How to culture astrocytes

Abstract

To provide a method for producing hyperproliferative cell, a hyperproliferative cell, a cellular secretion, a pharmaceutical composition and a method for suppressing a sympathetic nervous system.SOLUTION: Provided is a method for producing a hyperproliferative cell or an ectodermal precursor cell including: (i) preparing an ectodermal cell being a raw material; (ii) bringing the inhibitor in contact with an ectodermal cell being the raw material in a culture medium containing low molecular signal transduction pathway inhibitor to culture the inhibitor; and (iii) performing additional culture in a culture medium containing the inhibitor after the contact to obtain a cultured product including a hyperproliferative cell having higher cell proliferation capability than an ectodermal cell being the raw material. Also provided is a hyperproliferative cell which is equipped with characteristics of an ectodermal cell, and has proliferation capability that a cell number after a culture period of more than 28 days in which the inhibitor was brought in contact with the low molecular signal transduction pathway inhibitor exceeds 1.0 times the number of ectodermal cells cultured in the same culture condition over the same culture period other than the case where the cell is in non-contact with the inhibitor.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing highly proliferative cells, highly proliferative cells, and uses thereof.

Background Art

[0002] In the field of regenerative medicine, various cells in the living body, cells at various differentiation stages, or cells induced to differentiate in a specific direction are used. Considering use in the living body, it is desirable that these cells do not involve gene modification. So far, it has been reported that mature endodermal cells can be reprogrammed into stem cells or progenitor cells without gene modification by contacting them with specific low-molecular-weight compounds (International Publication No. 2017 / 119512 and International Publication No. 2020 / 080550).

Summary of the Invention

[0003] On the other hand, many cells obtained without gene modification, especially cells that are relatively close to mature cells, have limited proliferative ability and tend not to be maintained in or outside the living body for a long time. For this reason, there is still a demand for highly proliferative cells that are effective for long-term use of cell functions or mass production of useful substances produced by cells. In particular, ectodermal cells including nervous system cells have not been fully utilized unlike endodermal cells.

Problems to be Solved by the Invention

[0004] An object of the present disclosure is to provide a method for producing highly proliferative cells, highly proliferative cells, and uses thereof.

Means for Solving the Problems

[0005] As a result of intensive research, the present inventors have found that highly proliferative cells can be obtained by contacting ectodermal cells with a low-molecular-weight signal transduction pathway inhibitor.

[0006] That is, the present disclosure includes the following aspects: [1] A method for producing highly proliferative cells, comprising: (i) preparing ectodermal cells as a raw material; (ii) culturing the raw material ectodermal cells in a medium containing a low-molecular-weight signaling pathway inhibitor by contacting the inhibitor with the raw material ectodermal cells; and (iii) after the contacting, performing additional culturing in a medium containing the inhibitor to obtain a culture containing highly proliferative cells having a higher cell proliferation ability than the raw material ectodermal cells. A method for producing highly proliferative cells, comprising the above steps; [2] The highly proliferative cells have a proliferation ability such that the number of cells after a culture period of more than 28 days of contact with the inhibitor exceeds 1.0 times the number of cells of ectodermal cells cultured for the same culture period under the same culture conditions except for non-contact with the inhibitor. The production method according to [1]; [3] The production method according to [1] or [2], wherein the raw material ectodermal cells contain cells of the central nervous system; [4] The production method according to any one of [1] to [3], wherein the raw material ectodermal cells contain astrocytes; [5] The production method according to any one of [1] to [4], wherein the inhibitor contains at least one compound selected from the group consisting of a TGFβ receptor inhibitor and a ROCK inhibitor; [6] The production method according to [5], wherein the concentration of the TGFβ receptor inhibitor is in the range of 0.001 μM to 100 μM; [7] The production method according to [5] or [6], wherein the concentration of the ROCK inhibitor is in the range of 0.001 μM to 100 μM; [8] The production method according to any one of [1] to [7], wherein the highly proliferative cells express at least one gene specific to mature cells at the same level or higher than the raw material ectodermal cells, and express at least one gene specific to progenitor cells at the same level or higher than the raw material ectodermal cells; [9] The production method according to any one of [1] to [8], wherein at least one selected from the group consisting of Musashi1, Notch1, Nestin, and SOX2 is negative in the highly proliferative cells;

[10] A highly proliferative cell having the characteristics of ectodermal cells and having a proliferative ability such that the number of cells after a culture period of more than 28 days in contact with a small molecule signaling pathway inhibitor exceeds 1.0 times the number of ectodermal cells cultured for the same culture period under the same culture conditions except being not in contact with the inhibitor;

[11] The cell according to

[10] , wherein the highly proliferative cell comprises a cell in which at least one selected from the group consisting of Musashi1, Notch1, Nestin, and SOX2 is negative;

[12] The cell according to

[10] or

[11] , wherein the expression level of NG2 is higher than the expression level of NG2 in the ectodermal cells not in contact with the inhibitor;

[13] Obtaining a culture containing a highly proliferative cell obtained by the production method according to any one of [1] to [9] or a cell secretion secreted from the highly proliferative cell according to any one of

[10] to

[12] , and separating the cell secretion from the culture, a method for producing a cell secretion;

[14] The method according to

[13] , wherein the cell secretion is an exosome;

[15] A method for producing an ectodermal progenitor cell, The ectodermal progenitor cell is a highly proliferative cell, (i) preparing ectodermal cells as a raw material, (ii) contacting the inhibitor with the ectodermal cells as the raw material in a medium containing a small molecule signaling pathway inhibitor and culturing for a period of more than 28 days, and (iii) after the contact, performing additional culture in a medium containing the inhibitor to obtain a culture containing highly proliferative cells with enhanced cell proliferation ability compared to the ectodermal cells as the raw material A method for producing an ectodermal progenitor cell, comprising;

[16] The production method according to

[15] , further comprising isolating the highly proliferative cells from the culture;

[17] A cell secretion containing a protein and / or miRNA, The protein includes a combination of vinculin (P18206), integrin β-1, CD29 (P05556), pyruvate kinase M1 / 2 (P14618), and ephrin type-A receptor 2 (P29317). The miRNA includes a combination of hsa-miR-382-5p (MIMAT0000737), hsa-miR-155-5p (MIMAT0000646), hsa-miR-379-5p (MIMAT0000733), hsa-miR-16-5p (MIMAT0000069), hsa-miR-382-5p (MIMAT0000737), hsa-miR-16-5p (MIMAT0000069), hsa-miR-382-5p (MIMAT0000737), hsa-miR-21-5p (MIMAT0000076), hsa-let-7a-5p (MIMAT0000062), hsa-miR-16-5p (MIMAT0000069), hsa-miR-409-3p (MIMAT0001639), hsa-let-7a-5p (MIMAT0000062), and hsa-let-7f-5p (MIMAT0000067); a cell secretion. The cell secretion according to

[17] , which is an exosome. A cell secretion secreted from a highly proliferative cell obtained by the production method according to

[19] [1] or [2] or a highly proliferative cell according to any one of

[10] to

[12] , and The cell secretion according to

[17] or

[18] , and A pharmaceutical composition for preventing or treating a disorder related to peripheral nerve cells or central nerve cells, comprising any of the cell secretions. A cell secretion secreted from a highly proliferative cell obtained by the production method according to

[20] [1] or [2] or a highly proliferative cell according to any one of

[10] to

[12] , and The cell secretion according to

[17] or

[18] , and A method for suppressing the sympathetic nervous system, comprising bringing any of the cell secretions into contact with nerve cells.

Advantages of the Invention

[0007] According to the present disclosure, a method for producing highly proliferative cells, highly proliferative cells, and uses thereof can be provided.

Brief Description of the Drawings

[0008]

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Mode for Carrying Out the Invention

[0009] <1> Method for Producing Highly Proliferative Cells The method for producing highly proliferative cells according to the present disclosure is (i) preparing ectodermal cells as a raw material, (ii) culturing the inhibitor by contacting the inhibitor with the ectodermal cells as the raw material in a medium containing a low-molecular-weight signal transduction pathway inhibitor, and (iii) after the contact, performing additional culture in a medium containing the inhibitor to obtain a culture containing highly proliferative cells having a higher cell proliferation ability than the ectodermal cells as the raw material including.

[0010] In this specification, the term "cell" should be interpreted as a term including at least one cell unless otherwise specified. For this reason, unless otherwise specified, the cells meant by the term "cell" are not limited to a single cell and can be used synonymously with a cell population.

[0011] In this specification, the term "cell population" should be interpreted as a term including at least one type of cell unless otherwise specified. For this reason, unless otherwise specified, the cells meant by the term "cell population" are not limited to one type of cell.

[0012] The "ectoderm" is one of the three germ layers that occur during the development of metazoans. The ectoderm is derived from the outer layer of the embryo and develops into the skin, nervous system, and sensory organs. Examples of the skin include the epidermis, hair, nails, and skin glands. Examples of the nervous system include cranial nerves, spinal cord, and peripheral nerves. Examples of the sensory organs include visual organs, auditory organs, equilibrium organs, taste organs, olfactory organs, and tactile organs.

[0013] The "ectodermal cells" used in the present disclosure include, for example, cells of the nervous system, such as cells of the central nervous system and the peripheral nervous system. Therefore, in the present disclosure, the "starting ectodermal cells" can include, for example, cells of the central nervous system. The starting ectodermal cells may include astrocytes.

[0014] The ectodermal cells used in the production method of the present disclosure may be provided from any origin, for example, provided from mammals. Examples of mammals include, for example, humans, rats, mice, cats, dogs, guinea pigs, rabbits, sheep, horses, pigs, cows, and monkeys. The mammal is preferably a human, a rat, a mouse, a cat, or a dog, more preferably a human, a rat, or a mouse.

[0015] The "ectodermal cells" used in the present disclosure are cells that constitute "ectodermal tissues" and "ectodermal organs". Examples of "ectodermal tissues" and "ectodermal organs" include, for example, the central nervous system (brain and spinal cord), pituitary gland, peripheral nerves, enteric nerves, adrenal medulla, melanocytes, facial cartilage, dentin of teeth, epidermis, hair, nails, skin, sebaceous glands, salivary glands, sweat glands, mammary glands, nasal cavity, nasal mucosa, oral epithelium, oral cavity, eyes, bladder, and anus. The brain can be roughly divided into the cerebrum, cerebellum, and brainstem. The cerebrum can be further divided into the telencephalon and diencephalon, and the brainstem can be further divided into the midbrain, pons, and medulla oblongata. The central nervous system is composed of nerve cells and glial cells, and these cells can be selected as starting materials. Many mature cells that have completed the final differentiation stage are present in these tissues or organs.

[0016] The starting ectodermal cells can be selected from at least one selected from the group consisting of astrocytes, microglia, oligodendrocytes, oligodendrocyte progenitor cells (also referred to as polydendrocytes), ependymal cells, Schwann cells, and satellite cells, which are classified as glial cells, for example. These cells may be primary cells obtained from a living body, established cell lines, or ectodermal cells induced from pluripotent stem cells such as ES cells or induced pluripotent stem cells (iPS cells).

[0017] The cells used in the method of the present disclosure may be, for example, cells isolated and purified from a brain excised from a mammal.

[0018] For example, in the case of rats, it is preferable to use a brain excised from an adult rat aged 10 to 20 weeks, but a brain derived from a juvenile rat aged 2 months or less may also be used. In the case of humans, it can be obtained by biopsy or surgery. For example, both neurons and glial cells can be collected by biopsy. In the case of surgery, either an excised adult brain tissue piece or a brain excised from a fetal death can be used. Alternatively, after isolating and purifying neurons or glial cells from these excised brains, frozen cells can also be used.

[0019] The ectodermal cells as raw materials can be defined as cells showing the characteristics of ectodermal cells. Examples of the characteristics of ectodermal cells include cell morphology, expression of genes specific to the cells (sometimes referred to as "marker genes"), etc. That a cell is an ectodermal cell can be confirmed by the expression of at least one marker gene selected from the group consisting of, for example, GFAP, S100B, SLC1A2 (also known as "EAAT2" or "GLT-1"), and NG2 at any stage of the differentiation stage. The expression of the marker gene of ectodermal cells may be confirmed based on either the gene or the protein. The confirmation of the expression of the gene or the protein can be carried out by methods known in the art, such as the measurement of the expression level of the gene using quantitative PCR (sometimes described as "qPCR"), etc., and the measurement of the protein amount using immunoassay methods such as ELISA and flow cytometry.

[0020] In addition, in this specification, the expression of the marker gene can be confirmed using techniques known in the art. For example, it can be confirmed by measuring the expression level of the gene using quantitative PCR (sometimes described as "qPCR"), etc., and measuring the protein amount using immunoassay methods such as ELISA and flow cytometry.

[0021] The ectodermal cells used as the raw material prepared as described above are brought into contact with a small molecule signal transduction pathway inhibitor. The contact may be in vitro. There is no particular limitation on the small molecule signal transduction pathway inhibitor that can be used in this method, and any of them can be used. Examples of the small molecule signal transduction pathway inhibitor include a transforming growth factor (TGF) β receptor inhibitor, a ROCK [Rho-associated protein kinase] inhibitor, and a glycogen synthase kinase 3 (GSK3) inhibitor.

[0022] In the production method according to the present disclosure, the inhibitor may include at least one compound selected from the group consisting of a TGFβ receptor inhibitor, a ROCK inhibitor, and a GSK3 inhibitor. In the production method according to the present disclosure, the inhibitor may include at least one compound selected from the group consisting of a TGFβ receptor inhibitor and a ROCK inhibitor.

[0023] The TGFβ receptor inhibitor is not particularly limited as long as it has the effect of inhibiting the function of the TGFβ receptor. For example, 2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole (A-83-01), [2-(5-chloro-2-fluorophenyl)-4-(4-pyridylamino)]pteridine (SD-208), 3-(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (all of the above are from Merck), SB431542 (from Sigma Aldrich), and CultureSure® A-83-01 (from Fujifilm Wako Pure Chemical Corporation) can be mentioned. The TGFβ receptor inhibitor is preferably CultureSure® A-83-01. The TGFβ receptor inhibitor also includes a TGFβ receptor antagonist. These TGFβ receptor inhibitors may be used alone or in combination of two or more.

[0024] The ROCK inhibitor is not particularly limited as long as it has the effect of inhibiting the function of Rho-associated protein kinase. Examples of the ROCK inhibitor include GSK269962A (from Axon medchem), Fasudil hydrochloride (from Tocris Bioscience), CultureSure® Y-27632 (from Fujifilm Wako Pure Chemical Corporation), and H-1152 dihydrochloride (from Fujifilm Wako Pure Chemical Corporation). The ROCK inhibitor is preferably CultureSure® Y-27632. The ROCK inhibitor may be used alone or in combination of two or more.

[0025] The GSK3 inhibitor is not particularly limited as long as it has the effect of inhibiting the function of glycogen synthase kinase (GSK) 3. Examples of the GSK3 inhibitor include SB216763 (Selleck), CHIR 98014 (Axon medchem), CHIR 99021 (Axon medchem), SB415286 (Tocris Bioscience), and Kenpaullone (Cosmo Bio). The GSK3 inhibitor is preferably CHIR 99021. The GSK3 inhibitor may be used alone or in combination of two or more.

[0026] The small molecule signal transduction pathway inhibitor used in this production method can be at least one selected from the group consisting of a TGFβ receptor inhibitor and a ROCK inhibitor. That is, the small molecule signal transduction pathway inhibitor used in this production method is · a combination of a TGFβ receptor inhibitor and a ROCK inhibitor, · a TGFβ receptor inhibitor, or · a ROCK inhibitor and may be. In one aspect, the small molecule signal transduction pathway inhibitor used is a combination of a TGFβ receptor inhibitor and a ROCK inhibitor. In this specification, the term "small molecule signal transduction pathway inhibitor" may also be simply referred to as "inhibitor".

[0027] Examples of the aspects of the present disclosure include the following inhibitors: (1) At least one compound selected from the group consisting of 2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole (A-83-01), [2-(5-chloro-2-fluorophenyl)-4-(4-pyridylamino)]pteridine (SD-208), 3-(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, SB431542 and CultureSure® A-83-01; (2) At least one compound selected from the group consisting of 2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole (A-83-01), [2-(5-chloro-2-fluorophenyl)-4-(4-pyridylamino)]pteridine (SD-208), 3-(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, SB431542 and CultureSure® A-83-01, and a combination with at least one compound selected from the group consisting of GSK269962A, Fasudil hydrochloride, CultureSure® Y-27632 and H-1152 dihydrochloride; and (3) At least one compound selected from the group consisting of GSK269962A, Fasudil hydrochloride, CultureSure® Y-27632 and H-1152 dihydrochloride.

[0028] The concentration of the TGFβ receptor inhibitor in the medium is, for example, in the range of 0.001 μM to 100 μM, 0.01 μM to 50 μM, or 0.05 μM to 30 μM, and can be appropriately adjusted according to the type of TGFβ receptor used. In one aspect, the concentration of the TGFβ receptor inhibitor in the medium, such as CultureSure® A-83-01, is 0.1 μM to 3 μM.

[0029] The concentration of the ROCK inhibitor in the medium is, for example, in the range of 0.001 μM to 100 μM, 0.01 μM to 80 μM, or 0.1 μM to 50 μM, and can be appropriately adjusted according to the type of ROCK inhibitor used. In one aspect, the concentration of the ROCK inhibitor in the medium, such as CultureSure® Y-27632, is 1 μM to 30 μM.

[0030] The concentration of the GSK3 inhibitor in the medium is, for example, in the range of 0.001 μM to 100 μM, 0.01 μM to 80 μM, or 0.1 μM to 50 μM, and can be appropriately adjusted according to the type of GSK3 inhibitor used.

[0031] The above concentration ranges are applicable to both the cases where the TGFβ receptor inhibitor, the ROCK inhibitor, and the GSK3 inhibitor are used alone, and the cases where these inhibitors are used in combination. Further, when the above inhibitors are water-insoluble or sparingly water-soluble, they can be dissolved in a small amount of a low-toxic organic solvent (e.g., DMSO, etc.) and then added to the medium to achieve the above final concentration.

[0032] The contact between the ectodermal cells as raw materials and the small molecule signal transduction pathway inhibitor is carried out by culturing the ectodermal cells as raw materials in a medium containing the above inhibitor. Specifically, the culture is performed by adding the above inhibitor to the medium at an effective concentration.

[0033] After contacting the starting ectodermal cells with a small molecule signaling pathway inhibitor, the cells are further cultured in a medium containing the inhibitor. Here, the further culture is also referred to as "additional culture" or "second culture". In contrast, the culture until the starting ectodermal cells are contacted with the small molecule signaling pathway inhibitor, i.e., the culture of ectodermal cells in a medium not containing the small molecule signaling pathway inhibitor, is also referred to as "pre-culture" or "first culture". By performing additional culture after the contact with the inhibitor, highly proliferative cells with enhanced cell proliferation ability can be obtained. Note that during the additional culture, since the inhibitor is contained in the medium, the contact between the cells and the inhibitor continues.

[0034] In the present disclosure, as the medium used for pre-culture and additional culture, a medium widely used for culturing animal cells can be used as a basal medium, and a commercially available basal medium may be used. Examples of commercially available basal media include, but are not limited to, Astrocyte Growth Medium (AGM), Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), RPMI1640 medium, Medium 199, Ham's F12 medium, William's E medium, and NS basal medium (FUJIFILM Wako Pure Chemical Corporation). As the medium, the above-mentioned ones may be used alone or in combination of two or more.

[0035] Examples of additives added to the medium include cytokines, growth factors (e.g., epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2)), hormones (e.g., insulin, estradiol, progesterone, testosterone, and thyroxine), steroids (e.g., dexamethasone (Dex)), plasma-derived proteins (e.g., transferrin), various amino acids (e.g., L-glutamine and L-proline), various inorganic salts (e.g., selenite and NaHCO3), various vitamins (e.g., nicotinamide and ascorbic acid derivatives), N2 supplement (FUJIFILM Wako Pure Chemical Corporation), NS supplement (FUJIFILM Wako Pure Chemical Corporation), B-27 Plus Supplement (Thermo Fisher Scientific), various antibiotics (e.g., penicillin and streptomycin), antifungal agents (e.g., amphotericin), and buffers (e.g., Good buffers such as HEPES).

[0036] For the medium used in the pre-culture (first culture) and the additional culture (second culture), either a serum-supplemented medium or a serum-free medium may be used.

[0037] When using a serum-supplemented medium, for example, fetal bovine serum (FBS) can be used as the serum. Also, to facilitate exosome separation, FBS from which exosomes have been removed can be used. Examples of commercially available exosome-depleted media include FBS exosome-depleted, OneShot format (Gibco (registered trademark), Thermo Fisher Scientific). The concentration of serum in the medium is, for example, 0.5 - 25% (v / v), 1 - 25% (v / v), 1 - 20% (v / v), 1 - 15% (v / v), 2 - 15% (v / v), 2 - 10% (v / v), 3 - 10% (v / v), 3 - 8% (v / v), and 3 - 5% (v / v). In a specific embodiment, the concentration of serum in the medium is 3% (v / v).

[0038] When using serum-free medium, a serum replacement may be added. Examples of serum replacements include Bovine serum albumin (BSA), KnockOut Serum Replacement (KSR), Human serum albumin (HSA or Human albumin, serum; HAS), etc. As human serum albumin, human serum albumin isolated from human plasma or human serum albumin purified from rice expressing the human serum albumin gene (FUJIFILM Wako Pure Chemical Corporation) can be used. Usually, factors such as growth factors (hEGH, etc.), cytokines, and hormones are further added to the medium. Examples of these added factors include epidermal growth factor (EGF), insulin, transferrin, hydrocortisone 21-hemisuccinate or its salt, and dexamethasone (Dex), N2 supplement (FUJIFILM Wako Pure Chemical Corporation), NS supplement (FUJIFILM Wako Pure Chemical Corporation), and B-27 Serum Free Supplement (Thermo Fisher Scientific), but are not limited thereto.

[0039] The culture vessel used for culturing is not particularly limited as long as it is suitable for adherent culture. Examples include dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multiplates, multiwell plates, chamber slides, petri dishes, tubes, trays, and culture bags. As the culture vessel, a culture vessel with a surface processed so that cells cannot adhere can also be used for suspension culture of cells. Alternatively, in the case of adherent culture, one with an inner surface coated with a cell support substrate for the purpose of improving cell adhesion can be used. Examples of such cell support substrates include collagen, gelatin, Matrigel, poly-L-lysine, laminin, and fibronectin. Preferred cell support substrates are collagen or Matrigel.

[0040] The starting ectodermal cells are, for example, 1×10 2 ~1×10 6 cells / cm 2 、1×10 3 ~1×10 5 cells / cm 2 、or 1×10 3 ~1×10 4 cells / cm 2 and can be seeded onto a culture vessel at a cell density of.

[0041] For the culture, generally the conditions applicable to the culture of ectodermal cells can be directly applied. For the culture, a CO2 incubator can be used, and in that case, the culture temperature and CO2 concentration can be those commonly used, for example, 37°C and 5% (v / v).

[0042] The culture period is the period during which it is cultured with YA, and this period can be a continuous period or a discontinuous period, that is, a period obtained by summing a plurality of discontinuous periods. The culture period only needs to be more than 28 days, for example, it can be 5 weeks or more, 6 weeks or more, or 7 weeks or more. As the upper limit of the culture period, for example, it can be about 3 months or 100 days. Subculture can be appropriately performed according to the cell density in the culture vessel, the state of the medium, or the state of the cells, etc., and the subculture interval can be, for example, about 2 to 20 days.

[0043] Highly proliferating cells (which may be referred to as "HP cells" herein) obtained by this production method are cells with enhanced proliferative ability. Compared with control ectodermal cells cultured under the same conditions as those of the cells except that a medium not containing a low-molecular-weight signal transduction pathway inhibitor is used, they have high cell proliferation activity and the property of proliferating in a shorter time, that is, the property of having a shorter cell doubling time, the property of proliferating over a longer period, or the property of satisfying both of these. It means that the cells have such properties. The cell population contained in the culture obtained by this production method includes not only HP cells but also, in addition to HP cells, cells whose proliferative ability has not been enhanced and whose proliferative ability is the same as that of the original cells, that is, non-highly proliferating cells (which may be referred to as "non-HP cells" herein). The proportion of HP cells in the cell population obtained by this production method may be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more based on the number of cells.

[0044] Highly proliferating cells can have a proliferative ability such that the number of cells (which is referred to as "the number of highly proliferating cells N" herein) after a culture period exceeding 28 days (which is referred to as "period T" herein) in contact with an inhibitor is more than 1.0 times the number of cells of ectodermal cells (which is referred to as "the number of ectodermal cells N'" herein) cultured for the same culture period under the same culture conditions except for being not in contact with the inhibitor. As the culture conditions in this case, for example, a seeding density of 4×10 3 cells / cm 2 , a culture temperature of 37 °C, and a CO2 concentration of 5% can be used. Any of the media listed as the media used for the above pre-culture and additional culture can be used as the medium used when confirming the number of highly proliferating cells N. As the two types of media used when confirming the number of highly proliferating cells N and the number of ectodermal cells N' respectively, media having the same composition except for the presence or absence of the inhibitor can be used.

[0045] In one aspect, the period T is, for example, 90 days, 80 days, 70 days, 60 days, 50 days, 45 days, 42 days, 40 days, 35 days, 32 days, 30 days or 29 days.

[0046] In one aspect, the cell number N of the highly proliferative cells is not particularly limited as long as it exceeds 1.0 times the cell number N' of the ectodermal cells, and may be, for example, 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, or 1.5 times or more the cell number N'. Preferably, the cell number N of the highly proliferative cells is 1.5 times or more the cell number N' of the ectodermal cells.

[0047] The highly proliferative cells may be identified by the expression of cell-specific genes, that is, marker genes. The expression of the marker gene of the highly proliferative cells may be confirmed based on either genes or proteins as described above in this specification. The method for confirming the expression of genes or proteins can be performed by, for example, the presence or absence of expression, the specific expression level depending on the measurement method, and comparison with the expression level of the same gene or protein in specific cells.

[0048] In this specification, the expression of genes or proteins may be expressed as "positive" or "negative". Regarding the expression of genes or proteins, "positive" means the case where the expression of the target gene or protein is recognized, while "negative" means the case where the expression of the target gene or protein is not recognized. When expressing that the expression of a gene or protein is "high" or "positive", or "low" or "negative", specific cells different from the highly proliferative cells can be used as a comparison control. The specific cells serving as the comparison control, that is, the "control cells", may be cells with an extremely low expression level of the target gene or protein, may be the starting ectodermal cells, that is, the ectodermal cells not in contact with the inhibitor, or may be mature cells or their progenitor cells.

[0049] For example, when a highly proliferative cell is "positive" with respect to the expression of a marker gene, it can be meant that the expression level of the marker gene after contact with a small molecule signal transduction pathway inhibitor is 1.0 times or more the expression level of the marker gene in ectodermal cells cultured without contact with the inhibitor. In contrast, when a highly proliferative cell is "negative" with respect to the expression of a marker gene, it can be meant that the expression level of the marker gene after contact with the inhibitor is less than 1.0 times the expression level of the marker gene in ectodermal cells cultured without contact with the inhibitor.

[0050] The culture conditions of the highly proliferative cells and the ectodermal cells not in contact with the inhibitor provided for the method of confirming the expression level can be the same except for being in contact or not in contact with the inhibitor. For example, based on the cell state, proliferative ability, cell number, etc., the culture period, the same number of cell passages, and the same cell density can be appropriately adjusted so that the target cells are in the same or extremely similar state, and those skilled in the art can select based on the cell state.

[0051] Here, the expression level of the marker gene after a culture period of a certain period or more after contact with a small molecule signal transduction pathway inhibitor is expressed as the expression level relative to a control gene. As the control gene, for example, beta-actin (ACTB) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) can be used.

[0052] In one aspect, a highly proliferative cell can express at least one gene specific to mature cells at the same level or more than that of ectodermal cells used as a raw material, and at least one gene specific to progenitor cells at the same level or more than that of ectodermal cells used as a raw material.

[0053] As used herein, the term "progenitor cell" means a cell at a differentiation stage until it becomes a mature cell, which can be generated from a stem cell and can differentiate into a terminally differentiated cell that constitutes the body.

[0054] Genes specific to mature cells, such as genes highly expressed in astrocytes and the like, include, for example, GFAP, S100B, and SLC1A2. Genes specific to progenitor cells, such as genes highly expressed in oligodendrocyte progenitor cells, neural epithelial cells, and radial glial cells, include, for example, NG2, Notch1, Nestin, and SOX2.

[0055] Highly proliferative cells may be cells having any expression pattern of marker genes as long as they are cells having the high proliferative ability as described above. In other words, they may be a cell population containing cells having any characteristics. Highly proliferative cells may exhibit an expression pattern of marker genes as listed below. According to the expression patterns of these marker genes, the highly proliferative cells according to the present disclosure can be quickly identified and extracted.

[0056] Highly proliferative cells can include cells in which at least one selected from the group consisting of Musashi1 (MSI1), Notch1, Nestin, and SOX2 is negative.

[0057] For example, highly proliferative cells can include GFAP-positive cells. In another aspect, highly proliferative cells can include cells that are positive for GFAP and positive for NG2, or can express GFAP at the same level or higher than the ectodermal cells serving as a raw material, and can express NG2 at the same level or higher than the ectodermal cells serving as a raw material. In another aspect, highly proliferative cells can include cells that are positive for GFAP and S100B and positive for NG2, or can express GFAP and S100B at the same level or higher than the ectodermal cells serving as a raw material, and can express NG2 at the same level or higher than the ectodermal cells serving as a raw material.

[0058] In another aspect, the highly proliferative cells can include cells that express at least one gene specific to mature cells at the same level or higher than the ectodermal cells used as a raw material, and express at least one gene specific to progenitor cells at the same level or higher than the ectodermal cells used as a raw material.

[0059] In one aspect, the highly proliferative cells can include NG2-positive cells, or can include cells that express more than the expression level of NG2 in ectodermal cells not contacted with a small molecule signaling pathway inhibitor. That is, in this aspect, the highly proliferative cells may have an expression level of NG2 higher than the expression level of NG2 in ectodermal cells not contacted with a small molecule signaling pathway inhibitor.

[0060] For example, the highly proliferative cells can include cells that are positive for S100B and positive for NG2, or can express S100B at the same level or higher than the ectodermal cells used as a raw material, and can express NG2 at the same level or higher than the ectodermal cells used as a raw material.

[0061] In one aspect, at least one selected from the group consisting of Musashi1 (MSI1), Notch1, Nestin, and SOX2 is negative in the above-described cells included in the highly proliferative cells. That is, the expression level of at least one marker gene selected from the group consisting of Musashi1, Notch1, Nestin, and SOX2 in the cells included in the highly proliferative cells after contact with the inhibitor can be less than 1.0 times the expression level of the marker gene in ectodermal cells cultured without contact with a small molecule signaling pathway inhibitor. In a preferred aspect, two or more, three or more, or all of the above-described cells included in the highly proliferative cells are negative, selected from the group consisting of Musashi1, Notch1, Nestin, and SOX2.

[0062] In one aspect, the expression level of at least one selected from the group consisting of Musashi1, Notch1, Nestin, and SOX2 in a negative cell can be represented as the expression level relative to a control gene. In this case, "negative" means less than 1.0-fold the expression level of the control gene when, for example, beta-actin (ACTB) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are used as the control genes.

[0063] The cell population of highly proliferative cells according to the present disclosure is a heterogeneous cell population containing both mature cells and progenitor cells, regardless of the differentiation stage, as long as they are highly proliferative cells having a high proliferative ability, particularly highly proliferative ectodermal cells, or alternatively, a cell population in which a very large majority of multiple types of ectodermal cells with relatively close differentiation stages are present.

[0064] In one aspect, the cell population of highly proliferative cells obtained by the present production method may be a population containing highly proliferative ectodermal progenitor cells as a main component, or a population containing both highly proliferative ectodermal progenitor cells and highly proliferative ectodermal mature cells. Here, the "main component" in the cell population means cells that occupy at least 50% based on the number of cells in the cell population. In this specification, "very large majority" means that the number of target cells in the cell population is 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more based on the number of cells.

[0065] The proportion of ectodermal mature cells in the obtained cell population of highly proliferative cells may be less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% based on the number of cells. Therefore, the present disclosure also encompasses a method for enriching ectodermal cells with enhanced cell proliferative ability, which includes contacting low molecular weight signaling pathway inhibitors with the starting ectodermal cells. The contact of the starting ectodermal cells with the low molecular weight signaling pathway inhibitors can be carried out in vitro.

[0066] In this production method, when the ectodermal cells used as raw materials are astrocytes, the cell population of highly proliferative cells may contain cells of the ectodermal cell lineage related to astrocytes. Examples of cells of the ectodermal cell lineage related to astrocytes include astrocytes, radial glial cells, oligodendrocyte progenitor cells (also called polydendrocytes), oligodendrocytes, and neuroepithelial cells. In addition, the cell population of cells of the ectodermal cell lineage related to astrocytes may contain cells that have some of the characteristics found in ectodermal cells and cannot be classified into any of the above-described cells, as long as they are a cell population showing enhanced cell proliferation ability.

[0067] Another aspect of the present disclosure relates to highly proliferative cells. The highly proliferative cells may be the highly proliferative cells obtained by the above-described production method, or may be highly proliferative cells obtained by other production methods.

[0068] The highly proliferative cells according to one aspect of the present disclosure have the characteristics of ectodermal cells and have a proliferation ability such that the number of cells after a culture period of more than 28 days in contact with a small molecule signaling pathway inhibitor is more than 1.0 times the number of cells of ectodermal cells cultured under the same culture conditions for the same culture period except that they are not in contact with the small molecule signaling pathway inhibitor. As described above, by bringing the small molecule signaling pathway inhibitor into contact with the ectodermal cells, highly proliferative cells with enhanced cell proliferation ability can be obtained without gene introduction or gene modification into the ectodermal cells. As a result, compared with the inhibitor non-contact group, in the inhibitor contact group, culture can be carried out for a longer period, and regarding the number of cells obtained, the number of cells in the inhibitor contact group is larger than the number of cells in the inhibitor non-contact group.

[0069] In one aspect, a cell group obtained by culturing human primary mature astrocytes in contact with a combination of a TGFβ receptor inhibitor and a ROCK inhibitor (inhibitor contact group) is compared with a cell group obtained by culturing human primary mature astrocytes without contacting them with the combination of the inhibitors (inhibitor non-contact group). In the inhibitor contact group, culturing can be performed for a longer period. That is, in the inhibitor contact group, cell proliferation continues for a longer period compared to the inhibitor non-contact group. Also, regarding the number of cells finally obtained by proliferation, the number of cells in the inhibitor contact group is, for example, 2 times or more, 5 times or more, 10 times or more, 50 times or more, 100 times or more, 200 times or more, or 400 times that of the inhibitor non-contact group.

[0070] Thus, since the obtained highly proliferative cells have enhanced cell proliferation ability, a large number of cells can be obtained by long-term culture. Since the highly proliferative cells can produce cell secretions as described below, if a large number of cells are obtained by long-term culture, the amount of cell secretions that can be recovered can also increase.

[0071] Regarding the highly proliferative cells according to this aspect, the matters described as the production result of this production method are applied as they are. However, as long as the cells have the characteristics of ectodermal cells and have a proliferation ability such that the number of cells after a culture period of more than 28 days in contact with the inhibitor exceeds 1.0 times the number of ectodermal cells cultured for the same culture period under the same culture conditions except for not being in contact with the inhibitor, they are not limited to being the production result of this production method.

[0072] The highly proliferative cells of the present disclosure can include cells in which at least one selected from the group consisting of GFAP, NG2, and S100B is positive, as described above. The highly proliferative cells can include, for example, at least one selected from the group consisting of the following cells (I) to (VII): (I) GFAP-positive cells, for example, cells that express GFAP as much as or more than the ectodermal cells used as a raw material; (II) NG2-positive cells, for example, cells that express NG2 as much as or more than the ectodermal cells used as a raw material; (III) S100B-positive cells, for example, cells that express S100B at the same level as or higher than the ectodermal cells that are the raw material for S100B; (IV) Cells that are positive for GFAP and positive for NG2, for example, cells that express GFAP at the same level as or higher than the ectodermal cells that are the raw material for GFAP and express NG2 at the same level as or higher than the ectodermal cells that are the raw material for NG2; (V) Cells that are positive for GFAP and positive for S100B, for example, cells that express GFAP at the same level as or higher than the ectodermal cells that are the raw material for GFAP and express S100B at the same level as or higher than the ectodermal cells that are the raw material for S100B; (VI) Cells that are positive for S100B and positive for NG2, for example, cells that express S100B at the same level as or higher than the ectodermal cells that are the raw material for S100B and express NG2 at the same level as or higher than the ectodermal cells that are the raw material for NG2; (VII) Cells that are positive for GFAP, positive for S100B, and positive for NG2, for example, cells that express GFAP at the same level as or higher than the ectodermal cells that are the raw material for GFAP, express S100B at the same level as or higher than the ectodermal cells that are the raw material for S100B, and express NG2 at the same level as or higher than the ectodermal cells that are the raw material for NG2.

[0073] The highly proliferative cells of the present disclosure can also include cells that are positive for SLC1A2. Specifically, the highly proliferative cells can also include cells in which at least one of the above genes is positive and SLC1A2 is positive.

[0074] When the highly proliferative cells of the present disclosure include at least cells that are positive for GFAP, preferably, the expression level of the GFAP gene after contact with a low-molecular-weight signal transduction pathway inhibitor is 1.0 times or more and less than 4.0 times the expression level of the GFAP gene in ectodermal cells cultured without contact with the inhibitor.

[0075] The highly proliferative cells of the present disclosure can include cells in which at least one selected from the group consisting of GFAP, NG2, and S100B is positive and at least one selected from Musashi1, Notch1, Nestin, and SOX2 is negative. That is, in this aspect, in the cells of (I) to (VII) above, at least one selected from Musashi1, Notch1, Nestin, and SOX2 is negative.

[0076] For the isolation of highly proliferative cells, known means for isolating specific cells can be applied. For example, the fluorescence-activated cell sorting (FACS) method based on the expression of proteins derived from the aforementioned marker genes, and the magnetic cell separation method using Dynabeads or the like can be utilized. Since the obtained highly proliferative cells have a high proliferative ability, even if highly proliferative cells are substantially isolated by obtaining a cell population with an extremely high proportion of highly proliferative cells by performing long-term culture, it may be acceptable.

[0077] <2> Ectodermal progenitor cells and method for producing the same The highly proliferative cells obtained by the production method according to the present disclosure may include cells showing characteristics of a more immature differentiation stage than the starting material, i.e., the ectodermal cells serving as the raw material. Such immature stage ectodermal cells, i.e., ectodermal progenitor cells, have higher cell proliferation activity compared to control ectodermal cells cultured under the same culture conditions as those of the cells except using a medium not containing an inhibitor, and can have the property of proliferating in a shorter time, the property of proliferating over a longer period, or both properties. Such ectodermal progenitor cells showing high proliferation are sometimes referred to as highly proliferative progenitor cells in this specification. The "highly proliferative cells" in this specification may include highly proliferative progenitor cells. Thereby, there are advantages such as growing immature stage ectodermal cells over a longer period and obtaining, for example, a large amount of cell secretions produced by ectodermal progenitor cells in a shorter time compared to control ectodermal cells.

[0078] Another aspect of the present disclosure relates to a method for producing ectodermal progenitor cells, wherein the ectodermal progenitor cells are highly proliferative cells, and the production method comprises: (i) preparing ectodermal cells as a raw material; (ii) contacting the inhibitor with the ectodermal cells as the raw material in a medium containing a small molecule signaling pathway inhibitor and culturing for a period exceeding 28 days; and (iii) performing additional culturing in a medium containing the inhibitor after the contact to obtain a culture containing highly proliferative cells with enhanced cell proliferation ability compared to the ectodermal cells as the raw material. The method for producing ectodermal progenitor cells of the present disclosure may further comprise isolating the highly proliferative cells from the culture.

[0079] In the method for producing ectodermal progenitor cells of the present disclosure, as the ectodermal cells as a raw material and the small molecule signaling pathway inhibitor, for example, the ectodermal cells as a raw material and the small molecule signaling pathway inhibitor described with respect to the method for producing highly proliferative cells of the present disclosure can be used respectively.

[0080] For the isolation of highly proliferative progenitor cells, known means for isolating specific cells can be applied. For example, the fluorescence-activated cell sorting (FACS) method based on the expression of a protein derived from the aforementioned marker gene specific to progenitor cells, and the magnetic cell separation method using Dynabeads or the like can be utilized. Since the obtained highly proliferative progenitor cells have high proliferation ability, a cell population with an extremely high proportion of highly proliferative progenitor cells can be obtained by performing long-term culture, and highly proliferative progenitor cells may be substantially isolated.

[0081] In the highly proliferative cells obtained by the production method according to the present disclosure, when the starting material, the ectodermal cells serving as the raw material, contain astrocytes that have reached the final differentiation stage, the obtained highly proliferative cells may contain cells showing characteristics of a differentiation stage more immature than astrocytes. In addition to having an enhanced proliferative ability, such immature-stage ectodermal cells can further have the following characteristic (a) or (b): (a) Compared with astrocytes, the expression levels of the combinations of GFAP and S100B, GFAP and SLC1A2, S100B and SLC1A2, GFAP and NG2, NG2 and SLC1A2, or NG2 and S100B are increased. (b) Compared with astrocytes, the expression levels of the combinations of GFAP, S100B and NG2, GFAP, SLC1A2 and NG2, S100B, SLC1A2 and NG2, or GFAP, S100B, SLC1A2 and NG2 are increased.

[0082] Yet another aspect of the present disclosure relates to the ectodermal progenitor cells according to the present disclosure and their uses.

[0083] The ectodermal progenitor cells obtained by the above production method can be used, for example, in an evaluation method for a therapeutic agent for neuropathy. Specifically, the usefulness of a candidate drug for a therapeutic agent for neuropathy can be evaluated by bringing the candidate drug for a therapeutic agent for neuropathy into contact with the ectodermal progenitor cells. Accordingly, the present disclosure also relates to an evaluation method for a therapeutic agent for neuropathy, which includes bringing a candidate drug for a therapeutic agent for neuropathy into contact with the ectodermal progenitor cells. Furthermore, the present disclosure also relates to an evaluation method for a neuropathy model, which includes using the above-described ectodermal progenitor cells.

[0084] The present disclosure further relates to a method for producing ectodermal mature cells, which includes subjecting the ectodermal progenitor cells obtained by the production method according to the present disclosure to maturation conditions to induce differentiation of the ectodermal progenitor cells and obtaining ectodermal mature cells.

[0085] Here, "under maturation conditions" means culture conditions using known differentiation-inducing factors. Examples of differentiation-inducing factors include, for example, Fibroblast growth factor-2 (FGF-2); leukemia inhibitory factor (LIF) or ciliary neurotrophic factor (CNTF) and other IL-6 family cytokines; Bone morphogenetic protein (BMP) family cytokines such as BMP2 or BMP4, Notch family proteins; Wnt gene family proteins; sonic hedgehog protein; and retinoic acid. Furthermore, factors necessary for the proliferation or maintenance of mature cells include, for example, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), Neurotrophin-3 (NT-3), Neurotrophin-4 / 5 (NT-4 / 5), and platelet-derived growth factor (PDGF).

[0086] As the culture conditions using differentiation-inducing factors, known differentiation-inducing conditions applied in the differentiation-inducing method using differentiation-inducing factors can be applied, and the differentiation-inducing conditions can be appropriately selected according to the type of differentiation-inducing factor used.

[0087] <3>Use of inhibitors The inhibitor used in the above-described production method can be used to maintain or promote the proliferation ability of ectodermal cells. Therefore, a further aspect of the present disclosure relates to a proliferative regulator of ectodermal cells, which includes at least one selected from the group consisting of small molecule signaling pathway inhibitors, such as TGFβ receptor inhibitors and ROCK inhibitors. Specifically, the present disclosure also relates to highly proliferative cells obtained by the above-described production method, or a proliferative regulator used to maintain or promote the proliferation ability of ectodermal cells. Here, in the present specification, the term "proliferative regulation" can mean achieving at least one of cell proliferation maintenance and proliferation promotion. The terms "proliferation maintenance" and "proliferation promotion" are not particularly distinguished as long as the number of cells is maintained or increased over the culture period. As the use concentration of the proliferative regulator for cell proliferative regulation, the concentration described with respect to the use concentration in the above-described production method can be directly applied.

[0088] Furthermore, the present disclosure is a culture method of highly proliferative cells or ectodermal cells obtained by the above-described production method, which includes subculturing the cell population or the cells in the presence of at least one inhibitor selected from the group consisting of small molecule signaling pathway inhibitors, such as TGFβ receptor inhibitors and ROCK inhibitors. Here, the "culture method" shall include a method for regulating the proliferation of highly proliferative cells or ectodermal cells. As the use concentration of the inhibitor when used in this culture method, the concentration described with respect to the use concentration in the above-described production method can be directly applied.

[0089] <4>Cell Secretions and Their Production Methods Yet another aspect of the present disclosure relates to a method for producing a cell secretion, which includes obtaining a culture containing a cell secretion secreted from highly proliferative cells according to the present disclosure, and separating the cell secretion from the culture. The production method may further include any steps as necessary. Since the highly proliferative cells according to the present disclosure are highly proliferative cells with enhanced cell proliferation ability, compared with control cells cultured under the same culture conditions as the highly proliferative cells except using a medium without an inhibitor, they proliferate in a shorter time, proliferate over a longer period, or proliferate in a shorter time and over a longer period. Therefore, in this method for producing a cell secretion, the cell secretion secreted by the highly proliferative cells can be obtained in a large amount in a shorter time compared with the control cells.

[0090] Cell secretions are also referred to as "secretomes" and are not particularly limited as long as they are substances secreted from cells. Examples include extracellular vesicles such as exosomes, microvesicles, and apoptotic bodies; functional proteins such as cytokines, hormones, and antibodies. In one form, the cell secretion may be an extracellular vesicle, and among them, it may be an exosome.

[0091] The culture prepared in the method for producing a cell secretion according to the present disclosure contains a cell secretion secreted from highly proliferative cells obtained by any of the above aspects. The culture containing the cell secretion may be produced by implementing the method for producing highly proliferative cells according to the present disclosure, may be obtained by obtaining and culturing the highly proliferative cells according to the present disclosure, may be separately obtained from a culture obtained by implementing the production method according to the present disclosure, or may be separately obtained from a culture of the highly proliferative cells according to the present disclosure. As used herein, "culture" means a combination of cultured cells obtained by culturing cells and a culture supernatant (conditioned medium).

[0092] The method for producing a cell secretion according to the present disclosure includes separating the cell secretion from the culture. As the method for separating the cell secretion from the culture, known separation methods such as removal of the supernatant and centrifugation can be used. The cell secretion separated from the culture may be obtained in the form of a composition containing the cell secretion. When the cell secretion is a composition containing the cell secretion, i.e., a cell secretion-containing composition, the cell secretion-containing composition can include the cell secretion and an aqueous medium. The aqueous medium is not particularly limited as long as it is a known aqueous medium selected according to the type of the cell secretion and the like, and examples thereof include culture supernatant, water, buffer solutions (e.g., phosphate buffer, Good's buffer), physiological saline, and culture medium.

[0093] The method for producing a cell secretion can further include purifying or isolating the separated cell secretion. As the method for purifying or isolating the cell secretion, known methods can be applied without particular limitation according to the type of the cell secretion.

[0094] Examples of the method for isolating or purifying the cell secretion include filtration and concentration. For example, filtration can be performed using a membrane having a size or molecular weight cut-off value to isolate or purify the cell secretion. As another method, for example, tangential flow filtration or ultrafiltration can be used to filter or concentrate the cell secretion. The cell secretion can be used in the form of a composition containing the cell secretion or in the form of the isolated cell secretion obtained after isolation or purification. The cell secretion can be stored, refrigerated, or frozen in the form of the composition or the isolated cell secretion, or by subjecting it to known treatments such as spray drying and freeze-drying.

[0095] The cell secretions according to the present disclosure are specifically cell secretions containing proteins and / or miRNAs. The proteins are selected from those shown in FIGS. 4A and 4B, and the miRNAs are selected from those shown in FIGS. 5 to 10. In FIGS. 4A and 4B, the descriptions in parentheses following each protein name mean the accession numbers in UniProtKB / Swiss-Prot (https: / / www.uniprot.org / ). In FIGS. 5 to 10, the descriptions in parentheses following each miRNA name mean the accession numbers in miRBase (Release 21) (https: / / www.mirbase.org / ). The cell secretions may contain at least one selected from these proteins or at least one selected from these miRNAs, and may contain at least one selected from these proteins and at least one selected from these miRNAs.

[0096] In one embodiment, the cell secretion is, as a protein, · PA group: may contain at least a combination of vinculin (P18206), integrin β-1, CD29 (P05556), pyruvate kinase M1 / 2 (P14618), and ephrin type-A receptor 2 (P29317), · PB group: may contain at least a combination of vinculin (P18206), integrin β-1, CD29 (P05556), pyruvate kinase M1 / 2 (P14618), and ephrin type-A receptor 2 (P29317) and at least one selected from the group consisting of aminopeptidase N (P15144), annexin A2 (P07355), annexin A6 (P08133), myoferlin (Q9NZM1), and 5'-nucleotidase, CD73 (P21589), · PC group: may include a combination of vinculin (P18206), integrin β-1, CD29 (P05556), pyruvate kinase M1 / 2 (P14618), and ephrin type-A receptor 2 (P29317), and a combination of aminopeptidase N (P15144), annexin A2 (P07355), annexin A6 (P08133), myoferlin (Q9NZM1), and 5'-nucleotidase, CD73 (P21589), and at least one selected from the group consisting of moesin (P26038), integrin α-2, CD49b (P17301), integrin α-3, CD49c (P26006), 2',3'-cyclic nucleotide 3'-phosphodiesterase (P09543), F-actin capping protein β subunit (P79136), neuropilin 1 (O14786), and tenascin C (P24821), or · PD group: may include all the proteins described in FIGS. 4A and 4B.

[0097] In one embodiment, the cell secretion is, as miRNA, · RA group: may at least include a combination of hsa-miR-382-5p (MIMAT0000737), hsa-miR-155-5p (MIMAT0000646), hsa-miR-379-5p (MIMAT0000733), hsa-miR-16-5p (MIMAT0000069), hsa-miR-382-5p (MIMAT0000737), hsa-miR-16-5p (MIMAT0000069), hsa-miR-382-5p (MIMAT0000737), hsa-miR-21-5p (MIMAT0000076), hsa-let-7a-5p (MIMAT0000062), hsa-miR-16-5p (MIMAT0000069), hsa-miR-409-3p (MIMAT0001639), hsa-let-7a-5p (MIMAT0000062), and hsa-let-7f-5p (MIMAT0000067), · RB group: combination of hsa-miR-382-5p (MIMAT0000737), hsa-miR-155-5p (MIMAT0000646), hsa-miR-379-5p (MIMAT0000733), hsa-miR-16-5p (MIMAT0000069), hsa-miR-382-5p (MIMAT0000737), hsa-miR-16-5p (MIMAT0000069), hsa-miR-382-5p (MIMAT0000737), hsa-miR-21-5p (MIMAT0000076), hsa-let-7a-5p (MIMAT0000062), hsa-miR-16-5p (MIMAT0000069), hsa-miR-409-3p (MIMAT0001639), hsa-let-7a-5p (MIMAT0000062), and hsa-let-7f-5p (MIMAT0000067), and at least one selected from the group consisting of hsa-miR-151a-3p (MIMAT0000757), hsa-miR-93-5p (MIMAT0000093), hsa-miR-29a-3p (MIMAT0000086), hsa-miR-9-5p (MIMAT0000441), hsa-miR-25-3p (MIMAT0000081), hsa-miR-92a-3p (MIMAT0000092), hsa-miR-34a-5p (MIMAT0000255), hsa-miR-26a-5p (MIMAT0000082), hsa-miR-26b-5p (MIMAT0000083), hsa-miR-29a-3p (MIMAT0000086), hsa-miR-9-5p (MIMAT0000441), hsa-miR-155-5p (MIMAT0000646), hsa-miR-92a-3p (MIMAT0000092), hsa-miR-103a-3p (MIMAT0000101), and hsa-miR-26a-5p (MIMAT0000082) may be included at least, · RC groups: combinations of hsa-miR-382-5p (MIMAT0000737), hsa-miR-155-5p (MIMAT0000646), hsa-miR-379-5p (MIMAT0000733), hsa-miR-16-5p (MIMAT0000069), hsa-miR-382-5p (MIMAT0000737), hsa-miR-16-5p (MIMAT0000069), hsa-miR-382-5p (MIMAT0000737), hsa-miR-21-5p (MIMAT0000076), hsa-let-7a-5p (MIMAT0000062), hsa-miR-16-5p (MIMAT0000069), hsa-miR-409-3p (MIMAT0001639), hsa-let-7a-5p (MIMAT0000062), and hsa-let-7f-5p (MIMAT0000067); combinations of hsa-miR-151a-3p (MIMAT0000757), hsa-miR-93-5p (MIMAT0000093), hsa-miR-29a-3p (MIMAT0000086), hsa-miR-9-5p (MIMAT0000441), hsa-miR-25-3p (MIMAT0000081), hsa-miR-92a-3p (MIMAT0000092), hsa-miR-34a-5p (MIMAT0000255), hsa-miR-26a-5p (MIMAT0000082), hsa-miR-26b-5p (MIMAT0000083), hsa-miR-29a-3p (MIMAT0000086), hsa-miR-9-5p (MIMAT0000441), hsa-miR-155-5p (MIMAT0000646), hsa-miR-92a-3p (MIMAT0000092), hsa-miR-103a-3p (MIMAT0000101), and hsa-miR-26a-5p (MIMAT0000082); combinations of hsa-miR-128-3p (MIMAT0000424), hsa-miR-222-3p (MIMAT0000279), hsa-miR-31-5p (MIMAT0000089), hsa-miR-381-3p (MIMAT0000736), hsa-miR-128-3p (MIMAT0000424), hsa-miR-24-3p (MIMAT0000080)At least one selected from the group consisting of hsa-miR-26b-5p (MIMAT0000083), hsa-miR-24-3p (MIMAT0000080), hsa-miR-30a-3p (MIMAT0000088), hsa-miR-31-5p (MIMAT0000089), hsa-miR-128-3p (MIMAT0000424), hsa-miR-23a-3p (MIMAT0000078), hsa-miR-24-3p (MIMAT0000080), hsa-let-7d-5p (MIMAT0000065), and hsa-miR-654-3p (MIMAT0004814) may be included at least, or, · RD group: It may include all miRNAs described in FIGS. 5 to 10.

[0098] In another embodiment, the cell secretion may include, as a protein, the protein groups of the above-described PA group, PB group, PC group, or PD group, and, as an miRNA, the miRNA groups of the above-described RA group, RB group, RC group, or RD group. Thus, the cell secretion according to one embodiment can be used as a pharmaceutical composition having known actions due to these proteins and miRNAs.

[0099] In one aspect, the extracellular vesicle is a vesicle containing a lipid bilayer. The diameter of the extracellular vesicle is, for example, 50 nm to 5 μm, or 50 nm to 1000 nm. The diameter of an exosome, which is one of the extracellular vesicles, is, for example, 50 nm to 200 nm. Since exosomes contain various bioactive substances such as proteins, nucleic acids, carbohydrates, and lipids, they are expected to be used in the treatment and diagnosis methods of diseases, pharmaceuticals, cosmetics, etc.

[0100] Exosomes are secreted from adherent cells into the culture, particularly into the culture supernatant. Also, when non - adherent cells are present in the cell suspension, exosomes are secreted from non - adherent cells into the culture supernatant or the cell suspension. Exosomes derived from adherent cells or non - adherent cells are not particularly limited as long as they are obtained from ectodermal adherent cells or ectodermal non - adherent cells. For example, exosomes described in Journal of Controlled Release, Vol. 323, pp. 225 - 239 (2020) can be mentioned.

[0101] Exosomes can be isolated based on their molecular weight, size, shape, composition, or biological activity. Specifically, it can be isolated by fractionation of the precipitate by ultracentrifugation, fractionation of the fraction by density gradient ultracentrifugation, fractionation using size exclusion chromatography, ion exchange chromatography (for example, CIMmultus TM EV (manufactured by BIA separations) for fractionation, capture using a protein (for example, MagCapture TM Exosome Isolation Kit PS (manufactured by Fujifilm Wako Pure Chemical Corporation)) for fractionation by capture, fractionation by capture using an antibody, fractionation of the precipitate using a polymer such as polyethylene glycol, etc. These methods can be carried out singly or in combination.

[0102] The properties of exosomes can be used to track the activity of exosomes in the method for producing exosomes as cell secretions. For example, the activity of exosomes can be confirmed using static light scattering, dynamic light scattering, ultraviolet - visible detector, fluorescence detector, or differential refractive index detector.

[0103] From the perspective of obtaining exosomes with high purity, when isolating or purifying exosomes, usually, the culture medium containing the obtained highly proliferative cells can be replaced with a recovery medium for additional culture. Specifically, the recovery medium is a culture supernatant recovery medium. The additional culture means a short-term culture with the recovery medium performed after culturing with the culture medium. The period of the additional culture can be, for example, 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, or 60 hours or more, and can be, for example, 96 hours or less, or 72 hours or less. By performing the additional culture, the contamination rate of exosomes derived from a source different from the highly proliferative cells and contained in the culture medium can be reduced, and the purity of exosomes derived from the target highly proliferative cells can be increased.

[0104] Examples of the recovery medium include a serum-free medium and a medium that has been subjected to exosome removal treatment. Examples of commercially available media that have been subjected to exosome removal treatment include FBS exosome-depleted, OneShot format (Gibco (registered trademark), Thermo Fisher Scientific).

[0105] When using the recovery medium, after the additional culture, centrifugation is performed to separate the culture supernatant as an exosome-containing composition, and if necessary, the separated culture supernatant is subjected to the above-described exosome isolation or purification method to obtain exosomes.

[0106] <5>Uses of Highly Proliferative Cells and Cell Secretions Cell secretions separated from highly proliferative cells according to one embodiment, for example, exosomes separated or purified as described above, are expected to have various effects on various cell types, such as neurite outgrowth inhibitory effect, neurite extension effect, neurite network formation effect, nerve cell death prevention effect, and nerve cell proliferation promoting effect. Based on these functions, it is expected to be useful as a pharmaceutical composition for preventing or treating disorders related to peripheral nerve cells or central nerve cells. For example, the inhibitory effect on neurite outgrowth of cell secretions isolated from highly proliferative cells can suppress the differentiation from more undifferentiated cells to peripheral nerve cells, particularly sympathetic nerve cells, with respect to the differentiation of peripheral nerve cells. Therefore, by using the cell secretions according to the present disclosure on peripheral nerve cells, it can be expected to suppress the peripheral nervous system, particularly the sympathetic nervous system. Also, for example, by using cell secretions isolated from highly proliferative cells on central nerve cells, it can be expected to activate the central nervous system based on its neurite outgrowth promoting effect, neurite network formation promoting effect, nerve cell death prevention effect, nerve cell proliferation promoting effect, and the like.

[0107] Therefore, a pharmaceutical composition containing an effective amount of cell secretions as a treatment or prevention isolated from highly proliferative cells and a pharmaceutically acceptable carrier, as well as a treatment or prevention method including administering the pharmaceutical composition to a subject are also included in the scope of the present disclosure. As used herein, the term "treatment" includes not only the radical cure of a disorder, but also the improvement of symptoms of the disorder such as alleviation and remission.

[0108] The disorder to be treated or prevented may be any disorder related to peripheral nerve cells or central nerve cells, and is, for example, selected from the group consisting of cancer, pain, Alzheimer's disease, Parkinson's disease, depression, schizophrenia, and dementia. Examples of disorders related to peripheral nerve cells, particularly sympathetic nerve cells, include cancer, pain, etc. related to sympathetic nerve cells. Disorders related to central nerve cells are, for example, selected from the group consisting of Alzheimer's disease, Parkinson's disease, depression, schizophrenia, and dementia.

[0109] The pharmaceutically acceptable carrier is not particularly limited, and those known in the art are used, and examples include physiological saline and the like. Furthermore, in relation to the above effects, a method for suppressing the sympathetic nervous system, a method for suppressing neurite outgrowth, etc., including contacting cell secretions secreted from highly proliferative cells, specifically exosomes, with nerve cells, for example, sympathetic nerve cells, are also included in the scope of the present disclosure.

[0110] In this specification, a numerical range indicated using "~" indicates a range that includes the numerical values described before and after "~" as the minimum value and the maximum value, respectively. In the numerical ranges described stepwise in this specification, the upper limit value or the lower limit value of a numerical range at a certain step can be arbitrarily combined with the upper limit value or the lower limit value of a numerical range at another step.

[0111] This disclosure is based on the following Japanese patent applications and enjoys the benefits of the right of priority based on the said Japanese patent applications. Also, the entire contents of the following Japanese patent applications are incorporated herein by reference: Japanese Patent Application No. 2021-124172, filed on July 29, 2021, entitled "Method for Producing Highly Proliferative Cells, Highly Proliferative Cells and Their Uses".

[0112] All documents, patent applications, and technical standards described in this disclosure are incorporated herein by reference in their entirety.

Examples

[0113] Example 1: Long-Term Culture of Human Astrocytes in Inhibitor-Added Medium Using the materials, reagents, and culture products shown below, long-term culture of human astrocytes was performed.

[0114] <Materials> · Normal Human Astrocytes (NHA) (CC-2565, Lonza) <Reagents and Culture Products Used> · Culture medium: AGM Astrocyte Growth Medium Bullet Kit (CC-3186, Lonza) (Composition of AGM: basal medium, FBS (3% (v / v)), L-glutamine, ascorbic acid, hEGF, insulin, antibiotics), · Inhibitor Y: CultureSure® Y-27632 (034-24024, Fujifilm Wako Pure Chemical Corporation), final concentration: 10 μM, · Inhibitor A: CultureSure® A-83-01 (035-24113, Fujifilm Wako Pure Chemical Corporation), final concentration: 0.5 μM, · Cell detachment agent: Accutase (AT104, ICT), · Cell buffer: Phosphate-buffered saline solution (Dulbecco; calcium- and magnesium-free) (BNDSBN200, KAC), · Cell freezing medium: CELLBANKER 1 (CB011 TaKaRa (Nippon Zenyaku Kogyo Co., Ltd.)), · 60 mm cell culture dish (150462, Thermo Fisher Scientific), · 100 mm cell culture dish (150466, Thermo Fisher Scientific), · 150 mm cell culture dish (150468, Thermo Fisher Scientific), · Stericup Quick Release-GP Sterile Vacuum Filtration System (S2GPU02RE, Merck Millipore), · Inhibitor-free medium for collecting culture supernatant: DMEM (Dulbecco’s Modified Eagle Medium) (C11995500CP, Gibco®, Thermo Fisher Scientific) and N-2 Supplement (100×) (17502-048, Gibco®, Thermo Fisher Scientific)

[0115] <Procedure for long-term culture> Long-term culture was performed according to the following procedure. A cryotube of human astrocyte cells (NHA) was thawed in a 37 °C water bath. Subsequently, the lysed cells were transferred to a culture medium to make a total volume of 10 mL, centrifuged at 180×g for 3 minutes at room temperature, and then the medium was removed. The cells were divided into four groups: a group with only inhibitor Y added (hereinafter, NHA-Y group), a group with only inhibitor A added (hereinafter, NHA-A group), a group with both inhibitor Y and inhibitor A added (hereinafter, NHA-YA group), and a non-additive group (Normal group). The cells were suspended again in the culture medium and seeded into a 6-well plate (a 6-well multi-dish for cell culture (140675, Thermo Fisher Scientific. Hereinafter, omitted) at a seeding density of 4.2×10 3 cells / cm 2 . The next day, the medium was replaced with a culture medium containing (or not containing) each inhibitor for each group as shown in Table 2 (the concentration of each inhibitor was as described above), and the culture was continued. During the culture period, the cells were observed under a microscope and photographed.

[0116] The medium was changed every 1 or 2 days, and each time it was replaced with a fresh culture medium containing (or not containing) the inhibitor. When the cells reached 80 - 90% confluence, the cells were detached using a cell detachment agent, suspended in a culture medium containing (or not containing) each inhibitor, and subcultured into a 6-well plate at a seeding density of 4.2×10 3 cells / cm 2 . The cells were cultured in a culture medium containing (or not containing) each inhibitor for 41 days with subculturing.

[0117] The number of subcultures of the cells in each group and the number of days until the final subculture date are shown in Table 1 below, and the relationship between the culture days and the total cell number is shown in Table 2.

[0118]

Table 1

[0119]

Table 2

[0120] As shown in Table 1, in the Normal group, cell growth stopped 22 days after inhibitor contact, while in the NHA-YA group, cell growth continued for 37 days after contact at the maximum inhibition time. Furthermore, as can be seen from Table 2, regarding the number of cells obtained by growth, the number of cells in the NHA-YA group was approximately 400 times that of the Normal group. In all experimental groups, after the final passage date, observation was continued until the 41st day after the start of culture while changing the medium. The cell morphology (magnified 100 times) on the 41st day after the start of culture is shown in Figure 1. In Figure 1, "P" indicates the passage number. For example, "Y-P5" means cells at the 5th passage and cultured in a medium containing inhibitor Y.

[0121] Example 2: Recovery and Confirmation of Exosomes in Culture Supernatant <Recovery of Culture Supernatant of Human Astrocyte Cells> Following the following procedure, the culture supernatants were respectively recovered when human astrocyte cells were cultured using a YA-containing culture medium or an inhibitor-free medium.

[0122] (1) Recovery of Culture Supernatant in the Culture of Human Astrocyte Cells with YA-Containing Culture Medium The cryopreserved human astrocyte cells were thawed, washed once with the medium as in Example 1, then the cells were resuspended in the culture medium, and seeded into a 60 mm cell culture dish at a seeding density of 3.5×10 3 cells / cm 2 . The next day, the medium was changed to a YA-containing culture medium and the culture was continued. When reaching 80 - 90% confluence, the cells were detached using a cell detachment agent, suspended in the YA-containing culture medium, and subcultured into a 100 mm cell culture dish at a seeding density of 3.5×10 3 cells / cm 2 . The cell group cultured in the YA-containing culture medium was referred to as the NHA-YA group as in Example 1.

[0123] For the NHA-YA group, after subculturing 5 times and culturing in the YA-containing culture medium for a total of 60 days to ensure a sufficient number of cells, 8×10 were seeded into a 150 mm cell culture dish.3 cells / cm 2 They were seeded at a seeding density of. The number of cells seeded at this time was 1.16×10 6 cells per dish, and since 6 dishes were used, the total number of seeded cells was 6.96×10 6 cells.

[0124] On the next day, the medium was exchanged from the medium for YA-containing culture to the medium for recovery at a volume of 20 ml / 150 mm dish, and additional culture was started. As the medium for recovery, a medium obtained by removing only FBS from the medium for YA-containing culture was used. Two days after the start of the additional culture, the culture supernatant of the additional culture was collected from the dish, transferred to a tube, centrifuged at 2,000×g for 10 minutes at 4°C, filtered through a 0.22 μm filter system, and stored at 4°C. The cells remaining on the dish after the collection of the culture supernatant were detached using a cell detachment agent, and the cell count was performed. As a result, the total number of cells obtained as the NHA-YA group was 5.10×10 6 cells, and the survival rate with respect to the total number of seeded cells was 73.3%.

[0125] (2) Collection of culture supernatant in the culture of human astrocyte cells in a medium for inhibitor-free culture (control) The cryopreserved human astrocyte cells were thawed, washed once with a medium in the same manner as in Example 1, resuspended in a culture medium again, and suspended in a 60 mm cell culture dish at 3.5×10 3 cells / cm 2 They were seeded at a seeding density of. The cell group cultured in a culture medium containing no inhibitor was referred to as the Normal group in the same manner as in Example 1.

[0126] For the Normal group, subculturing was performed twice, and the cells were cultured in a culture medium containing no inhibitor for a total of 26 days, and seeded in a 150 mm cell culture dish at 8×10 3 cells / cm 2 They were seeded at a seeding density of. The number of cells seeded at this time was 1.16×10 6 cells per dish, and since 2 dishes were used, the total number of seeded cells was 2.32×10 6 cells.

[0127] The next day, the medium was changed from the culture medium to the recovery medium with N-2 Supplement added to DMEM at a volume of 20 ml / 150 mm dish to start additional culture. Two days after the start of the additional culture, the culture supernatant of the additional culture was collected from the dish, transferred to a tube, centrifuged at 2,000×g for 10 minutes at 4°C, filtered through a 0.22 μm filter system, and stored at 4°C. The cells remaining on the dish after the collection of the culture supernatant were detached using a cell detachment agent, and the cell number was counted. As a result, the total cell number obtained as the Normal group was 8.25×10 5 cells, and the survival rate with respect to the total number of seeded cells was 35.6%. After centrifuging the supernatant at 2,000×g for 10 minutes at 4°C, it was filtered through a 0.22 μm filter system and stored at 4°C. The cell morphology before and after the exchange to the recovery medium is shown in Figure 2.

[0128] <Measurement of the number of cells, particles, and particle size distribution in the culture supernatant> The culture supernatants of the additional cultures of the Normal group and the NHA-YA group recovered above were each diluted 5-fold with a cell buffer (filtered through a 0.22 μm filter before use), and then the number of particles and the particle size distribution were confirmed using a NanoSight LM10 (Malvern Panalytical). As a blank, the number of particles in the cell buffer was also measured, and the number of particles in the culture supernatant was calculated by subtracting the number of particles in the cell buffer from the measured values of each culture supernatant. The confirmation of the number of particles and the particle size distribution was performed twice each. An example of the number of particles and the particle size of the particles contained in the recovered culture supernatant of the additional culture is shown in Table 3, and the particle size distribution is shown in Figures 3A and 3B, respectively. In Figures 3A and 3B, the number of particles (10 6 per mL) is shown on the vertical axis, and the particle size (nm) is shown on the horizontal axis.

[0129]

Table 3

[0130] As is clear from Table 3 and Figure 3B, multiple peaks were confirmed in the culture supernatant of the NHA-YA group at a particle size of 100 nm to 200 nm, indicating that exosomes were contained. Also, it was confirmed that the amount of exosomes contained in the culture supernatant of the NHA-YA group was larger compared to the Normal group. Subsequently, by purification methods such as tangential flow filtration and ultrafiltration, as described later, particles with a peak at a particle size of 100 nm to 200 nm could be isolated or purified.

[0131] Example 3-1: Confirmation of ectodermal cell markers by quantitative PCR (1) Regarding the cell population of astrocytes used as the starting material in the culture of Example 1 and the obtained ectodermal cell population, the expression levels of the following ectodermal cell marker genes were examined. Detection primers were prepared based on the sequences registered under the accession numbers of RefSeq, which is the NCBI database described below. ·Notch1 (including vX1) (NM_017617) ·Nestin (NM_006617) ·SOX2 (SRY-box transcription factor 2) (NM_003106) ·S100B (S100 calcium binding protein B) (NM_006272) ·NG2 (Chondroitin sulfate proteoglycan 4) (NM_001897) ·GFAP (Glial fibrillary acidic protein) (v1.v4, vX1, vX3) (NM_002055)

[0132] <Synthesis of cDNA> RNA was extracted from the following samples according to a conventional method, and cDNA was synthesized using the RNA. The preparation of the RNA sample and the setting of the reaction conditions followed the protocol attached to the product. Samples: · Normal Human Astrocytes (NHA) (Primary cells purchased, revived, proliferated, frozen, then revived again and cultured for 4 days. No subculture.) · NHA - YA (Primary cells purchased, revived, proliferated, frozen, then revived again and cultured in medium containing YA for 28 days and frozen again, then revived once more and cultured in medium containing YA for 14 days. During the 14 - day culture period, subcultured once.)

[0133] Reagent: High - Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems) Equipment: SimpliAmp Thermal Cycler (Applied Biosystems)

[0134] <Quantitative PCR> The prepared detection primers are shown in Table 4 below. Using these primers, the expression of ectodermal cell marker genes was examined by quantitative PCR. The preparation of RNA samples and the setting of reaction conditions followed the protocol attached to the product.

[0135]

Table 4

[0136] Reagent: Platinum SYBR Green qPCR SuperMix - UDG (Invitrogen) Equipment: StepOnePlus Real - Time PCR System (Applied Biosystems)

[0137] The results of confirming the expression of each marker of Notch1, Nestin, and SOX2, which are markers of neuroepithelial cells, and each marker of Nestin, which is a marker of radial glial cells, by quantitative PCR, as well as the results of confirming the expression of GFAP and S100B, which are markers of astrocytes, and NG2, which is a marker of oligodendrocyte progenitor cells, are shown in Table 5 and Table 6, respectively.

[0138] In Table 5 and Table 6, the expression levels of each marker gene in NHA-YA (expression level relative to the control gene) are shown as relative values when the expression level in NHA is set to "1". Table 5 shows the expression levels of each marker when the ACTB gene is used as the control gene, and Table 6 shows the expression levels of each marker when the GAPDH gene is used as the control gene.

[0139]

Table 5

[0140]

Table 6

[0141] As shown in Table 5 and Table 6, in the obtained cell population (NHA-YA), higher expression was observed in both GFAP and S1OB, which are markers of astrocytes, and NG2, which is a marker of oligodendrocyte progenitor cells, compared to the astrocytes (NHA) used as the raw material.

[0142] From these results, it was found that the obtained cell population (NHA-YA) has the characteristics of both mature astrocytes and oligodendrocyte progenitor cells, which are progenitor cells, and has a higher proliferative ability than astrocytes. This NHA-YA can proliferate more in a shorter time than astrocytes. Also, it can be seen that it is advantageous to use NHA-YA to obtain more exosomes in a shorter time.

[0143] Example 3-2: Confirmation of ectodermal cell markers by quantitative PCR (2) For the cell group of astrocytes used as the starting material in the culture of Example 1 and the obtained ectodermal cell population, in the same manner as in Example 3-1, the expression levels of the following ectodermal cell marker genes were measured by quantitative PCR. The primers for detecting each marker gene were prepared based on the sequences registered with the accession numbers of RefSeq, which is the database of NCBI described below. · SLC1A2 (solute carrier family 1 member 2) (including v1, v2, v3, vX1 to vX7) (NM_004171) · SLC1A3 (solute carrier family 1 member 3) (v1 to v5, vX1, vX2, vX3) (NM_004172) · OLIG2 (oligodendrocyte transcription factor 2) (NM_005806) · PAX6 (paired box 6) (v1 to v48) (NM_000280) · ALDH1L1 (aldehyde dehydrogenase 1 family member L1) (v1, v2, v3, vX1, vX2, vX3) (NM_001270364) · Musashi1 (musashi RNA binding protein 1; MSI1) (v1, vX1 to vX10) (NM_002442)

[0144] The prepared detection primers are shown in Table 7 below. Using these primers, the expression of the ectodermal cell marker gene was examined by quantitative PCR. The preparation of the RNA sample and the setting of the reaction conditions were in accordance with the protocol attached to the product. As for the measuring instrument for quantitative PCR, for Musashi1, SimpliAmp Thermal Cycler (Applied Biosystems) or CFX96 Touch Real-time PCR Detection system (Bio-Rad) was used, and for the other marker genes, the same measuring instrument as that used in Example 3-1 was used.

[0145]

Table 7

[0146] The results are shown in Tables 8 and 9. In Tables 8 and 9, the expression levels of each marker gene in NHA-YA (the expression level relative to the control gene) are shown as relative values when the expression level in NHA is set to "1". Table 8 shows the expression levels of each marker when the ACTB gene is used as the control gene, and Table 9 shows the expression levels of each marker when the GAPDH gene is used as the control gene.

[0147]

Table 8

[0148]

Table 9

[0149] Example 4: Proteome Analysis of NHA-YA-derived Exosomes <Recovery of NHA-YA-derived Exosomes> A cryotube containing a cell culture solution containing NHA was thawed in a 37°C water bath. Subsequently, the thawed cells were transferred to a medium for YA-containing culture to make a total volume of 10 mL, centrifuged at 180×g for 3 minutes at 25°C, and then the medium was removed. The obtained NHA was suspended in the medium for YA-containing culture and seeded into a 6-well plate at a seeding density of 4.2×10 3 cells / cm 2 . Subculture was performed for 11 days in the medium for YA-containing culture with two passages.

[0150] Thereafter, the medium was changed from the medium for YA-containing culture to a recovery medium at a volume of 20 mL / 150 mm dish, and additional culture was performed at 37°C for 72 hours. As the recovery medium, a medium containing FBS [3% (v / v)] from which exosomes were removed instead of FBS in the medium for YA-containing culture was used. The additional culture was performed at a seeding density of 1.16×10 5 cells / 150 mm dish. After the additional culture, the culture supernatant of the additional culture was recovered from the dish, transferred to a tube, centrifuged at 2,000×g for 10 minutes at 4°C, and then filtered through a 0.22 μm filter system to prepare an exosome-containing solution.

[0151] <Proteome Analysis of Exosomes 1> The exosome-containing solution obtained above was precipitated with trichloroacetic acid, and then a Tris buffer solution of 5 mM dithiothreitol was added, followed by reduction treatment at 35 °C for 2 hours. To the reduced sample, a Tris buffer solution of 14 mM iodoacetamide was added, and the reaction was carried out at 25 °C for 30 minutes under light-shielded conditions. Subsequently, trypsin treatment was performed at 37 °C for 20 hours to decompose the exosomes. The obtained decomposition products of exosomes were solvent-exchanged using a cation exchange column, followed by desalting and concentration, and then subjected to LC-MS / MS analysis. An EASY-nLC 1200 System (Thermo Fisher Scientific Inc., USA) was used for the HPLC device, and an EASY-Spray column, 15 cm × 75 μm ID, 3 μm particles, 100 Å pore size (Thermo Fisher Scientific Inc., USA) was used for the column. Based on the product ion measurement data of the spectra obtained by LC-MS / MS analysis, database search was performed using the MASCOT server (https: / / www.matrixscience.com / help.html). Subsequently, the obtained results were compared with the following two databases, and a total of 1798 protein fragments were detected by combining the exosome fraction and the medium components. · Human-derived proteins: SwissProt (number of sequences 20376) · Bovine-derived proteins: UniProtKB (number of sequences 47043)

[0152] From all 1285 marker proteins detected on SwissProt in the exosome fraction, those with a Quantitative Value (number of detected spectra. with correction) of 5 or more were selected, and 520 marker proteins were extracted. From the 520 extracted proteins, characteristic proteins were selected. It was confirmed that the selected proteins were not components in the medium, and when they were bovine-derived proteins, the identification probability with human-derived proteins was examined, and those with a high identification probability were selected. The following describes the steps for selecting characteristic proteins.

[0153] a: Step of selecting proteins characteristic of the brain (a1) The 520 proteins extracted above were searched using Tissue Enrich (https: / / tissueenrich.gdcb.iastate.edu). Here, the dataset of the Human Protein Atlas and all tissue-specific genes were targeted for the search; (a2) Twenty-two genes specific to the brain were selected; (a3) Among the 22 genes selected in (a2) above, 12 genes not detected in the culture samples were selected; (a4) Among the 12 genes selected in (a3) above, 8 genes highly likely to be human proteins were selected; (a5) Among the 8 genes selected in (a4) above, 5 genes highly related to diseases were selected. The proteins encoded by the 5 genes selected by the above steps (a1) to (a5) are shown in Figure 4.

[0154] b: Step of selecting proteins characteristic of membrane proteins, extracellular regions, etc. (b1) Among the 520 proteins extracted above, 409 proteins were selected as the proteins bracketed by "membrane" in the GO of Scaffold Proteome viewer; (b2) Among the 520 proteins extracted above, 443 proteins were selected as the proteins bracketed by "extracellular region" in the GO of Scaffold Proteome viewer; (b3) Among the 520 proteins extracted above, 15 proteins were selected as the proteins not bracketed by either "membrane" or "extracellular region" in the GO of Scaffold Proteome viewer; (b4) Among the proteins extracted in the above steps (b1), (b2), and (b3), 35 proteins were selected as proteins not contained in the culture medium sample. Among the 35 selected proteins, 14 overlap with the proteins selected by the following steps (c1) to (c3). The 35 proteins selected by the above steps (b1) to (b4) are shown in Figure 4.

[0155] <Proteomic Analysis of Exosomes 2> Characteristic proteins were selected in selection step c, which is different from selection steps a and b above. c: Selection steps for characteristic proteins (c1) 103 sequences with a Quantitative Value of 20 or more in the exosome fraction were selected from a total of 1562 sequences combining SwissProt_Homo sapiens and UniProtKB_Bos taurus; (c2) Among the 103 sequences selected in (c1) above, 65 sequences with a Quantitative Value of 10 or less in the culture medium sample were selected; (c3) For the 65 sequences selected in step (c2) above, 20 sequences related to central nervous system diseases or presumed to be involved in the growth, development, differentiation, morphogenesis, migration, metabolism, etc. of the brain and nerve cells were selected by the paper search site PubMed (https: / / pubmed.ncbi.nlm.nih.gov / ). Among the 20 selected sequences, 14 overlap with the proteins selected by the above steps (b1) to (b4). The 20 proteins selected by the above steps (c1) to (c3) are shown in Figure 4.

[0156] A total of 46 proteins could be selected by the above selection steps a to c (see Figure 4).

[0157] Example 5: miRNA Analysis of NHA-YA Derived Exosomes <Recovery of NHA-YA Derived Exosomes> A cryotube containing a cell culture solution containing NHA was thawed in a 37°C water bath. Subsequently, the lysed cells were transferred to a medium for culturing containing YA to a total volume of 10 mL, centrifuged at 180×g for 3 minutes at 25°C, and then the medium was removed. The obtained NHA was suspended in the medium for culturing containing YA and subcultured at a seeding density of 4.2×10 3 cells / cm 2 . Culturing was performed in the medium for culturing containing YA for 47 days with 4 passages.

[0158] Thereafter, the medium was exchanged from the medium for culturing containing YA to a recovery medium at a volume of 20 mL / 150 mm dish, and additional culturing was performed at 37°C for 48 hours. As the recovery medium, the recovery medium used in Example 4 was used. The additional culturing was performed at a seeding density of 1.16×10 6 cells / 150 mm dish. After the additional culturing, the culture supernatant of the additional culturing was recovered from the dish, transferred to a tube, centrifuged at 2,000×g for 10 minutes at 4°C, and then filtered through a 0.22 μm filter system to prepare an exosome-containing solution.

[0159] <Extraction of RNA from exosomes derived from NHA-YA> 205 mL of the exosome-containing solution obtained above was centrifuged at 250,000×g for 70 minutes at 4°C using an ultracentrifuge Optima XE-90 (Beckman Coulter), and then the supernatant was removed to obtain an exosome fraction. Subsequently, RNA was extracted from the obtained exosome fraction using a miRNeasy Mini Kit (217004, Qiagen). As a result, 41.8 ng of total RNA was recovered from 230 μL of the exosome fraction.

[0160] <RNA analysis> The RNA obtained above was quality-checked using the Agilent RNA 6000 pico kit (Agilent Technologies) and the Agilent small RNA kit (Agilent Technologies) with an Agilent 2100 Bioanalyzer. After quality-check, an RNA library was prepared from the total RNA obtained above using a directional (Stranded) RNA library preparation kit, the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. mRNA-Seq analysis was performed using the prepared RNA library, and 6,538 mRNAs were detected.

[0161] Similarly, a miRNA library was prepared from the total RNA obtained above using a miRNA library preparation kit, the QIAseq miRNA Library Kit (Qiagen), and the QIAseq miRNA NSG 96 Index IL (Qiagen). miRNA-Seq analysis was performed using the prepared miRNA library, and 370 miRNAs were detected.

[0162] Quality-check of the sequencing library was performed using the High Sensitivity DNA kit (Agilent Technologies) with an Agilent 2100 Bioanalyzer.

[0163] NGS was performed using the NextSeq500, Illumina (single-end, 75 bp, average read count of approximately 10 million reads).

[0164] After performing quality assessment of reads (FastQC) on the data after the sequence, the data was aligned (mapped) to the reference genome (Human hg38) at the GeneGlobe:Data Analysis Center (QIAGEN), the expression levels were normalized by Trimmed mean of M values (TMM), and an Excel-formatted file containing the annotation information of each miRNA and the classification summary of the Small RNA composition was created (analysis tool: StrandNGS v4.0, R v3.6.2; annotation information: compliant with miRBase Release21). Thereafter, after extracting genes with variable expression as data analysis, GO analysis and Pathway analysis were performed from target gene prediction.

[0165] <Selection of Functional Marker miRNAs> 1. miRNAs as Parkinson's Disease Markers Referring to the following literature, 42 miRNAs with decreased expression in the human brain tissue of Parkinson's disease were selected. By administering exosomes containing the miRNA to the cells of Parkinson's disease patients, the function of the miRNA is complemented externally in Parkinson's disease patients, and a therapeutic effect for Parkinson's disease is expected. Literature: MicroRNAs in Parkinson’s disease and emerging therapeutic targets. Neural Regeneration Research, 12(12), pp.1945 - 1959 (2017)

[0166] Next, 16 miRNAs detected as exosomal miRNAs derived from NHA - YA were selected from among them. The 16 selected miRNAs are shown in Figure 5.

[0167] 2. miRNAs as Alzheimer's Disease Markers Using IMOTA (https: / / ccb-web.cs.uni-saarland.de / imota / ), miRNAs associated with the following proteins known to be related to the pathological conditions of Alzheimer's disease, particularly multiple proteins contributing to the pharmacological actions of Alzheimer's disease therapeutics, were selected.

[0168] 2-1. miRNA as an Alzheimer's disease marker (1) Using IMOTA, 70 miRNAs associated with amyloid precursor protein (APP) in the cerebral cortex were selected. APP is a major component of senile plaques, which are considered one of the causes of Alzheimer's disease onset, i.e., deposits of amyloid-β protein.

[0169] Subsequently, 23 miRNAs detected as exosomal miRNAs derived from NHA-YA were selected from these. The 23 selected miRNAs are shown in Figure 6.

[0170] 2-2. miRNA as an Alzheimer's disease marker (2) Using IMOTA, 42 miRNAs associated with BACE1 (β-site APP cleaving enzyme) in the cerebral cortex were selected. At the onset of Alzheimer's disease, BACE1 cleaves the N-terminal portion of APP, producing abnormal amyloid-β protein.

[0171] Subsequently, 20 miRNAs detected as exosomal miRNAs derived from NHA-YA were selected from these. The 20 selected miRNAs are shown in Figure 7.

[0172] 2-3. miRNA as an Alzheimer's disease marker (3) Using IMOTA (https: / / ccb-web.cs.uni-saarland.de / imota / ), 66 miRNAs associated with N-methyl-D-aspartate receptor (NMDA receptor) in the cerebral cortex were selected. When Alzheimer's disease develops, abnormal proteins accumulate in the brain, causing excessive release of substances that excite nerves. Excessive activation of NMDA receptors by this excitatory substance impairs neurotransmission and memory.

[0173] Next, 27 miRNAs detected as exosomal miRNAs derived from NHA-YA were selected from among them. The 27 selected miRNAs are shown in Figure 8.

[0174] 2-4. miRNA as an Alzheimer's disease marker (4) Using IMOTA (https: / / ccb-web.cs.uni-saarland.de / imota / ), 58 miRNAs associated with glycogen synthase kinase-3-β (GSK-3β) in the cerebral cortex were selected. GSK-3β is an enzyme that phosphorylates various proteins and plays diverse roles in maintaining cell life and regulating physiological functions through multiple pathway controls. In Alzheimer's disease, it promotes the deposition of amyloid-β protein in the brain and the accumulation of tau protein in nerve cells. As a result, it induces apoptosis of nerve cells.

[0175] Next, 24 miRNAs detected as exosomal miRNAs derived from NHA-YA were selected from among them. The 24 selected miRNAs are shown in Figure 9.

[0176] 3. miRNA as a depression marker Referring to the following literature, 24 miRNAs with decreased expression in the plasma of individuals with depression were selected. By administering exosomes containing the miRNA to the cells of depression patients, the function of the miRNA is complemented externally in depression patients, and a therapeutic effect for depression is expected. Literature: MicroRNAs expressed in depression and their associated pathways: A systematic review and a bioinformatics analysis (Journal of Chemical Neuroanatomy 100 (2019) 101650)

[0177] Next, 13 miRNAs detected as NHA-YA-derived exosomal miRNAs were selected from among these. The 13 selected miRNAs are shown in Fig. 10.

[0178] Example 6: Inhibitory test of neurite outgrowth of PC-12 cells by NHA-YA-derived exosomes As a functional evaluation test using NHA-YA-derived exosomes, an inhibitory test of neurite outgrowth was performed on a cell line derived from rat adrenal pheochromocytoma, PC-12 cells (RCB0009, RIKEN Bank). As a control, exosomes derived from the NHA cells used in Example 1 were used.

[0179] <Reagents and culture products> The following reagents and culture products were used. · Growth medium: Composition: basal medium, 10% (w / v) FBS, 10% (w / v) HS, antibiotics Basal medium: DMEM, high glucose, pyruvate (11995-073; Gibco) FBS: Fetal Bovine Serum, qualified, Brazil (10270-106; Gibco) HS: Horse Serum, heat inactivated, New Zealand origin (26050-088; Gibco) Antibiotics: Antibiotic-Antimycotic (100X) (15240-062; Gibco) · Cell detachment agent 1: Accutase (AT104, ICT) · Cell detachment agent 2: TrypLE Express Enzyme (1X), phenol red-free (12604-013, Gibco), · Cell buffer: Phosphate-buffered saline solution (Dulbecco; calcium and magnesium-free) (BNDSBN200, KAC), · Assay medium: Composition: Basal medium, additives, antibiotics, Basal medium: Advanced DMEM (12491-015, Gibco), Additives: GlutaMAX Supplement (100X) (35050-061, Gibco), Antibiotics: Antibiotic-Antimycotic (100X) (15240-062, Gibco), · Positive control reagent: Rat-derived nerve growth factor (NGF)-β (N2513-.1MG, Sigma-Aldrich), · Culture medium: AGM Astrocyte Growth Medium Bullet Kit (CC-3186, Lonza) (Composition of AGM: Basal medium, FBS (3% (v / v)), L-glutamine, ascorbic acid, hEGF, insulin, antibiotics) (Same as used in Example 1), · Inhibitor Y: CultureSure® Y-27632 (034-24024, Fujifilm Wako Pure Chemical Corporation), final concentration: 10 μM (Same as used in Example 1), · Inhibitor A: CultureSure® A-83-01 (035-24113, Fujifilm Wako Pure Chemical Corporation), final concentration: 0.5 μM (Same as used in Example 1), · Exosome-depleted fetal bovine serum: Exosome-Depleted Fetal Bovine Serum Qualified One shot (A2720803, Gibco), · 0.22 μm filter system: Stericup Quick Release-GP Sterile Vacuum Filtration System (S2GPU02RE, Merck Millipore), · Medium for collecting culture supernatant: The one with 3% (w / v) exosome-depleted fetal bovine serum (A2720803; Gibco) added instead of fetal bovine serum (FBS), which is a component in AGM Astrocyte Growth Medium Bullet Kit (CC-3186, Lonza), · 96-well plate: Collagen I-coated 96-well plate (4860-010, IWAKI), · T-75 cell culture flask (430641, Corning), · 100 mm cell culture dish: 150466, Thermo Fisher Scientific (the same as used in Example 1), · 150 mm cell culture dish: 150468, Thermo Fisher Scientific (the same as used in Example 1).

[0180] <Preparation of samples for evaluation> The following samples for evaluation were prepared according to the following procedure and used in the neurite outgrowth inhibition test. (1) Control medium (CM): Obtained by ultracentrifuging the medium for collecting culture supernatant. (2) NHA AGM P6: Obtained by ultracentrifuging the culture supernatant of NHA at passage 6. (3) NHA-YA P6: Obtained by ultracentrifuging the culture supernatant of NHA-YA at passage 6. The above (2) and (3) were prepared by adding to the assay medium so that the final concentration of the exosome amount was 10 μg / mL. Also, the ultracentrifugation used in the preparation of each of the above (1) to (3) samples for evaluation was performed at 250,000×g for 70 minutes at 4°C using an ultracentrifuge (main body: Optima XE-90, rotor: SW41 Ti; Beckman Coulter).

[0181] <Recovery and confirmation of exosomes in culture supernatant> Using the materials, reagents, and culture products shown below, the culture supernatants were collected when human astrocyte cells were cultured using a YA-containing culture medium or an inhibitor-free culture medium, respectively. 1. Materials As NHA, the same materials as those used in Example 1 were used.

[0182] [[ID=⑧]]2. Collection of culture supernatants of human astrocyte cells The collection of the culture supernatant was performed according to the following procedure. The frozen tube of human astrocyte cells (NHA) was thawed in a 37°C water bath. Then, the thawed cells were transferred to a culture medium to make a total volume of 10 mL, centrifuged at 180×g for 3 minutes at room temperature, and the medium was removed. The cells were suspended in the culture medium and seeded into a T-75 culture flask (Corning) at a seeding density of 1.25×10 4 cells / cm 2 . Thereafter, the cells were cultured at 37°C for about 12 hours to allow the cells to adhere, and then the medium was replaced with a culture medium for either the "inhibitor Y + inhibitor A addition group (hereinafter, NHA-YA group)" or the "non-addition group (NHA group)" (the concentration of each inhibitor was as described above), and the culture was continued.

[0183] The medium was replaced every 1 or 2 days with fresh inhibitor-containing (or inhibitor-free) culture medium. After culturing for 7 days, the cells (passage number: 1) were detached using cell detachment agent 1, suspended in an inhibitor-containing culture medium, i.e., a YA-containing culture medium, and an inhibitor-free culture medium, and subcultured in a 150 mm cell culture dish at a seeding density of 3.3×10 4 cells / cm 2 . After culturing for 9 days, in the same manner, subculture for 5 days was repeated at a seeding density of 3.3 - 6.6×10 4 cells / cm 2 , and a total of 5 subcultures were performed over 31 days in total. The cell group cultured in the YA-containing culture medium as in Example 1 was designated as the NHA-YA group, and the cell group cultured in the inhibitor-free culture medium was designated as the Normal group.

[0184] As the 6th passage, the NHA-YA group was seeded at a density of 3.3×10 4 cells / cm 2 onto 5 pieces of 150 mm cell culture dishes, and the Normal group was seeded at a density of 2.3×10 4 cells / cm 2 . The total number of seeded cells was 5.0×10 6 cells in the NHA-YA group and 3.5×10 6 cells in the Normal group.

[0185] After seeding, the cells were cultured for 4 days. On the 35th day after induction (after contact with the inhibitor), it was confirmed that the cells reached 40 - 60% confluence. The medium was exchanged from the culture medium to the medium for collecting culture supernatant at a volume of 20 mL / 150 mm dish, and additional culture was started. 48 hours after the start of additional culture, the culture supernatant of the additional culture was collected from the dish. Then, the collected culture supernatant was filtered through a 0.22 μm filter system to obtain an exosome-containing culture supernatant with cell residues removed. The obtained filtered exosome-containing culture supernatant was stored at 4°C. Next, fresh medium for collecting culture supernatant was added to the cells remaining in the dish, and the cells were cultured for 48 hours. In the same manner, a filtered exosome-containing culture supernatant was obtained. The same operation was performed a total of 3 times, and approximately 300 mL of the filtered exosome-containing culture supernatant was collected. On the 41st day after induction, after collecting the culture supernatant, the cells remaining on the dish were detached using Cell Detachment Agent 1, and the cell number was counted. The total number of obtained cells was 4.42×10 7 cells in the NHA-YA group and 1.51×10 7 cells in the NHA group. The survival rate relative to the total number of seeded cells was 94.1% in the NHA-YA group and 84.0% in the Normal group.

[0186] As the control medium (Control medium (CM)) for exosome function evaluation, the medium for collecting culture supernatant was added to a 150 mm cell culture dish without seeded cells at a volume of 20 mL / 150 mm dish, and the culture supernatant was collected under the same conditions as during cell culture.

[0187] 3. Concentration of exosomes in the culture supernatant Transfer 180 mL of the culture supernatants of the NHA-YA group, NHA group, and CM collected above into tubes, and centrifuge at 250,000×g for 70 minutes at 4°C using the ultracentrifuge. After removing the supernatant, to wash the obtained precipitate, add phosphate buffered saline solution to the precipitate and then centrifuge at 250,000×g for 70 minutes at 4°C. Remove the supernatant to obtain the washed precipitate. The washed precipitate was resuspended in phosphate buffered saline solution to approximately 1 / 1000 of the culture supernatant volume.

[0188] 4. Measurement of the number of particles and protein amount in the suspension The suspension derived from the culture supernatant concentrated by the above method was diluted 100-fold with phosphate buffered saline solution filtered through a 0.22 μm filter before use, and then the number of particles in the diluted solution was measured by ZetaView X20 (Particle Metrix). The total number of particles in the suspension was calculated by multiplying the number of particles obtained by the measurement by the dilution factor, that is, 100. The number of particles in the assay medium was measured after diluting 100-fold with phosphate buffered saline solution, and the number of particles in the culture supernatant was calculated by subtracting the number of particles in the obtained assay medium from the measured value of each culture supernatant. In addition, the absorbance at 280 nm was measured by NanoDrop (Thermo Scientific) to measure the protein concentration in the suspension. The total protein amount in the suspension was calculated by multiplying the protein concentration obtained by the measurement by the volume of the suspension. The measurement results are shown in Table 10. The notation "aE+b" in the column of "Total number of particles" in Table 10 means a×10 b as shown in Table 10, it was found that the NHA-YA group contained the most total protein amount, and the number of particles was also significantly higher compared to the control medium (CM).

[0189]

Table 10

[0190] <Evaluation experiment protocol> The frozen tube of the above PC-12 cells was thawed in a 37°C water bath. Subsequently, the thawed cells were transferred to a growth medium to make a total of 10 mL, centrifuged at 180×g for 5 minutes at room temperature, and then the medium was removed. Subsequently, the cells were suspended in the growth medium and seeded in a 100 mm cell culture dish at a seeding density of approximately 5,300 cells / cm 2 . Cultivation was carried out at 37°C while changing the medium every 2 or 3 days, and the cells were grown with passages. The obtained PC-12 cells were detached using Cell Detachment Agent 2, washed with the assay medium, and then resuspended in the assay medium at a density of 3.96×10 4 cells / mL. The suspended cells were seeded in a 96-well plate at a density of 1.98×10 3 cells / 50 μL medium, that is, at a seeding density of 6000 cells / cm 2 . After allowing the cells to adhere for approximately 12 hours, the medium was replaced with each of the above evaluation samples. Thereafter, cultivation was carried out at 37°C, and the status of the PC-12 cells on the 3rd, 4th, and 5th days after the start of cultivation was observed with a microscope (BZ-X710, KEYENCE), and observation images were obtained. The length of neurite-like processes and the number of cells in the images were measured using the Multi-point tool (number of cells) and Straight tool (elongated process length) of the image analysis software Fiji (ImageJ2 ver. 2.3.0). Microsoft Excel was used for statistical processing.

[0191] As a result, in the above (2) and (3) containing exosomes, no significant neurite outgrowth was observed in the PC-12 cell line, which is a sympathetic nerve cell, compared with (1), indicating that the outgrowth of neurites was suppressed. Therefore, it was suggested that the exosomes of the present disclosure do not have the function of elongating the neurites of PC-12 cells, but rather have the function of suppressing the outgrowth of neurites. Thereby, the exosomes of the present disclosure can suppress the sympathetic nervous system.

Claims

1. 1. A method for culturing astrocytes, comprising: Simultaneous inhibition of Rho-associated protein kinase and TGFβ receptor function in culture for periods of more than 28 days. method.

2. Cultivating in a medium containing 1 μM to 100 μM Y-27632 and 0.1 μM to 100 μM A-83-01; The method of claim 1.

3. Cultivating in a medium containing 10 μM to 100 μM Y-27632 and 0.5 μM to 100 μM A-83-01; The method of claim 1.

4. The culture for more than 28 days is performed with intermittent passages. The method of claim 1.

5. Freezing and dormancy are required during subculture. The method of claim 4.

6. The astrocytes are pre-cultured in a medium that does not contain a small molecule signaling pathway inhibitor. The method of claim 1.

7. Culturing astrocytes by the method of claim 1; and purifying or isolating the cell secretions secreted from the astrocytes; A method for producing a cell secretion comprising:

8. Culturing astrocytes by the method of claim 1; Further culturing the astrocytes in a recovery medium; and Purifying or isolating from the recovery medium the cell secretions secreted by the astrocytes into the recovery medium; A method for producing a cell secretion comprising:

9. 1. A method for producing a cell secretion product comprising a combination of vinculin (P18206), integrin beta-1, CD29 (P05556), pyruvate kinase M1 / 2 (P14618), and ephrin type-A receptor 2 (P29317), comprising: Culturing astrocytes by the method of claim 1; and causing the astrocytes to secrete a cell secretion; A method comprising:

10. hsa-miR-382-5p (MIMAT0000737), hsa-miR-155-5p (MIMAT0000646), hsa-miR-379-5p (MIMAT0000733), hsa-miR-16- 5p (MIMAT0000069), hsa-miR-382-5p (MIMAT0000737), hsa-miR-16-5p (MIMAT0000069), hsa-miR-382-5p (MIMAT00007 37), hsa-miR-21-5p (MIMAT0000076), hsa-let-7a-5p (MIMAT0000062), hsa-miR-16-5p (MIMAT0000069), hsa-miR-409-3p (MIMAT0001639), hsa-let-7a-5p (MIMAT0000062), and hsa-let-7f-5p (MIMAT0000067), Culturing astrocytes by the method of claim 1; and causing the astrocytes to secrete a cell secretion; A method comprising:

11. A method for producing a pharmaceutical composition used for the prevention or treatment of a disorder associated with peripheral nerve cells or central nerve cells, comprising: Obtaining extracellular vesicles as the cell secretions by the method according to claim 9 or 10; and preparing a pharmaceutical composition comprising the extracellular vesicles and a carrier; A method comprising:

12. 1. A method for producing a composition for use in suppressing the sympathetic nervous system, comprising: Obtaining extracellular vesicles as the cell secretions by the method according to claim 9 or 10; and preparing a composition comprising the extracellular vesicles; A method comprising:

13. A method for producing a pharmaceutical or cosmetic product, comprising: Obtaining exosomes as the cell secretions by the method according to claim 9 or 10; and Preparing a pharmaceutical or cosmetic containing the exosome; A method comprising:

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