age, weight, general health, sex and diet, administration time, route of administration and secretion rate of the therapeutic agent, duration of treatment, and drugs used concurrently. Taking all of the above factors into consideration, it is important to include an amount that can obtain the maximum effect with the minimum amount without side effects. For example, the daily dose of astrocytes according to the present invention is 1.0 × 10⁻⁶ 4 ~1.0×10 11 Cells / kg body weight, preferably 1.0 × 10⁶ 5 ~1.0×10 9 The cells / kg body weight can be administered in one or several divided doses. However, it should be understood that the actual dose of the active ingredient should be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and sex, and therefore, the aforementioned dose does not limit the scope of the present invention in any way.

[0119] The present invention provides a therapeutic method comprising administering a therapeutically effective amount of the cell therapy agent according to the present invention to a mammal. The term "mammal" as used herein refers to a mammal that is the subject of treatment, observation, or experimentation, preferably a human.

[0120] Furthermore, the present invention provides a food composition for the prevention or improvement of neurodegenerative diseases, comprising CPI-455 or a food-grade salt thereof as an active ingredient, and the food composition may also be a functional food composition.

[0121] In this invention, the term "food-grade acceptable salt" includes salts derived from food-grade acceptable organic acids, inorganic acids, or bases. When using CPI-455 of this invention as a food additive, it can be added as is, or it can be used with other foods or food components and can be used appropriately by conventional methods. The amount of active ingredient mixed can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, CPI-455 of this invention can be added in an amount of 15% by weight or less, or 10% by weight or less, relative to the raw materials. However, in the case of long-term intake for health and hygiene purposes, or for health regulation purposes, the amount may be less than the aforementioned range, and since there are no safety concerns, the active ingredient can be used in amounts greater than the aforementioned range.

[0122] There are no special restrictions on the types of food mentioned above. Examples of foods to which the substance may be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the usual sense.

[0123] The health beverage composition according to the present invention may contain various flavorings or natural carbohydrates as additional ingredients, as in ordinary beverages. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrates is generally about 0.01 to 0.20 g, or about 0.04 to 0.10 g, per 100 mL of the composition of the present invention.

[0124] In addition to the above, the compositions of the present invention may contain various nutrients, vitamins, electrolytes, flavorings, colorings, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Furthermore, the compositions of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not of great importance, but is generally selected in the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.

[0125] In this specification, "health functional foods" is the same term as "foods for special health use (FoSHU)," and refers to foods that have high medical and therapeutic effects, processed to efficiently exhibit biological regulatory functions in addition to providing nutrition. Such foods may be manufactured in various forms such as tablets, capsules, powders, granules, liquids, and pills in order to obtain effects useful for the prevention or improvement of neurodegenerative diseases.

[0126] The health functional food of the present invention can be manufactured by methods commonly used in the industry, and during the manufacturing process, raw materials and ingredients commonly added in the industry can be added. Furthermore, unlike general pharmaceuticals, it has the advantage of being made from food as a raw material and not having the side effects that can occur with long-term use of pharmaceuticals, and it is highly portable. [Examples]

[0127] The following are preferred embodiments to aid in understanding the present invention. However, the following embodiments are provided only to facilitate understanding of the present invention and do not limit the scope of the present invention.

[0128] <Experimental Method> 1. Cell culture, chemical treatment, and sampling All animal experiments were approved by the Animal Experiment Ethics Committee of Chung-Ang University and conducted in accordance with the National Institute of Health's guidelines for the management and use of laboratory animals.

[0129] Neural stem cells (NSCs) were cultured and harvested as previously reported (Faseb j 2018, 32 (2), 1108-1119). Specifically, cells were harvested by isolating the cerebral cortex from 14-day-old Sprague-Dawley rat embryos (Orient Bio, Seongnam, South Korea). The cells were incubated for 6 days at 37°C and 5% CO2 in Dulbecco's modified Eagle's media (DMEM / F12) (Cat no. 12500-062, Gibco, Grand Island, NY, USA) containing Ham's nutrient mixture F-12, which was mixed with 1% antibiotic-antifungal agent (Cat no. 15240-062, Gibco), 2% B-27 supplement (Cat no. 17504-044, Gibco), 20 ng / ml EGF (Cat no. GF144, Chemicon, Burlington, MA, USA), and 20 ng / ml FGF2 (Cat no. GF003AF, Chemicon), to form neurospheres. Subsequently, the nerve globules were dissociated into individual cells using Accutase (Cat no. SCR005, Sigma-Aldrich, St. Louis, MO, USA). The cells were then cultured on culture plates for 1 day in a medium enriched with EGF and FGF2. The NSCs were then treated in DMEM / F12 medium mixed with a final concentration of 1% antibiotic-antimicrobial agent, 2% B-27 supplement, and either DMSO (Cat no. 472301, Sigma-Aldrich) or CPI-455 (Cat no. HY-100421, Medchemexpress, Princeton, NJ, USA) dissolved in DMSO. The medium was changed every 2 days. Cells for Western blotting and immunohistochemistry were cultured for 4 days after CPI-455 treatment. Cells for RT-qPCR and ChIP-qPCR were cultured for 3 days after CPI-455 treatment.

[0130] 2. Western blot The cells were washed with PBS and lysed on ice using NP-40 lysis buffer. The lysates were centrifuged at 16000g for 30 minutes to purify the total protein. The proteins were separated by electrophoresis on an 8-10% polyacrylamide gel and transferred to immobilion-P PVDF membrane (Cat no. PVH00010, Millipore, Burlington, MA, USA). The membranes were blocked with 5% skim milk using TBS / 0.03% Tween 20 or 5% bovine serum albumin using TBS / 0.1% Tween 20 (TBS / 0.1% Tween 20).

[0131] The following primary antibodies were used to treat the membrane overnight: TUBB3 (1:3000; Cat no. T5076, Sigma-Aldrich), GFAP (1:9000; Cat No. G9269, Sigma-Aldrich), OLIG2 (1:2000; Cat No. AB9610, Millipore), GAPDH (1:1000; Cat no. sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA), STAT3 (1:2000; Cat no. 4904, Cell Signaling Technology, Danvers, MA, USA), and pSTAT3 (1:2000; Cat no. 9145, Cell Signaling Technology).

[0132] The following secondary antibodies were used for 1 hour: Anti-mouse IgG F(ab')2 (1:5000; Cat no. ADI-SAB-100, Enzo, Farmingdale, NY, USA) and Anti-Rabbit IgG (1:5000; Cat no. ADI-SAB-300-J, Enzo).

[0133] Chemiluminescence was detected in a novel CP-BU film (Agfa, Mortsel, Belgium) using Western blotting luminol reagent (Cat no. sc-2048, Santa Cruz Biotechnology). Band intensity was measured using ImageJ version 2.3.0. GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) was measured as a housekeeping protein.

[0134] 3.Immunocytochemistry (ICC) Immunocytochemistry was performed as previously reported (Faseb j 2018, 32(2), 1108-1119). Specifically, cells were fixed in 4% paraformaldehyde solution (Cat no. P231, Biosesang, Seongnam, South Korea) for 30 minutes. Blockade was performed by treating cells with 5% goat serum (Cat no. S26, Millipore) diluted in 0.2% Triton X-100 (Cat no. 0694-1L, VWR International, Radnor, PA, USA).

[0135] Cells were stained for 1 hour with the following primary antibodies: TUBB3 (1:1000; Cat no. T5076, Sigma-Aldrich) and GFAP (1:500; Cat no. Z0334, Dako, Santa Clara, CA, USA).

[0136] Next, the following secondary antibodies were used to treat the goat for 30 minutes: Alexa488 goat anti-mouse IgG (1:1000; Cat no. A21131, Invitrogen, Waltham, MA, USA) and Cy3 goat anti-rabbit IgG (1:1000; Cat no. 111-165-144, Jackson, West Grove, PA, USA).

[0137] Nuclei were stained by treating PBS solution with 0.01% DAPI (4',6-diamidino-2-phenylindole) (Cat No. D9542, Sigma-Aldrich) for 5 minutes. Fluorescence images were captured using a Leica DM IL inverse fluorescence microscope and LAS X software (Leica, Wetzlar, Germany).

[0138] 4.RT-qPCR (Reverse transcriptase-quantitative polymerase chain reaction)

[0139] RNA was extracted using TRIzol® reagent (Cat no. 15596018, Thermo Fisher, Waltham, MA, USA) and dissolved in diethylpyrocarbonate-treated water (Cat no. C-9030, Bioneer, Daejeon, South Korea). Complementary DNA (cDNA) was synthesized using QuantiTect reverse transcriptase kit (Cat no. 205311, Qiagen, Hilden, Germany) with 1 ug of RNA per sample. cDNA was then converted to iQ TM The mixture was combined with SYBR® Green supermix (Cat no. 1708880, Bio-Rad, Hercules, CA, USA) and its corresponding forward and reverse primers (Cosmo Genetech, Seoul, South Korea). The primers used are shown in Table 1. qPCR was performed for 40 cycles using a CFX Connect thermal cycler (Bio-Rad) under the following conditions: 10 seconds at 95°C, 15 seconds at 58°C, and 20 seconds at 72°C.

[0140] [Table 1]

[0141] 5. Chromatin immunoprecipitation (ChIP) Chromatin immunoprecipitation was performed using a modified method from Nature 2007, 450(7168), 415-419, as described in Faceb J 2018, 32(2), 1108-1119. Specifically, cells were fixed with 1% formaldehyde (Cat no. BP531, Fisher Scientific, Waltham, MA, USA) for 10 minutes and collected by scraping. The cell suspension was centrifuged at 700 g for 5 minutes and lysed with buffer. Chromatin was fragmented into 0.5–1 kb sizes using a VCX-500 sonicator (Sonics & Materials, Newtown, CT, USA) equipped with a 2 mm stepped microtip. 1% volume of each sample was separated as 1% input. Chromatin was immunoprecipitated using Rabbit H3K4me3 antibody (Cat no. 07-473, Millipore) and protein A-agarose bead (Cat No. 16-157, Millipore). Chromatin was reverse-crosslinked overnight in 65°C at 200 mM NaCl incubation. DNA fragments were purified using the QIAquick PCR Purification Kit (Cat no. 28104, Qiagen). qPCR was performed as described in 4.RT-qPCR. A 1% input from each sample was reverse-crosslinked and purified, and qPCR was performed in parallel with the immunoprecipitated samples.

[0142] 6.Statistical analysis The bar graph results are shown as mean ± SEM or SD. To assess the statistical significance of results between two groups, a Two-tailed Student's t-test was performed, with a threshold of p<0.05 vs. the control group. To assess the statistical significance of results between three or more groups, a One-way ANOVA was performed, followed by Dunnett's multiple comparisons test using GraphPad Prism 7.00.

[0143] Example 1. CPI-455 induces astrocyte differentiation in NCS. NSCs proliferate in the presence of epidermal growth factor (EGF) and fibrous growth factor-2 (FGF2), and differentiate into neurons and astrocytes in the absence of these growth factors in the culture medium. To investigate the effect of CPI-455 on NSC differentiation, brain cortical tissue was collected from 14-day-old (E14) Sprague-Dawley rat embryos, and NSCs were grown in the presence of EGF and FGF2 for one week. Subsequently, NSCs without growth factors were treated with either CPI-455 or a control group of DMSO (0.1%) to induce differentiation for four days.

[0144] Western blotting targeting the neuronal labeling protein TUBB3, the astrocyte labeling protein GFAP, and the oligodendrocyte labeling protein OLIG2 in differentiating neurons showed that CPI-455 significantly increased GFAP protein expression compared to the control group, as shown in Figure 1(a), but there was no significant change in TUBB3 or OLIG2 expression. RT-qPCR results, as shown in Figure 1(b), showed a significant increase in gfap mRNA expression in cells treated with 50 μM CPI-455 compared to the control group. This suggests that treatment with CPI-455 during NSC differentiation increases astrocytogenesis.

[0145] Furthermore, to investigate whether GFAP induction was due to an increase in astrocytes or an increase in protein expression in individual cells, immunocytochemistry (ICC) was performed to assess the astrocyte ratio during NSC differentiation. As a result, as shown in Figures 2(a) and 2(b), we observed a significant increase in the number of GFAP-positive cells when NSCs were treated with 50 μM CPI-455 during differentiation. In contrast, there was no significant difference in the ratio of TUBB3-positive cells between the control group and the CPI-455-treated group. There was also no significant difference in the number of DAPI-positive cells between the control group and the CPI-455-treated cells. This suggests that CPI-455 treatment during NSC differentiation enhances astrocyte generation without inducing cytotoxicity.

[0146] Example 2. Inducing astrocyte generation with the BMP2-STAT3 signal using CPI-455 To confirm the mechanism by which astrocytes are generated in NSCs treated with CPI-455, Western blotting was performed to measure STAT3 expression and phosphorylation. As shown in Figure 3(a), treatment with 50 μM CPI-455 significantly increased phosphorylated STAT3 (pSTAT3) relative to total STAT3 in differentiating neurons. The JAK (Janus kinase) / STAT pathway is one of the signaling pathways involved in various cellular events, including cell differentiation, inflammation, and regeneration. It has been previously reported that the JAK / STAT pathway is involved in regulating astrocyte differentiation in NSCs. STAT3 is activated during CNTF (ciliary neurotrophic factor)-induced astrocyte generation, and disruption of STAT3 using dominant-negative mutations has been shown to suppress astrocyte generation. Furthermore, STAT3 has been reported as a regulator of proliferation, differentiation, and self-regeneration in many stem cells and cancer cells. Induction of STAT3 phosphorylation means that the JAK / STAT3 signaling pathway is activated in the CPI-455-induced astrocyte generation process.

[0147] The cell fate and differentiation of NSCs are regulated by various cytokines. The JAK / STAT pathway can be activated by IL-6 (interleukin-6) series cytokines, including LIF (leukemia inhibitory factor) and CNTF. BMP2, a TGF-β (transforming growth factor-beta) series cytokine, can also induce astrocyte differentiation in fetal mouse brain cells and NSCs. Therefore, to investigate the expression of cytokines associated with astrocyte differentiation and STAT3 signaling in CPI-455-treated neurons, we checked the levels of il-6, lif, cntf, and bmp2 by RT-qPCR. As a result, as shown in Figure 3(b), we confirmed a significant increase in bmp2 in neurons treated with 50 μM CPI-455.

[0148] Interestingly, among the cytokines investigated that are known to induce astrocyte generation in NSCs, bmp2 was the only cytokine mRNA that showed a significant increase. Since BMP2 has been reported to induce developmental changes toward astrocyte generation through activation of the Smad pathway, CPI-455-induced astrocyte generation may be due to activation of the BMP2 signaling pathway. Generally, LIF and BMP2 synergistically induce astrocyte generation through activation of JAK-STAT3 and the Smad pathway, respectively. However, in CPI-455-induced astrocyte generation, increased STAT3 phosphorylation and astrocyte generation were observed along with increased bmp2 expression without increased lif expression. This discrepancy between lif expression and STAT3 phosphorylation was presumed to be due to a non-standard BMP2-STAT3 signaling pathway involving STAT3 phosphorylation. In fact, the BMP2-STAT3 pathway has been reported as a pathway that can induce astrocyte generation without LIF stimulation. Recombinant BMP2 treatment of human oral cancer cells also increased STAT3 phosphorylation, supporting the claim that BMP2 stimulation increases STAT3 phosphorylation during signal transduction.

[0149] Considering these results regarding STAT3 phosphorylation and BMP2 signaling, it was hypothesized that the astrocyte-generating effect of CPI-455 is induced by activation of the Gfap promoter via a non-standard BMP2-STAT3 signaling pathway.

[0150] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the present invention pertains will understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the present invention. Therefore, the embodiments described above should be understood in all respects as illustrative and not limiting. [Industrial applicability]

[0151] This invention demonstrates a technology for inducing the differentiation of stem cells into astrocytes using CPI-455. This not only allows for differentiation into astrocytes without the use of existing stem cell differentiation promoters, but is also expected to enable the effective treatment of neurodegenerative diseases using the differentiated astrocytes, thus possessing potential for industrial application.

Claims

1. A composition for inducing the differentiation of stem cells into astrocytes, characterized by containing CPI-455 as an active ingredient.

2. The differentiation-inducing composition according to claim 1, characterized in that the stem cells are one or more selected from the group consisting of embryonic stem cells, adult stem cells, embryonic germ stem cells, and embryonic tumor stem cells.

3. The differentiation-inducing composition according to claim 1, characterized in that the CPI-455 is contained in the overall composition at a concentration of 0.1 to 100 μM.

4. The CPI-455 is characterized by satisfying one or more of the following features, as described in claim 1, for differentiation induction: (a) Promote the phosphorylation of STAT3 (Signal transmitter and activator of transcription 3); and (b) Increases the secretion of BMP2 (Bone morphogenic protein 2).

5. A method for differentiating stem cells into astrocytes, comprising the step of treating them with the composition described in claim 1 and culturing the stem cells.

6. The method according to claim 5, characterized in that the astrocyte expresses GFAP (Glial fibrillary acid protein).

7. A kit for inducing differentiation of stem cells into astrocytes, characterized by comprising CPI-455 or the composition for inducing differentiation of stem cells into astrocytes described in claim 1.

8. The application of CPI-455 or the composition for inducing differentiation of stem cells into astrocytes according to claim 1, for use in inducing differentiation of stem cells into astrocytes.

9. A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, characterized by containing CPI-455 as an active ingredient.

10. The aforementioned neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis, adrenoleukodystrophy, Alexander disease, Alpas disease, ataxia vasodilator, Batten disease, bovine spongiform encephalopathy (BSE), Canavan disease, corticobasal degeneration, Creutzfeldt-Jakob disease, Lewy body dementia, fatal familial insomnia, frontotemporal lobar degeneration, Huntington's disease, Kennedy disease, Krabbe disease, Lyme disease, Machado-Joseph disease, multiple sclerosis, and many other conditions. The pharmaceutical composition according to claim 9, characterized in that it is one or more selected from the group consisting of systemic atrophy, neuroacanthocytosis, Niemann-Pick disease, Parkinson's disease, Pick disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelin-disintegrating sclerosis, spinocerebellar ataxia, subacute combined degeneration of the spinal cord, spinal syphilis, Tay-Sachs disease, toxic encephalopathy, transmissible spongiform encephalopathy, and unstable hedgehog syndrome.

11. A method for preventing or treating neurodegenerative diseases, characterized by comprising the step of administering CPI-455, or a composition containing it as an active ingredient, to an individual in need.

12. CPI-455, or compositions containing it as an active ingredient, for the prevention or treatment of neurodegenerative diseases.

13. Uses of CPI-455, or compositions containing it as an active ingredient, for the manufacture of therapeutic agents for neurodegenerative diseases.

14. A cell therapy agent for the prevention or treatment of neurodegenerative diseases, comprising astrocytes produced by the method described in claim 5.

15. The aforementioned neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis, adrenoleukodystrophy, Alexander disease, Alpas disease, ataxia vasodilator, Batten disease, bovine spongiform encephalopathy (BSE), Canavan disease, corticobasal degeneration, Creutzfeldt-Jakob disease, Lewy body dementia, fatal familial insomnia, frontotemporal lobar degeneration, Huntington's disease, Kennedy disease, Krabbe disease, Lyme disease, Machado-Joseph disease, multiple sclerosis, and many other conditions. The cell therapy agent according to claim 14, characterized in that it is one or more selected from the group consisting of systemic atrophy, neuroacanthocytosis, Niemann-Pick disease, Parkinson's disease, Pick disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelin-disintegrating sclerosis, spinocerebellar ataxia, subacute combined degeneration of the spinal cord, spinal syphilis, Tay-Sachs disease, toxic encephalopathy, transmissible spongiform encephalopathy, and unstable Hedgehog syndrome.

16. A method for preventing or treating a neurodegenerative disease, characterized by comprising the step of administering the cell therapy agent described in claim 14 to an individual in need thereof.

17. The cell therapy agent according to claim 14 is used for the prevention or treatment of neurodegenerative diseases.

18. A manufacturing application for a cell therapy agent produced by the method of claim 5.

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