Highly proliferative cells, cell secretions, ectodermal progenitor cells, and method for producing pharmaceutical compositions

Abstract

A method for producing a highly proliferative cell, which is a method for producing a highly proliferative cell or an epiblastic progenitor cell, the method comprising (i) preparing an epiblastic cell that serves as a raw material, (ii) performing culture in a culture medium containing a low-molecular-weight signaling pathway inhibitor while bringing the inhibitor into contact with the epiblastic cell that serves as the raw material, and (iii) performing additional culture in a culture medium containing the inhibitor after the above-mentioned contact to produce a culture containing a highly proliferative cell having increased cell proliferation capability compared with the epiblastic cell that serves as the raw material; a highly proliferative cell which has the characteristic properties of an epiblastic cell and has such a proliferation capability that the number of cells after the elapse of a culture period of longer than 28 days while contacting with a low-molecular-weight signaling pathway inhibitor is more than 1.0 time the number of cells of the epiblastic cell that is cultured for the same culture period under the same culture conditions except that the cells are not in contact with the inhibitor; a cell secretion derived from the aforementioned highly proliferative cell or a highly proliferative cell produced by the highly proliferative cell production method; a cell secretion containing a specific protein and / or miRNA; a pharmaceutical composition which contains each of the cell secretions and is used for the prevention or treatment of a disorder associated with a peripheral nerve cell or a central nerve cell; and a method for depressing a sympathetic nervous system, the method comprising bringing the highly proliferative cell produced by the production method or a cell secretion secreted from the highly proliferative cell into contact with a nerve cell.

Description

[Technical Field]

[0001] The present invention relates to a method for producing hyperproliferative cells, hyperproliferative cells, and uses thereof. [Background technology]

[0002] In the field of regenerative medicine, various cells in vivo, cells at various stages of differentiation, or cells induced to differentiate in a specific direction are utilized. Considering their use in vivo, it is desirable for these cells not to be genetically modified. It has been reported that contacting endodermal mature cells with specific low-molecular-weight compounds can reprogram the mature cells into stem cells or progenitor cells without genetic modification (WO 2017 / 119512 and WO 2020 / 080550). Summary of the Invention

[0003] On the other hand, many cells obtained without genetic modification, especially those relatively close to mature cells, have limited proliferation capacity and tend not to be maintained in vivo or in vitro for long periods. Therefore, there remains a need for highly proliferative cells that are effective for long-term utilization of cell functions or for mass production of useful substances produced by cells. In particular, ectodermal cells, including cells of the nervous system, have not been fully utilized, unlike endodermal cells. [Problem to be solved by the invention]

[0004] An object of the present disclosure is to provide a method for producing hyperproliferative cells, hyperproliferative cells, and uses thereof. [Means for solving the problem]

[0005] As a result of extensive research, the present inventors have found that highly proliferative cells can be obtained by contacting ectodermal cells with a small molecule signaling pathway inhibitor.

[0006] That is, the present disclosure includes the following aspects: [1] A method for producing highly proliferative cells, comprising: (i) providing a starting ectodermal cell; (ii) contacting the ectodermal cells as the source material with a small molecule signaling pathway inhibitor in a medium containing the inhibitor, and culturing the inhibitor; and (iii) After the contact, further culturing is performed in a medium containing the inhibitor, thereby obtaining a culture containing highly proliferative cells having enhanced cell proliferation ability compared to the starting ectodermal cells. a method for producing highly proliferative cells, comprising: [2] The production method according to [1], wherein the number of the highly proliferative cells after a culture period of more than 28 days in contact with the inhibitor is more than 1.0 times the number of ectodermal cells cultured under the same culture conditions but without contact with the inhibitor for the same culture period; [3] The method according to [1] or [2], wherein the ectodermal cells used as the starting material include cells of the central nervous system; [4] The method according to any one of [1] to [3], wherein the ectodermal cells used as the raw material include astrocytes; [5] The method according to any one of [1] to [4], wherein the inhibitor comprises 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 within the range of 0.001 μM to 100 μM; [7] The method according to [5] or [6], wherein the concentration of the ROCK inhibitor is within the range of 0.001 μM to 100 μM; [8] The 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 that of the starting ectodermal cells, and express at least one gene specific to progenitor cells at the same level or higher than that of the starting ectodermal cells; [9] The method according to any one of [1] to [8], wherein the highly proliferative cells are negative for at least one selected from the group consisting of Musashi1, Notch1, Nestin, and SOX2;

[10] Highly proliferative cells that have characteristics of ectodermal cells and have a proliferation potential in which 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 ectodermal cells cultured under the same culture conditions but without contact with the inhibitor for the same culture period;

[11] The cell according to

[10] , wherein the highly proliferative cells include cells that are negative for at least one selected from the group consisting of Musashi1, Notch1, Nestin, and SOX2;

[12] The cell according to

[10] or

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

[13] A method for producing a cell secretion, comprising obtaining a culture containing a cell secretion secreted from the highly proliferative cells obtained by the production method according to any one of [1] to [9] or the highly proliferative cells according to any one of

[10] to

[12] , and separating the cell secretion from the culture;

[14] The method according to

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

[15] A method for producing ectodermal progenitor cells, comprising: the ectodermal progenitor cells are hyperproliferative cells; (i) providing a starting ectodermal cell; (ii) contacting the source ectodermal cells with a small molecule signaling pathway inhibitor in a medium containing the inhibitor for a period of more than 28 days; and (iii) After the contact, further culturing is performed in a medium containing the inhibitor, thereby obtaining a culture containing highly proliferative cells having enhanced cell proliferation ability compared to the starting ectodermal cells. a method for producing ectodermal progenitor cells,

[16] The method of

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

[17] A cell secretion containing a protein and / or miRNA, The proteins include a combination of vinculin (P18206), integrin beta-1, CD29 (P05556), pyruvate kinase M1 / 2 (P14618), and ephrin type-A receptor 2 (P29317); The miRNAs are 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(MI MAT0000737), 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) in cell secretions;

[18] Cell secretions as described in

[17] , which are exosomes;

[19] A cell secretion product secreted from a highly proliferative cell obtained by the production method according to [1] or [2] or a highly proliferative cell according to any one of

[10] to

[12] .

[17] or

[18] , and a pharmaceutical composition for use in the prevention or treatment of disorders relating to peripheral nerve cells or central nerve cells, comprising any one of the cell secretions of

[20] A cell secretion product secreted from a highly proliferative cell obtained by the production method according to [1] or [2] or a highly proliferative cell according to any one of

[10] to

[12] .

[17] or

[18] , and A method for inhibiting the sympathetic nervous system, comprising contacting a nerve cell with a cell secretion of any one of the above. [Effects of the Invention]

[0007] According to the present disclosure, a method for producing hyperproliferative cells, hyperproliferative cells, and uses thereof can be provided. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the morphology of ectodermal cells after long-term culture according to Example 1. [Figure 2] FIG. 2 shows the cell morphology of ectodermal cells before and after medium exchange with a recovery medium according to Example 2. [Figure 3A] FIG. 3A shows the particle size distribution of particles in the culture supernatant collected after the additional culture of the Normal group according to Example 2. [Figure 3B] FIG. 3B shows the particle size distribution of particles in the culture supernatant collected after the additional culture of the NHA-YA group according to Example 2. [Figure 4A] FIG. 4A shows proteins selected by proteome analysis of NHA-YA-derived exosomes according to Example 4. [Figure 4B] FIG. 4B shows proteins selected by proteome analysis of NHA-YA-derived exosomes according to Example 4. [Figure 5] FIG. 5 shows miRNAs selected as Parkinson's disease markers by miRNA analysis of NHA-YA-derived exosomes according to Example 5. [Figure 6] FIG. 6 shows miRNAs selected as Alzheimer's disease markers by miRNA analysis of NHA-YA-derived exosomes according to Example 5. [Figure 7] FIG. 7 shows miRNAs selected as Alzheimer's disease markers by miRNA analysis of NHA-YA-derived exosomes according to Example 5. [Figure 8] FIG. 8 shows miRNAs selected as Alzheimer's disease markers by miRNA analysis of NHA-YA-derived exosomes according to Example 5. [Figure 9] FIG. 9 shows miRNAs selected as Alzheimer's disease markers by miRNA analysis of NHA-YA-derived exosomes according to Example 5. [Figure 10] FIG. 10 shows miRNAs selected as depression markers by miRNA analysis of NHA-YA-derived exosomes according to Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0009] <1> Method for producing highly proliferative cells The method for producing highly proliferative cells according to the present disclosure includes: (i) providing a starting ectodermal cell; (ii) contacting the ectodermal cells as the source material with a small molecule signaling pathway inhibitor in a medium containing the inhibitor, and culturing the inhibitor; and (iii) After the contact, further culturing is performed in a medium containing the inhibitor, thereby obtaining a culture containing highly proliferative cells having enhanced cell proliferation ability compared to the starting ectodermal cells. Includes:

[0010] As used herein, the term "cell" should be construed as including at least one cell, unless otherwise specified. Therefore, unless otherwise specified, the term "cell" is not limited to a single cell, and may be used synonymously with a cell population.

[0011] As used herein, the term "cell population" should be construed as including at least one type of cell, unless otherwise specified. Therefore, unless otherwise specified, the cells referred to in the term "cell population" are not limited to one type of cell.

[0012] The "ectoderm" is one of the three germ layers that arise during metazoan development. The ectoderm is derived from the outer layer of the embryo and develops into the skin, nervous system, and sensory organs. Examples of skin include the epidermis, hair, nails, and skin glands; examples of the nervous system include the cranial nerves, spinal cord, and peripheral nerves; and examples of sensory organs include the organs of vision, hearing, balance, taste, smell, and touch.

[0013] As used herein, "ectodermal cells" include, for example, cells of the nervous system, such as cells of the central nervous system and cells of the peripheral nervous system. Thus, in this disclosure, "source ectodermal cells" can include, for example, cells of the central nervous system. Source ectodermal cells may include astrocytes.

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

[0015] As used herein, "ectodermal cells" refer to cells that constitute "ectodermal tissues" and "ectodermal organs." Examples of "ectodermal tissues" and "ectodermal organs" include the central nervous system (brain and spinal cord), pituitary gland, peripheral nerves, enteric nerves, adrenal medulla, melanocytes, facial cartilage, dental dentin, epidermis, hair, nails, skin, sebaceous glands, salivary glands, sweat glands, mammary glands, nasal cavity, nasal mucosa, oral epithelium, oral cavity, eyes, urinary bladder, and anus. The brain can be broadly divided into the cerebrum, cerebellum, and brainstem. The cerebrum is further divided into the telencephalon and diencephalon, and the brainstem is further divided into the midbrain, pons, and medulla oblongata. The central nervous system is composed of neurons and glial cells, and these cells can be selected as starting materials. These tissues or organs contain many mature cells that have completed the terminal differentiation stage.

[0016] The ectodermal cells used as raw materials can be at least one selected from the group consisting of glial cells such as astrocytes, microglia, oligodendrocytes, oligodendrocyte precursor cells (also called polydendrocytes), ependymal cells, Schwann cells, and satellite cells. These cells may be primary cells obtained from a living organism, 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 methods of the present disclosure may be, for example, cells isolated and purified from the brain removed from a mammal.

[0018] For example, in the case of rats, brains excised from adult rats aged 10 to 20 weeks are preferably used, but brains from infant rats aged 2 months or less can also be used. In the case of humans, brains can be obtained by biopsy or surgery. For example, both neurons and glial cells can be collected by biopsy. In the case of surgery, excised brain tissue pieces from adults or brains excised from dead fetuses can be used. Alternatively, neurons or glial cells can be isolated and purified from these excised brains, and then frozen.

[0019] Ectodermal cells as raw materials can be defined as cells that exhibit characteristics of ectodermal cells. Characteristics of ectodermal cells include cell morphology and the expression of cell-specific genes (sometimes referred to as "marker genes"). Ectodermal cells can be identified by the expression of at least one marker gene selected from the group consisting of GFAP, S100B, SLC1A2 (also known as "EAAT2" or "GLT-1"), and NG2 at any stage of differentiation. Expression of ectodermal cell marker genes can be confirmed based on either the gene or the protein. Gene or protein expression can be confirmed by methods known in the art, such as the presence or absence of expression, specific expression levels depending on the measurement method, or comparison with the expression levels of the same gene or protein in specific cells.

[0020] In this specification, the expression of a marker gene can be confirmed using techniques known in the art, for example, by measuring the expression level of the gene using quantitative PCR (sometimes referred to as "qPCR"), or by measuring the amount of protein using immunoassays such as ELISA or flow cytometry.

[0021] The source ectodermal cells prepared as described above are contacted with a small molecule signaling pathway inhibitor. The contact may be in vitro. There are no particular limitations on the small molecule signaling pathway inhibitors that can be used in this method; any can be used. Examples of small molecule signaling pathway inhibitors include transforming growth factor (TGF) β receptor inhibitors, ROCK (Rho-associated protein kinase) inhibitors, and glycogen synthase kinase 3 (GSK3) inhibitors.

[0022] In the production method according to the present disclosure, the inhibitor may comprise 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 comprise 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 an effect of inhibiting the function of the TGFβ receptor, and examples thereof include 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 from Merck), SB431542 (Sigma-Aldrich), and the like. Examples of TGFβ receptor inhibitors include CultureSure® A-83-01 (Fujifilm Wako Pure Chemical Industries, Ltd.) and CultureSure® A-83-01 (Fujifilm Wako Pure Chemical Industries, Ltd.). The TGFβ receptor inhibitor is preferably CultureSure® A-83-01. TGFβ receptor inhibitors also include TGFβ receptor antagonists. 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 ROCK inhibitors include GSK269962A (Axon Medchem), Fasudil hydrochloride (Tocris Bioscience), CultureSure® Y-27632 (Fujifilm Wako Pure Chemical Corporation), and H-1152 dihydrochloride (Fujifilm Wako Pure Chemical Corporation). The ROCK inhibitor is preferably CultureSure® Y-27632. One type of ROCK inhibitor may be used alone, or two or more types may be used in combination.

[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 GSK3 inhibitors 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. One GSK3 inhibitor may be used alone, or two or more GSK3 inhibitors may be used in combination.

[0026] The small molecule signaling 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. Combination of TGFβ receptor inhibitors and ROCK inhibitors TGFβ receptor inhibitors, or ROCK inhibitors In one embodiment, the small molecule signaling pathway inhibitor used is a combination of a TGFβ receptor inhibitor and a ROCK inhibitor. In this specification, the term "small molecule signaling pathway inhibitor" may also be simply referred to as "inhibitor."

[0027] Embodiments of the present disclosure include, for example, 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; in 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, within 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 adjusted appropriately depending on the type of TGFβ receptor used. In one embodiment, the concentration of the TGFβ receptor inhibitor in the medium, for example, CultureSure (registered trademark) A-83-01, is 0.1 μM to 3 μM.

[0029] The concentration of the ROCK inhibitor in the medium is, for example, within 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 adjusted appropriately depending on the type of ROCK inhibitor used. In one embodiment, the concentration of the ROCK inhibitor, for example, CultureSure (registered trademark) Y-27632, in the medium is 1 μM to 30 μM.

[0030] The concentration of the GSK3 inhibitor in the medium is, for example, within 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 adjusted appropriately depending on the type of GSK3 inhibitor used.

[0031] The above concentration ranges are applicable to both the use of the TGFβ receptor inhibitor, ROCK inhibitor, and GSK3 inhibitor alone and the use of these inhibitors in combination. In addition, if the inhibitor is water-insoluble or poorly water-soluble, it can be dissolved in a small amount of a low-toxicity organic solvent (e.g., DMSO) and then added to the medium to the above final concentration.

[0032] The contact of the starting ectodermal cells with the small molecule signaling pathway inhibitor is achieved by culturing the starting ectodermal cells in a medium containing the inhibitor, specifically by adding the 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. This further culture is also referred to as "additional culture" or "second culture." In contrast, the culture up to the time 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 a small molecule signaling pathway inhibitor, is also referred to as "preculture" or "first culture." After contact with the inhibitor, additional culture is performed to obtain highly proliferative cells with enhanced cell proliferation ability. Even during the additional culture, the medium contains the inhibitor, so the cells continue to be in contact with the inhibitor.

[0034] In the present disclosure, the medium used for pre-culture and additional culture can be a medium widely used for culturing animal cells, and commercially available basal media may also 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 Minimum Essential Medium (DMEM), RPMI 1640 medium, 199 medium, Ham's F12 medium, William's E medium, and NS basal medium (Fujifilm Wako Pure Chemical Industries, Ltd.). The above media may be used alone or in combination of two or more.

[0035] Examples of additives that can be added to the culture 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), antimycotics (e.g., amphotericin), and buffers (e.g., Good's buffer such as HEPES).

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

[0037] When using a serum-supplemented medium, fetal bovine serum (FBS) can be used as the serum. Furthermore, exosome-depleted FBS can be used to facilitate exosome isolation. Commercially available exosome-depleted media include FBS exosome-depleted, OneShot format (Gibco®, Thermo Fisher Scientific). The serum concentration 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 serum concentration in the medium is 3% (v / v).

[0038] When using a serum-free medium, a serum substitute may be added. Examples of serum substitutes include bovine serum albumin (BSA), knockout serum replacement (KSR), and human serum albumin (HSA or human albumin, serum; HAS). Human serum albumin may be isolated from human plasma or purified from rice expressing the human serum albumin gene (Fujifilm Wako Pure Chemical Industries, Ltd.). Typically, growth factors (e.g., human EGFR), cytokines, hormones, and other factors are added to the medium. These added factors include, but are not limited to, epidermal growth factor (EGF), insulin, transferrin, hydrocortisone 21-hemisuccinate or a salt thereof, dexamethasone (Dex), N2 Supplement (Fujifilm Wako Pure Chemical Industries, Ltd.), NS Supplement (Fujifilm Wako Pure Chemical Industries, Ltd.), and B-27 Serum Free Supplement (Thermo Fisher Scientific).

[0039] The culture vessel used for the culture is not particularly limited as long as it is suitable for adherent culture, and examples include dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, and culture bags. For cell suspension culture, culture vessels with surfaces treated to prevent cell adhesion can also be used. Alternatively, for adherent culture, vessels whose inner surfaces are coated with a cell support substrate can be used to improve cell adhesion. 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 The cells can be seeded onto the culture vessel at a cell density of 0.1 to 1.0 μg / ml.

[0041] The conditions generally used for culturing ectodermal cells can be applied to the culture. A CO2 incubator can be used for the culture, and the culture temperature and CO2 concentration can be those generally used, such as 37°C and 5% (v / v).

[0042] The culture period is the period during which the cells are cultured with YA, and this period may be a continuous period or a discontinuous period, i.e., the sum of multiple discontinuous periods. The culture period may be longer than 28 days, and may be, for example, 5 weeks or more, 6 weeks or more, or 7 weeks or more. The upper limit of the culture period may be, for example, about 3 months or 100 days. Passage may be performed appropriately depending on the cell density in the culture vessel, the state of the medium, the state of the cells, etc., and the passaging interval may be, for example, about 2 to 20 days.

[0043] Highly proliferating cells (sometimes referred to herein as "HP cells") obtained by this production method are cells with enhanced proliferation potential. Compared with control ectodermal cells cultured under the same culture conditions as the cells in question, except for the use of a medium containing no small molecule signaling pathway inhibitor, these cells possess the properties of higher cell proliferation activity, shorter proliferation time (i.e., shorter cell doubling time), longer proliferation time, or both. The cell population contained in the culture obtained by this production method can contain not only HP cells, but also cells whose proliferation potential is not enhanced and is unchanged from that of the original cells, i.e., non-highly proliferating cells (sometimes referred to herein as "non-HP cells"). 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 by cell number.

[0044] Highly proliferative cells can have a proliferation potential such that the cell number (herein referred to as "cell number N of highly proliferative cells") after a culture period of more than 28 days (herein referred to as "period T") in contact with an inhibitor is more than 1.0 times the cell number (herein referred to as "cell number N' of ectodermal cells") of ectodermal cells cultured under the same culture conditions but without contact with the inhibitor for the same culture period. In this case, the culture conditions may be, for example, 4 x 10 3 cells / cm 2 The seeding density can be 100 μg / ml, the culture temperature can be 37°C, and the CO2 concentration can be 5%. Any of the media listed above for use in pre-culture and additional culture can be used as the culture medium used to confirm the cell number N of highly proliferative cells. The two types of culture medium used to confirm the cell number N of highly proliferative cells and the cell number N' of ectodermal cells can be media with the same composition except for the presence or absence of an inhibitor.

[0045] In one embodiment, 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 embodiment, the cell number N of the highly proliferative cells is not particularly limited as long as it is more than 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] Highly proliferative cells may be identified by the expression of cell-specific genes, i.e., marker genes. As described above, the expression of marker genes in highly proliferative cells may be confirmed based on either genes or proteins, and the expression of genes or proteins may be confirmed by the presence or absence of expression, specific expression levels depending on the measurement method, or comparison with the expression levels of the same genes or proteins in specific cells.

[0048] As used herein, gene or protein expression may be expressed as "positive" or "negative." With respect to gene or protein expression, "positive" refers to the presence of expression of the target gene or protein, whereas "negative" refers to the absence of expression of the target gene or protein. When describing gene or protein expression as "high expression" or "positive," or "low expression" or "negative," specific cells other than highly proliferative cells can be used as a control. The specific cells used as a control, i.e., "control cells," may be cells with extremely low expression of the target gene or protein, or may be source ectodermal cells, i.e., ectodermal cells not contacted with an inhibitor, or may be mature cells or their precursor cells.

[0049] For example, a highly proliferative cell being "positive" with respect to marker gene expression can mean that the expression level of the marker gene after contact with a small molecule signaling pathway inhibitor is 1.0-fold or more compared to the expression level of the marker gene in ectodermal cells cultured without contact with the inhibitor. In contrast, a highly proliferative cell being "negative" with respect to marker gene expression can mean that the expression level of the marker gene after contact with the inhibitor is less than 1.0-fold compared to the expression level of the marker gene in ectodermal cells cultured without contact with the inhibitor.

[0050] The culture conditions for the highly proliferative cells and ectodermal cells not in contact with an inhibitor used in the method for confirming expression levels can be the same except for whether they are in contact with an inhibitor or not. However, these can be appropriately adjusted based on the state, proliferation, and number of cells, for example, to ensure that the target cells are in the same or nearly the same state, such as the same culture period, the same number of cell passages, and a similar cell density, and those skilled in the art can select these conditions based on the state of the cells.

[0051] Here, the expression level of the marker gene after a certain period of culture in contact with a small molecule signaling pathway inhibitor is expressed as the expression level relative to that of a control gene, such as beta-actin (ACTB) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

[0052] In one aspect, the hyperproliferative cells are capable of expressing at least one gene characteristic of a mature cell at the same or greater level than the ectodermal cells from which they are derived, and at least one gene characteristic of a progenitor cell at the same or greater level than the ectodermal cells from which they are derived.

[0053] As used herein, the term "progenitor cell" refers to a cell at a differentiation stage leading up to a mature cell, which is capable of developing from a stem cell and differentiating into a terminally differentiated cell that constitutes the body.

[0054] Genes specific to mature cells, such as those expressed at relatively high levels in astrocytes, include GFAP, S100B, and SLC1A2. Genes specific to progenitor cells, such as those expressed at relatively high levels in oligodendrocyte progenitor cells, neuroepithelial cells, and radial glial cells, include NG2, Notch1, Nestin, and SOX2.

[0055] Highly proliferative cells may be cells characterized by any marker gene expression pattern, or in other words, cell populations containing cells with any characteristic, as long as they have the high proliferation potential described above. Highly proliferative cells may exhibit the following marker gene expression patterns. Highly proliferative cells according to the present disclosure can be rapidly identified and extracted based on the expression patterns of these marker genes.

[0056] The hyperproliferative cells can include cells that are negative for at least one selected from the group consisting of Musashi1 (MSI1), Notch1, Nestin, and SOX2.

[0057] For example, the highly proliferative cells can comprise GFAP-positive cells. In another embodiment, the highly proliferative cells can comprise cells that are GFAP-positive and NG2-positive, or can express GFAP at or above the level of the ectodermal cells from which they are derived and can express NG2 at or above the level of the ectodermal cells from which they are derived. In another embodiment, the highly proliferative cells can comprise cells that are GFAP- and S100B-positive and NG2-positive, or can express GFAP and S100B at or above the level of the ectodermal cells from which they are derived and can express NG2 at or above the level of the ectodermal cells from which they are derived.

[0058] In another aspect, the hyperproliferative cells can include cells that express at least one gene characteristic of a mature cell at the same or greater level than the ectodermal cells from which they are derived, and that express at least one gene characteristic of a progenitor cell at the same or greater level than the ectodermal cells from which they are derived.

[0059] In one embodiment, the hyperproliferative cells may include NG2-positive cells or cells that express NG2 at a level higher than that of ectodermal cells not contacted with a small molecule signaling pathway inhibitor. That is, in this embodiment, the hyperproliferative cells may express NG2 at a level higher than that of ectodermal cells not contacted with a small molecule signaling pathway inhibitor.

[0060] For example, highly proliferative cells can include cells that are positive for S100B and positive for NG2, or can express S100B at or above the level of the ectodermal cells from which they are derived and express NG2 at or above the level of the ectodermal cells from which they are derived.

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

[0062] In one embodiment, the expression level of cells negative for at least one selected from the group consisting of Musashi1, Notch1, Nestin, and SOX2 can be expressed as the expression level relative to a control gene. In this case, "negative" means that the expression level of the control gene is less than 1.0-fold when using, for example, beta-actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

[0063] The cell population of highly proliferative cells according to the present disclosure may be a heterogeneous cell population containing both mature cells and progenitor cells, regardless of the differentiation stage, as long as the cell population is made up of highly proliferative cells with high proliferation potential, particularly highly proliferative ectodermal cells, or a cell population in which the vast majority of cells are made up of multiple types of ectodermal cells at relatively similar differentiation stages.

[0064] In one embodiment, the cell population of highly proliferative cells obtained by the present production method may be a population containing highly proliferative ectodermal progenitor cells as the major constituent, or a population containing both highly proliferative ectodermal progenitor cells and highly proliferative ectodermal mature cells. Here, the term "major constituent" in a cell population refers to cells that account for at least 50% of the cell population by number. As used herein, the term "significant majority" refers to cells of interest that account for 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more of the cell population by number.

[0065] The proportion of mature ectodermal cells in the resulting 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% by cell number. Therefore, the present disclosure also encompasses a method for enriching ectodermal cells with enhanced cell proliferation potential, which comprises contacting starting ectodermal cells with a small molecule signaling pathway inhibitor. Contacting starting ectodermal cells with a small molecule signaling pathway inhibitor can be carried out in vitro.

[0066] In this production method, when the starting ectodermal cells are astrocytes, the cell population of highly proliferative cells may contain cells of an astrocyte-related ectodermal cell lineage. Examples of cells of an astrocyte-related ectodermal cell lineage include astrocytes, radial glial cells, oligodendrocyte precursor cells (also known as polydendrocytes), oligodendrocytes, and neuroepithelial cells. In addition, the cell population of an astrocyte-related ectodermal cell lineage may contain cells that possess some of the characteristics of ectodermal cells but cannot be classified as any of the above-mentioned cells, so long as the cell population exhibits enhanced cell proliferation potential.

[0067] Another aspect of the present disclosure relates to hyperproliferative cells, which may be obtained by the aforementioned production method or by other production methods.

[0068] According to one aspect of the present disclosure, highly proliferative cells have characteristics of ectodermal cells and exhibit a proliferation potential such that the number of cells after contact with a small molecule signaling pathway inhibitor for a culture period of more than 28 days is greater than 1.0-fold the number of ectodermal cells cultured under identical culture conditions for the same culture period, except that the cells are not contacted with the small molecule signaling pathway inhibitor. As described above, by contacting ectodermal cells with a small molecule signaling pathway inhibitor, highly proliferative cells with enhanced proliferation potential can be obtained without gene transfer or genetic modification of the ectodermal cells. As a result, the inhibitor-contacted group can be cultured for a longer period of time than the inhibitor-uncontacted group, and the number of cells obtained is greater in the inhibitor-contacted group than in the inhibitor-uncontacted group.

[0069] In one embodiment, a cell population obtained by culturing human primary mature astrocytes in contact with a combination of a TGFβ receptor inhibitor and a ROCK inhibitor (inhibitor-exposed group) can be cultured for a longer period of time than a cell population obtained by culturing human primary mature astrocytes without contacting the inhibitor combination (inhibitor-unexposed group). That is, cell proliferation continues for a longer period in the inhibitor-exposed group than in the inhibitor-unexposed group. Furthermore, the number of cells ultimately obtained by proliferation in the inhibitor-exposed group is, for example, 2-fold or more, 5-fold or more, 10-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, or 400-fold greater than the number of cells in the inhibitor-unexposed group.

[0070] The resulting highly proliferative cells have an increased proliferation potential, and therefore a large number of cells can be obtained by long-term culture. Since highly proliferative cells are capable of producing cell secretions as described below, the amount of cell secretions that can be recovered can also increase if a large number of cells are obtained by long-term culture.

[0071] The highly proliferative cells of this aspect are subject to the same description as for the product produced by the production method of the present invention, except that they are not limited to being the product produced by the production method of the present invention, as long as they have the characteristics of ectodermal cells and have a proliferation potential such that the number of cells after a culture period of more than 28 days in contact with an inhibitor is greater than 1.0 times the number of ectodermal cells cultured under the same culture conditions but for the same culture period, except for the absence of the inhibitor.

[0072] As described above, the hyperproliferative cells of the present disclosure can include cells that are positive for at least one selected from the group consisting of GFAP, NG2, and S100B. For example, the hyperproliferative cells can include at least one selected from the group consisting of the following cells (I) to (VII): (I) GFAP-positive cells, e.g., cells that express GFAP at the same level or higher than the ectodermal cells from which they are derived; (II) NG2-positive cells, e.g., cells that express NG2 at the same level or higher than the ectodermal cells from which they are derived; (III) S100B-positive cells, e.g., cells that express S100B at or above the level of the ectodermal cells from which they are derived; (IV) GFAP-positive and NG2-positive cells, for example, cells that express GFAP at or above the level of the ectodermal cells from which they are derived and that express NG2 at or above the level of the ectodermal cells from which they are derived; (V) GFAP-positive and S100B-positive cells, for example, cells that express GFAP at or above the level of the ectodermal cells from which they are derived and that express S100B at or above the level of the ectodermal cells from which they are derived; (VI) S100B-positive and NG2-positive cells, for example, cells that express S100B at or above the level of the ectodermal cells from which they are derived and that express NG2 at or above the level of the ectodermal cells from which they are derived; (VII) GFAP-positive, S100B-positive, and NG2-positive cells, for example, cells that express GFAP at or above the level of the ectodermal cells from which they are derived, express S100B at or above the level of the ectodermal cells from which they are derived, and express NG2 at or above the level of the ectodermal cells from which they are derived.

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

[0074] When the highly proliferative cells of the present disclosure include at least GFAP-positive cells, the expression level of the GFAP gene after contact with a small molecule signaling pathway inhibitor is preferably 1.0 to 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 that are positive for at least one selected from the group consisting of GFAP, NG2, and S100B, and negative for at least one selected from Musashi1, Notch1, Nestin, and SOX2. That is, in this embodiment, the cells of (I) to (VII) above are negative for at least one selected from Musashi1, Notch1, Nestin, and SOX2.

[0076] Highly proliferative cells can be isolated by any known means for isolating specific cells, such as fluorescence-activated cell sorting (FACS) based on the expression of proteins derived from the aforementioned marker genes, or magnetic cell separation using Dynabeads, etc. Because the resulting highly proliferative cells have a high proliferation capacity, long-term culture may be performed to obtain a cell population with an extremely high proportion of highly proliferative cells, thereby substantially isolating the highly proliferative cells.

[0077] <2> Ectodermal progenitor cells and method for producing same The highly proliferative cells obtained by the production method of the present disclosure may include cells exhibiting characteristics of a more immature differentiation stage than the starting ectodermal cells. These immature ectodermal cells, i.e., ectodermal progenitor cells, may have higher cell proliferation activity and may proliferate in a shorter time, for a longer period of time, or both, compared to control ectodermal cells cultured under the same culture conditions as the cells except for using a medium containing no inhibitors. These highly proliferative ectodermal progenitor cells are sometimes referred to herein as highly proliferative progenitor cells. The term "highly proliferative cells" herein may also encompass highly proliferative progenitor cells. This has the advantage of allowing immature ectodermal cells to be grown for a longer period of time, thereby enabling, for example, the production of large amounts of cell secretions produced by ectodermal progenitor cells in a shorter time than 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, the method comprising: (i) providing a starting ectodermal cell; (ii) contacting the source ectodermal cells with a small molecule signaling pathway inhibitor in a medium containing the inhibitor for a period of more than 28 days; and (iii) After the contact, further culturing is performed in a medium containing the inhibitor, thereby obtaining a culture containing highly proliferative cells having enhanced cell proliferation ability compared to the starting ectodermal cells. The method for producing ectodermal progenitor cells of the present disclosure may further comprise isolating the hyperproliferative cells from the culture.

[0079] In the method for producing ectodermal progenitor cells disclosed herein, the raw material ectodermal cells and the small molecule signaling pathway inhibitor may be, for example, the raw material ectodermal cells and the small molecule signaling pathway inhibitor described in relation to the method for producing highly proliferative cells disclosed herein.

[0080] Highly proliferative progenitor cells can be isolated by any known means for isolating specific cells, such as fluorescence-activated cell sorting (FACS) based on the expression of proteins derived from the aforementioned marker genes specific to progenitor cells, or magnetic cell separation using Dynabeads, etc. Because the resulting highly proliferative progenitor cells have a high proliferation potential, long-term culture may be performed to obtain a cell population with an extremely high proportion of highly proliferative progenitor cells, thereby substantially isolating the highly proliferative progenitor cells.

[0081] In the case of highly proliferative cells obtained by the production method of the present disclosure, when the starting ectodermal cells contain astrocytes that have reached a terminal differentiation stage, the obtained highly proliferative cells may contain cells that exhibit characteristics of a more immature differentiation stage than astrocytes. In addition to having enhanced proliferation potential, such immature ectodermal cells may further possess the following characteristics (a) or (b): (a) Compared to astrocytes, the expression levels of the combination of GFAP and S100B, the combination of GFAP and SLC1A2, the combination of S100B and SLC1A2, the combination of GFAP and NG2, the combination of NG2 and SLC1A2, or the combination of NG2 and S100B are increased. (b) Compared to astrocytes, the expression levels of the combination of GFAP, S100B and NG2, the combination of GFAP, SLC1A2 and NG2, the combination of S100B, SLC1A2 and NG2, or the combination of GFAP, S100B, SLC1A2 and NG2 are increased.

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

[0083] The ectodermal progenitor cells obtained by the above-described production method can be used, for example, in a method for evaluating a therapeutic agent for a neurological disorder. Specifically, the usefulness of a candidate therapeutic agent for a neurological disorder can be evaluated by contacting the candidate therapeutic agent for a neurological disorder with the ectodermal progenitor cells. Therefore, the present disclosure also relates to a method for evaluating a therapeutic agent for a neurological disorder, which comprises contacting a candidate therapeutic agent for a neurological disorder with the ectodermal progenitor cells. Furthermore, the present disclosure also relates to a method for evaluating a neurological disease model, which comprises using the above-described ectodermal progenitor cells.

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

[0085] Here, "maturation conditions" refers to culture conditions using known differentiation-inducing factors. Examples of differentiation-inducing factors include fibroblast growth factor-3 (FGF-2); IL-6 family cytokines such as leukemia inhibitory factor (LIF) or ciliary neurotrophic factor (CNTF); bone morphogenetic protein (BMP) family cytokines such as BMP2 or BMP4; Notch family proteins; Wnt gene family proteins; sonic hedgehog proteins; and retinoic acid. Furthermore, factors necessary for the proliferation or maintenance of mature cells include 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 a differentiation-inducing factor, known differentiation-inducing conditions used in differentiation-inducing methods using differentiation-inducing factors can be applied, and the differentiation-inducing conditions can be appropriately selected depending on the type of differentiation-inducing factor used.

[0087] <3> Inhibitor Uses The inhibitors used in the aforementioned production methods can be used to maintain or promote the proliferation of ectodermal cells. Accordingly, a further aspect of the present disclosure relates to a proliferation regulator for ectodermal cells, comprising at least one small molecule signaling pathway inhibitor, e.g., a TGFβ receptor inhibitor and a ROCK inhibitor. Specifically, the present disclosure also relates to a proliferation regulator used to maintain or promote the proliferation of highly proliferative cells obtained by the aforementioned production methods, or to a proliferation regulator used to maintain or promote the proliferation of ectodermal cells. Herein, the term "proliferation regulation" can mean achieving at least one of maintaining and promoting cell proliferation. The terms "maintaining proliferation" and "proliferation promotion" are not particularly distinguishable, as long as the cell number is maintained or increased over the culture period. The concentrations of the proliferation regulator used to regulate cell proliferation can be the same as those described for the aforementioned production methods.

[0088] The present disclosure also relates to a method for culturing highly proliferative cells or ectodermal cells obtained by the aforementioned production method, comprising subculturing the cell population or the cells in the presence of a small molecule signaling pathway inhibitor, such as at least one inhibitor selected from the group consisting of a TGFβ receptor inhibitor and a ROCK inhibitor. The "culturing method" herein encompasses a method for regulating the proliferation of highly proliferative cells or ectodermal cells. The inhibitor concentrations used in this culture method can be the same as those described for the aforementioned production method.

[0089] <4> Cell secretions and methods for producing same Yet another aspect of the present disclosure relates to a method for producing a cell secretion, comprising obtaining a culture containing a cell secretion secreted from the highly proliferative cells of the present disclosure and separating the cell secretion from the culture. This production method may further include any optional steps, as necessary. The highly proliferative cells of the present disclosure are highly proliferative cells with enhanced cell proliferation ability, and therefore, compared to control cells cultured under the same culture conditions as the highly proliferative cells except for using a medium containing no inhibitor, they proliferate in a shorter time, for a longer period of time, or both for a shorter time and for a longer period of time. Therefore, this method for producing a cell secretion allows the cell secretion secreted by the highly proliferative cells to be obtained in large quantities in a shorter time than the control cells.

[0090] Cell secretions, also known as "secretomes," are not particularly limited as long as they are substances secreted from cells, and examples include extracellular vesicles such as exosomes, microvesicles, and apoptotic vesicles; and functional proteins such as cytokines, hormones, and antibodies. In one embodiment, the cell secretions may be extracellular vesicles, and in particular, may be exosomes.

[0091] The culture prepared in the method for producing a cell secretion according to the present disclosure contains a cell secretion secreted from the highly proliferative cells obtained by any of the above-described embodiments. The culture containing the cell secretion may be produced by carrying out the method for producing highly proliferative cells according to the present disclosure, or may be obtained by obtaining and culturing the highly proliferative cells according to the present disclosure, or may be obtained separately from a culture obtained by carrying out the production method according to the present disclosure, or may be obtained separately from a culture of highly proliferative cells according to the present disclosure. As used herein, the term "culture" refers to 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. The cell secretion can be separated from the culture by known separation methods, such as removing the supernatant and centrifugation. 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 contain the cell secretion and an aqueous medium. The aqueous medium is not particularly limited as long as it is a known aqueous medium selected depending on the type of cell secretion, and examples thereof include culture supernatant, water, buffer (e.g., phosphate buffer, Good's buffer), physiological saline, and culture medium.

[0093] The method for producing a cell secretion product may further include purifying or isolating the separated cell secretion product. As a method for purifying or isolating a cell secretion product, any known method can be applied depending on the type of cell secretion product, without particular limitation.

[0094] Methods for isolating or purifying cell secretions include, for example, filtration and concentration. For example, cell secretions can be isolated or purified by filtration using a membrane with a size or molecular weight cutoff value. As another method, for example, tangential flow filtration or ultrafiltration can be used to filter or concentrate cell secretions. The cell secretions can be used in the form of a composition containing the cell secretions, or in the form of isolated cell secretions obtained after isolation or purification. The cell secretions can be stored, refrigerated, or frozen in the form of a composition or isolated cell secretions, or by subjecting them to known processes such as spray drying and freeze drying.

[0095] Specifically, the cell secretions according to the present disclosure are cell secretions containing proteins and / or miRNAs, wherein 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 description in parentheses following each protein name indicates its accession number in UniProtKB / Swiss-Prot (https: / / www.uniprot.org / ). In FIGS. 5 to 10, the description in parentheses following each miRNA name indicates its accession number 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 comprises, as a protein: PA group: may include at least a combination of vinculin (P18206), integrin beta-1, CD29 (P05556), pyruvate kinase M1 / 2 (P14618), and ephrin type-A receptor 2 (P29317); PB group: may include a combination of vinculin (P18206), integrin beta-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: A combination of vinculin (P18206), integrin β-1, CD29 (P05556), pyruvate kinase M1 / 2 (P14618), and ephrin type-A receptor 2 (P29317) with a combination of aminopeptidase N (P15144), annexin A2 (P07355), annexin A6 (P08133), myoferlin (Q9NZM1), and 5'-nucleotidase, CD73 (P21589) with a combination of moiety. and at least one selected from the group consisting of escin (P26038), integrin alpha-2, CD49b (P17301), integrin alpha-3, CD49c (P26006), 2',3'-cyclic nucleotide 3'-phosphodiesterase (P09543), F-actin capping protein beta subunit (P79136), neuropilin 1 (O14786), and tenascin C (P24821), or PD group: may contain all proteins listed in Figures 4A and 4B.

[0097] In one embodiment, the cell secretion contains, as miRNA: ·RA group: 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(MIMAT000 0737), 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: hsa-miR-382-5p (MIMAT0000737), hsa-miR-155-5p (MIMAT0000646), hsa-miR-379-5p (MIMAT0000733), hsa-miR-16-5p (M IMAT0000069), hsa-miR-382-5p(MIMAT0000737), hsa-miR-16-5p(MIMAT0000069), hsa-miR-382-5p(MIMAT0000737), hsa-miR- The combination of 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) with 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), ·RC group: 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(M IMAT0000062), hsa-miR-16-5p(MIMAT0000069), hsa-miR-409-3p(MIMAT0001639), hsa-let-7a-5p(MIMAT0000062), and hsa-let-7f-5p(MIMAT0000067) in combination with 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-2 6b-5p(MIMAT0000083), hsa-miR-29a-3p(MIMAT0000086), hsa-miR-9-5p(MIMAT0000441), hsa-miR-155-5p(MIMAT0000646), hsa-miR-92a-3p(MIMAT 0000092), hsa-miR-103a-3p(MIMAT0000101), and hsa-miR-26a-5p(MIMAT0000082) in combination with 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),and 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), or RD group: may contain all of the miRNAs listed in Figures 5 to 10.

[0098] In another embodiment, the cell secretion product may contain, as a protein, the above-mentioned proteins of group PA, group PB, group PC, or group PD, and, as a miRNA, the above-mentioned miRNAs of group RA, group RB, group RC, or group RD. This allows the cell secretion product of one embodiment to be used as a pharmaceutical composition having known effects attributable to these proteins and miRNAs.

[0099] In one embodiment, the extracellular vesicles are vesicles comprising a lipid bilayer. The diameter of the extracellular vesicles is, for example, 50 nm to 5 μm, or 50 nm to 1000 nm. The diameter of exosomes, which are one type of extracellular vesicles, is, for example, 50 nm to 200 nm. Since exosomes contain various physiologically active substances such as proteins, nucleic acids, carbohydrates, and lipids, they are expected to be used in disease treatment and diagnosis methods, pharmaceuticals, cosmetics, and the like.

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

[0101] Exosomes can be isolated based on their molecular weight, size, shape, composition, or biological activity. Specifically, exosomes can be isolated by ultracentrifugation, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography (e.g., CIMmultus). TM EV separation (BIA separations), protein separation (e.g., MagCapture TM Isolation can be achieved by capturing and separating using the Exosome Isolation Kit PS (Fujifilm Wako Pure Chemical Industries, Ltd.), capturing and separating using antibodies, separating precipitates using polymers such as polyethylene glycol, etc. These methods can be performed alone or in combination.

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

[0103] To obtain highly pure exosomes, during exosome isolation or purification, the culture medium containing the obtained highly proliferative cells is typically replaced with a recovery medium, followed by additional culture. The recovery medium is specifically a medium for recovering culture supernatant. The additional culture refers to short-term culture in the recovery medium after culturing in the culture medium. The additional culture period 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. The additional culture reduces the contamination rate of exosomes derived from sources other than the highly proliferative cells and may be contained in the culture medium, thereby increasing the purity of exosomes derived from the desired highly proliferative cells.

[0104] Examples of recovery media include serum-free media and exosome-depleted media. Commercially available exosome-depleted media include FBS exosome-depleted, OneShot format (Gibco®, Thermo Fisher Scientific).

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

[0106] <5> Uses of hyperproliferative cells and cell secretions In one embodiment, cell secretions isolated from highly proliferative cells, such as exosomes isolated or purified as described above, are expected to have various effects on various cell types, such as neurite outgrowth inhibition, neurite outgrowth, neurite network formation, neuronal cell death prevention, and neuronal proliferation promotion. Based on these functions, they are expected to be useful as pharmaceutical compositions for the prevention or treatment of disorders related to peripheral neurons or central neurons. For example, the neurite outgrowth inhibitory effect of cell secretions isolated from highly proliferative cells can inhibit the differentiation of peripheral nerve cells from less differentiated cells into peripheral nerve cells, particularly sympathetic nerve cells. Therefore, the use of cell secretions according to the present disclosure on peripheral nerve cells is expected to suppress the peripheral nervous system, particularly the sympathetic nervous system. Furthermore, for example, the use of cell secretions isolated from highly proliferative cells on central nerve cells is expected to activate the central nervous system based on their neurite outgrowth effect, neurite network formation effect, neuronal cell death prevention effect, and neuronal proliferation promotion effect.

[0107] Therefore, the scope of the present disclosure also includes a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a cell secretion isolated from hyperproliferative cells and a pharmaceutically acceptable carrier, as well as a therapeutic or prophylactic method comprising administering the pharmaceutical composition to a subject. As used herein, the term "treatment" includes not only complete cure of a disorder, but also 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 or central nerve cells, for example, selected from the group consisting of cancer, pain, Alzheimer's disease, Parkinson's disease, depression, schizophrenia, and dementia. Disorders related to peripheral nerve cells, particularly sympathetic nerve cells, include, for example, cancer and pain related to sympathetic nerve cells. Disorders related to central nerve cells, for example, are 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 any known carrier in the art may be used, such as physiological saline, etc. Furthermore, in relation to the above-mentioned effects, methods for suppressing the sympathetic nervous system, methods for suppressing neurite outgrowth, etc., which include contacting a cell secretion, specifically an exosome, secreted from a highly proliferative cell with a nerve cell, for example, a sympathetic nerve cell, are also encompassed within the scope of the present disclosure.

[0110] In this specification, numerical ranges indicated using "to" indicate ranges that include the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In numerical ranges described in stages in this specification, the upper limit or lower limit of a numerical range in one stage can be arbitrarily combined with the upper limit or lower limit of a numerical range in another stage.

[0111] This disclosure is based on and benefits from the priority rights of the following Japanese patent applications, the entire contents of which 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 uses thereof."

[0112] All publications, patent applications, and technical standards mentioned in this disclosure are incorporated by reference in their entirety into this disclosure. [Example]

[0113] Example 1: Long-term culture of human astrocytes in inhibitor-supplemented medium Long-term culture of human astrocytes was performed using the materials, reagents, and culture products listed below.

[0114] <Material> ·Normal Human Astrocytes (NHA) (CC-2565, Lonza) <Reagents and culture products used> Culture medium: AGM Astrocyte Growth Medium Bullet Kit (CC-3186, Lonza) (AGM composition: basal medium, FBS (3% (v / v)), L-glutamine, ascorbic acid, hEGF, insulin, antibiotics), Inhibitor Y: CultureSure® Y-27632 (034-24024, Fujifilm Wako Pure Chemical Industries, Ltd.), final concentration: 10 μM Inhibitor A: CultureSure® A-83-01 (035-24113, Fujifilm Wako Pure Chemical Industries, Ltd.), 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.)), Cell culture dish 60mm (150462, Thermo Fisher Scientific), Cell culture dish 100mm (150466, Thermo Fisher Scientific), ·Cell culture dish 150mm (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 (100x) (17502-048, Gibco®, Thermo Fisher Scientific)

[0115] <Procedure for long-term culture> Long-term culture was performed according to the following procedure. A frozen tube of human astrocyte cells (NHA) was thawed in a 37°C water bath. Next, the lysed cells were transferred to a total of 10 mL of culture medium and centrifuged at 180 × g for 3 minutes at room temperature, after which the medium was removed. The cells were divided into four groups: a group with inhibitor Y only (hereinafter referred to as the NHA-Y group), a group with inhibitor A only (hereinafter referred to as the NHA-A group), a group with inhibitor Y and inhibitor A added (hereinafter referred to as the NHA-YA group), and a group without any addition (Normal group). They were again suspended in culture medium and plated at 4.2 × 10 cells per well into a 6-well plate (Cell Culture Multi-Dish 6 well (140675, Thermo Fisher Scientific; hereafter omitted). 3 cells / cm 2 The next day, the medium was replaced with culture medium containing (or not containing) each inhibitor (the concentration of each inhibitor was as described above) for each group as shown in Table 2, 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 with fresh culture medium containing (or not containing) the inhibitor. When the cells reached 80-90% confluence, they were detached using a cell detacher, suspended in culture medium containing (or not containing) the inhibitor, and plated at 4.2 × 10 cells per well in a 6-well plate. 3 cells / cm 2 The cells were cultured in the culture medium containing (or not containing) each inhibitor for 41 days with alternating passages.

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

[0118] [Table 1]

[0119] [Table 2]

[0120] As shown in Table 1, proliferation in the Normal group ceased 22 days after exposure to the inhibitor, whereas cell proliferation in the NHA-YA group continued for 37 days after exposure at maximum inhibition. Furthermore, as can be seen from Table 2, the number of cells obtained through proliferation in the NHA-YA group was approximately 400 times that of the Normal group. In all experimental groups, observations were continued until day 41 after the start of culture, with medium changes from the final passage day. Cell morphology on day 41 after the start of culture (magnified 100x) is shown in Figure 1. In Figure 1, "P" indicates the passage number; for example, "Y-P5" refers to a cell group of cells at passage 5 cultured in medium containing inhibitor Y.

[0121] Example 2: Recovery and identification of exosomes in culture supernatant <Recovery of culture supernatant from human astrocyte cells> According to the following procedure, human astrocyte cells were cultured in a YA-containing culture medium or an inhibitor-free medium, and the culture supernatant was collected.

[0122] (1) Culture supernatant collection of human astrocyte cells cultured in YA-containing culture medium Frozen human astrocyte cells were thawed and washed once with the medium in the same manner as in Example 1, and then the cells were resuspended in the culture medium and plated in a 60 mm cell culture dish at 3.5 × 10 3 cells / cm 2 The next day, the medium was replaced with YA-containing culture medium, and the culture was continued. When the cells reached 80-90% confluence, they were detached using a cell detacher, suspended in YA-containing culture medium, and plated at a density of 3.5 × 10 cells per 100 mm cell culture dish. 3 cells / cm 2 The cell group cultured in the YA-containing culture medium is referred to as the NHA-YA group, as in Example 1.

[0123] The NHA-YA group was subcultured five times and cultured in YA-containing culture medium for a total of 60 days, and a considerable number of cells were obtained.3 cells / cm 2 The number of cells seeded was 1.16 × 10 per dish. 6 Since six dishes were used, the total number of cells seeded was 6.96 × 10 6 It was a cell.

[0124] The next day, the medium was replaced from the YA-containing culture medium with a recovery medium at a volume of 20 ml / 150 mm dish, and additional culture was initiated. The recovery medium was the YA-containing culture medium minus FBS. Two days after the start of additional culture, the culture supernatant of the additional culture was collected from the dish and transferred to a tube. It was centrifuged at 2,000 × g for 10 minutes at 4°C, filtered through a 0.22 μm filter system, and stored at 4°C. After collecting the culture supernatant, the cells remaining on the dish were detached using a cell detachment agent, and the cell number was counted. The total number of cells obtained for the NHA-YA group was 5.10 × 10 6 The survival rate of the total number of cells seeded was 73.3%.

[0125] (2) Culture supernatant collection for human astrocyte cells cultured in inhibitor-free culture medium (control) Frozen human astrocyte cells were thawed and washed once with the medium in the same manner as in Example 1, and then the cells were resuspended in the culture medium and placed in a 60 mm cell culture dish at 3.5 × 10 3 cells / cm 2 The cell group cultured in the inhibitor-free culture medium is referred to as the Normal group, as in Example 1.

[0126] The normal group was subcultured twice and cultured in inhibitor-free culture medium for a total of 26 days. 3 cells / cm 2 The number of cells seeded was 1.16 × 10 per dish. 6 Since two dishes were used, the total number of cells seeded was 2.32 × 10 6 It was a cell.

[0127] The next day, the medium was replaced with a recovery medium (DMEM with N-2 Supplement) at a volume of 20 ml per 150 mm dish to initiate additional culture. Two days after the start of additional culture, the culture supernatant of the additional culture was collected from the dish and transferred to a tube. It was centrifuged at 2,000 × g for 10 minutes at 4°C, filtered through a 0.22 μm filter system, and stored at 4°C. After collecting the culture supernatant, the cells remaining on the dish were detached using a cell detacher, and the cell number was counted. The total number of cells obtained as the normal group was 8.25 × 10 5 The survival rate of the total number of cells was 35.6%. The supernatant was centrifuged at 2000 × g for 10 minutes at 4°C, filtered through a 0.22 μm filter system, and stored at 4°C. Figure 2 shows the cell morphology before and after replacing the medium with recovery medium.

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

[0129] [Table 3]

[0130] As is clear from Table 3 and FIG. 3B, multiple peaks were confirmed in the culture supernatant of the NHA-YA group at a particle size of 100 nm to 200 nm, and it was found that exosomes were included. 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. Thereafter, 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 group of astrocytes used as the starting material for the culture in 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 with 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) (purchased primary cells were put to sleep, expanded, frozen, and then re-cultured for 4 days. No subculture.) NHA-YA (purchased primary cells were put to sleep, expanded, and frozen, then put to sleep again and cultured in a medium containing YA for 28 days, then frozen, then put to sleep again and cultured in a medium containing YA for 14 days. The cells were passaged once during the 14-day culture period.

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

[0134] <Quantitative PCR> The detection primers prepared are shown in Table 4. These primers were used to examine the expression of ectodermal cell marker genes by quantitative PCR. RNA samples were prepared and reaction conditions were set according to the protocol provided with the product.

[0135] [Table 4]

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

[0137] The results of quantitative PCR to confirm the expression of the neuroepithelial cell markers Notch1, Nestin, and SOX2, as well as the radial glial cell marker Nestin, and the astrocyte markers GFAP and S100B, and the oligodendrocyte precursor cell marker NG2 are shown in Tables 5 and 6, respectively.

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

[0139] [Table 5]

[0140] [Table 6]

[0141] As shown in Tables 5 and 6, the resulting cell population (NHA-YA) showed higher expression of both the astrocyte markers GFAP and S100B and the oligodendrocyte precursor cell marker NG2 compared to the astrocytes used as the source material (NHA).

[0142] These results indicate that the resulting cell population (NHA-YA) possesses both the characteristics of mature astrocytes and the characteristics of precursor oligodendrocyte progenitor cells, and has a higher proliferation potential than astrocytes. NHA-YA can proliferate more rapidly than astrocytes. Furthermore, the use of NHA-YA is advantageous for obtaining a larger amount of exosomes in a shorter time.

[0143] Example 3-2: Confirmation of ectodermal cell markers by quantitative PCR (2) The expression levels of the following ectodermal cell marker genes were measured by quantitative PCR for the astrocyte cell population used as the starting material for the culture in Example 1 and the resulting ectodermal cell population, as in Example 3-1. Primers for detecting each marker gene were prepared based on the sequences registered under the accession numbers in the NCBI database RefSeq, listed 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~v5, vX1, vX2, vX3)(NM_004172) ·OLIG2(oligodendrocyte transcription factor 2)(NM_005806) ·PAX6(paired box 6)(v1~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~vX10)(NM_002442)

[0144] The prepared detection primers are shown in Table 7 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 were in accordance with the protocol attached to the product. As the measuring instrument for quantitative PCR, for Musashi1, a SimpliAmp Thermal Cycler (Applied Biosystems) or a CFX96 Touch Real-time PCR Detection system (Bio-Rad) was used, and for the other marker genes, the same measuring instrument as used in Example 3-1 was used.

[0145]

Table 7

[0146] The results are shown in Table 8 and Table 9. In Table 8 and Table 9, 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 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> The frozen tube containing the cell culture medium containing NHA was thawed in a water bath at 37°C. The lysed cells were then transferred to a total volume of 10 mL of YA-containing culture medium, centrifuged at 180 × g for 3 minutes at 25°C, and the medium was then removed. The resulting NHA was suspended in YA-containing culture medium and placed in a 6-well plate at 4.2 × 10 3 cells / cm 2 The cells were subcultured at a seeding density of 1000. The cells were cultured in YA-containing culture medium for 11 days with two alternate passages.

[0150] The medium was then replaced with 20 mL of recovery medium per 150 mm dish, and additional culture was performed at 37°C for 72 hours. The recovery medium was a medium containing exosome-depleted FBS [3% (v / v)] instead of the FBS in the YA-containing culture medium. The additional culture was performed at 1.16 × 10 5 After the additional culture, the culture supernatant was collected from the dish and transferred to a tube. After centrifugation at 2,000 × g for 10 minutes at 4°C, the culture supernatant was filtered through a 0.22 μm filter system to prepare an exosome-containing solution.

[0151] <Exosome proteome analysis 1> The exosome-containing solution obtained above was precipitated with trichloroacetic acid, followed by reduction at 35°C for 2 hours with the addition of 5 mM dithiothiazol in Tris buffer. The reduced sample was then added with 14 mM iodoacetamide in Tris buffer and incubated at 25°C for 30 minutes in the dark. The exosomes were then digested with trypsin at 37°C for 20 hours. The resulting exosome digest was subjected to solvent exchange using a cation exchange column, desalted, concentrated, and subjected to LC-MS / MS analysis. The HPLC system used was an EASY-nLC 1200 System (Thermo Fisher Scientific Inc., USA), and the column used was an EASY-Spray column, 15 cm x 75 μm ID, 3 μm particles, 100 Å pore size (Thermo Fisher Scientific Inc., USA). Based on the product ion measurement data of the spectra obtained by LC-MS / MS analysis, a database search was performed using the MASCOT server (https: / / www.matrixscience.com / help.html). The results were then compared with the following two databases, and a total of 1,798 protein fragments were detected from the exosome fraction and medium components. Human-derived proteins: SwissProt (20376 sequences) Bovine protein: UniProtKB (sequence number: 47043)

[0152] Of the 1,285 marker proteins detected on SwissProt in the exosome fraction, those with a quantitative value (number of detected spectra, corrected) of 5 or greater were selected, and 520 marker proteins were extracted. From these 520 extracted proteins, characteristic proteins were selected, and it was confirmed that the selected proteins were not components of the culture medium. If the selected proteins were bovine-derived proteins, the probability of identification as human-derived proteins was examined, and those with a high probability of identification were selected. The steps for selecting characteristic proteins are described below.

[0153] a: Selection step of proteins characteristic of the cerebrum (a1) The 520 proteins extracted above were searched in Tissue Enrich (https: / / tissueenrich.gdcb.iastate.edu), where the Human Protein Atlas dataset and all tissue-specific genes were searched; (a2) 22 genes specific to the cerebrum were selected; (a3) Of the 22 genes selected in (a2) above, 12 genes that were not detected in the culture medium samples were selected; (a4) Of the 12 genes selected in (a3) ​​above, 8 genes were selected that were likely to be human proteins; (a5) Of the eight genes selected in (a4) above, five genes highly associated with disease were selected. The proteins encoded by the five genes selected in steps (a1) to (a5) above are shown in FIG.

[0154] b: Selection step of proteins characteristic of membrane proteins, extracellular regions, etc. (b1) Of the 520 proteins extracted above, 409 proteins were selected as proteins categorized as “membrane” in the GO of the Scaffold Proteome viewer; (b2) Of the 520 proteins extracted above, 443 proteins were selected as proteins categorized as "extracellular region" in GO in the Scaffold Proteome viewer; (b3) Of the 520 proteins extracted above, 15 proteins were selected as proteins that were not categorized into either "membrane" or "extracellular region" in the GO of the 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 proteins 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 step of 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 sequences 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).​​​​​​ A cryotube containing a cell culture solution containing NHA was thawed in a 37°C water bath. Next, the thawed cells were transferred to a culture 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 a culture medium for culturing containing YA, and subcultured at a seeding density of 4.2×10 3 cells / cm 2 . Subculture was performed for 47 days in a culture medium for culturing containing YA with 4 passages.

[0158] Thereafter, the medium was changed from the culture medium for culturing containing YA to a recovery medium at a volume of 20 mL / 150 mm dish, and additional culture was performed at 37°C for 48 hours. The recovery medium used in Example 4 was used as the recovery medium. The additional culture was performed at a seeding density of 1.16×10 6 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.

[0159] <Extraction of RNA from NHA-YA-derived exosomes> 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. Next, RNA was extracted from the obtained exosome fraction using 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) on an Agilent 2100 Bioanalyzer. After quality check, an RNA library was prepared from the total RNA obtained above using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. mRNA-Seq analysis was performed using the prepared RNA library, detecting 6538 mRNAs.

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

[0162] The quality of the sequence library was checked using a High Sensitivity DNA kit (Agilent Technologies) and an Agilent 2100 Bioanalyzer.

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

[0164] The sequencing data was subjected to read quality assessment (FastQC) and then aligned (mapped) to the reference genome (Human hg38) using GeneGlobe:Data Analysis Center (QIAGEN). Expression levels were normalized using Trimmed Mean of M values ​​(TMM). An Excel file containing annotation information for each miRNA and a classification summary of small RNA composition was created (analysis tools: StrandNGS v4.0, R v3.6.2; annotation information: miRBase Release 21 compliant). Subsequently, differentially expressed genes were extracted, followed by GO analysis and pathway analysis based on target gene prediction.

[0165] <Selection of functional marker miRNAs> 1. miRNA as a marker for Parkinson's disease Based on the following literature, we selected 42 miRNAs whose expression is reduced in human brain tissues with Parkinson's disease. By administering exosomes containing these miRNAs to the cells of Parkinson's disease patients, the function of these miRNAs can be exogenously complemented in Parkinson's disease patients, and this is expected to have a therapeutic effect on Parkinson's disease. Literature: MicroRNAs in Parkinson's disease and emerging therapeutic targets. Neural Regeneration Research, 12(12), pp.1945-1959 (2017)

[0166] Next, we selected 16 miRNAs that were detected as exosomal miRNAs derived from NHA-YA. 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 / ), we selected miRNAs associated with the following proteins, which are known to be associated with proteins related to the pathology of Alzheimer's disease, particularly multiple proteins that contribute to the pharmacological actions of Alzheimer's disease therapeutics.

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

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

[0170] 2-2. miRNA as a marker for Alzheimer's disease (2) Using IMOTA, we identified 42 miRNAs associated with BACE1 (β-site APP cleaving enzyme) in the cerebral cortex. During Alzheimer's disease, BACE1 cleaves the N-terminal portion of APP to produce abnormal amyloid β protein.

[0171] Next, 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 a marker for Alzheimer's disease (3) Using IMOTA (https: / / ccb-web.cs.uni-saarland.de / imota / ), we selected 66 miRNAs associated with NMDA receptors (N-methyl-D-aspartate receptors) in the cerebral cortex. During the onset of Alzheimer's disease, abnormal proteins accumulate in the brain, causing excessive release of neuroexcitatory substances. These excitatory substances overactivate NMDA receptors, impairing neurotransmission and memory.

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

[0174] 2-4. miRNA as a marker for Alzheimer's disease (4) Using IMOTA (https: / / ccb-web.cs.uni-saarland.de / imota / ), we selected 58 miRNAs associated with glycogen synthase kinase-3β (GSK-3β) in the cerebral cortex. GSK-3β is an enzyme that phosphorylates various proteins and plays a variety of roles in maintaining cell life and regulating physiological functions by controlling multiple pathways. In Alzheimer's disease, it promotes the deposition of amyloid beta protein in the brain and the accumulation of tau protein in neurons, which in turn induces neuronal apoptosis.

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

[0176] 3. miRNAs as depression markers Based on the following literature, we selected 24 miRNAs whose expression levels are reduced in the plasma of individuals with depression. By administering exosomes containing these miRNAs to the cells of patients with depression, the function of these miRNAs can be exogenously complemented in patients with depression, which is expected to have a therapeutic effect on depression. 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 were selected from these and detected as exosomal miRNAs derived from NHA-YA. The 13 selected miRNAs are shown in Figure 10.

[0178] Example 6: Inhibition of neurite outgrowth in PC-12 cells by NHA-YA-derived exosomes To evaluate the functionality of NHA-YA-derived exosomes, we performed a neurite outgrowth inhibition test on PC-12 cells (RCB0009, RIKEN Bank), a cell line derived from rat adrenal pheochromocytoma. As a control, we used the exosomes derived from the NHA cells used in Example 1.

[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), Antibiotic: 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), Antibiotic: 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) (AGM composition: basal medium, FBS (3% (v / v)), L-glutamine, ascorbic acid, hEGF, insulin, antibiotics) (the same as used in Example 1), Inhibitor Y: CultureSure (registered trademark) Y-27632 (034-24024, Fujifilm Wako Pure Chemical Industries, Ltd.), final concentration: 10 μM (the same as used in Example 1), Inhibitor A: CultureSure® A-83-01 (035-24113, Fujifilm Wako Pure Chemical Industries, Ltd.), final concentration: 0.5 μM (the same as used in Example 1), 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), Culture supernatant collection medium: AGM Astrocyte Growth Medium Bullet Kit (CC-3186, Lonza) contains 3% (w / v) exosome-depleted bovine serum (A2720803; Gibco) instead of fetal bovine serum (FBS), a component of the kit. 96-well plate: Collagen I coated 96-well plate (4860-010, IWAKI), ·Cell culture flask T-75 (430641, Corning), Cell culture dish 100 mm: 150466, Thermo Fisher Scientific (same as used in Example 1), Cell culture dish 150 mm: 150468, Thermo Fisher Scientific (same as used in Example 1).

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

[0181] <Recovery and identification of exosomes in culture supernatant> Using the materials, reagents, and culture products listed below, human astrocyte cells were cultured in YA-containing culture medium or inhibitor-free medium, and the culture supernatant was collected. 1.Material The same material as used in Example 1 was used as the NHA.

[0182] 2. Collection of Human Astrocyte Cell Culture Supernatant The culture supernatant was collected according to the following procedure. A frozen tube of human astrocyte cells (NHA) was thawed in a 37°C water bath. The lysed cells were then transferred to a total of 10 mL of culture medium and centrifuged at 180 × g for 3 minutes at room temperature, after which the medium was removed. The cells were suspended in culture medium and placed in a T-75 culture flask (Corning) at 1.25 × 10 4 cells / cm 2 The cells were then cultured at 37°C for approximately 12 hours to allow the cells to adhere, after which the medium was replaced with either the "Inhibitor Y + Inhibitor A added group (hereafter referred to as NHA-YA group)" or the "No added group (NHA group)" culture medium (the concentrations of each inhibitor were as described above), and the culture was continued.

[0183] The medium was changed every 1 or 2 days with fresh culture medium containing (or not containing) the inhibitor. After 7 days of culture, the cells (passage number: 1) were detached using cell detachment agent 1 and suspended in either inhibitor-containing culture medium (YA-containing culture medium) or inhibitor-free culture medium. 3.3 × 10 cells were placed in a 150 mm cell culture dish. 4 cells / cm 2 After 9 days of culture, the cells were subcultured at a seeding density of 3.3–6.6 × 10 cells / ml onto a 150 mm cell culture dish in the same manner. 4 cells / cm 2 The cells were subcultured for 5 days at a seeding density of 100 μg / ml for 31 days in total. As in Example 1, the cell group cultured in the YA-containing culture medium is referred to as the NHA-YA group, and the cell group cultured in the inhibitor-free culture medium is referred to as the Normal group.

[0184] At the 6th passage, 3.3 × 10 cells of the NHA-YA group were cultured in five 150 mm cell culture dishes. 4 cells / cm 2 , Normal group 2.3 × 10 4 cells / cm 2 The total number of cells seeded was 5.0 × 10 in the NHA-YA group. 6 cells, 3.5 × 10 in the normal group 6 It was a cell.

[0185] After seeding, the cells were cultured for 4 days. On day 35 after induction (after exposure to the inhibitor), the cells were confirmed to have reached 40-60% confluence. The medium was then replaced with 20 mL of medium for culture supernatant collection at a volume of 20 mL per 150 mm dish, and additional culture was initiated. Forty-eight hours after the start of additional culture, the culture supernatant was collected from the dish and then filtered through a 0.22 μm filter system to remove cellular debris and obtain exosome-containing culture supernatant. The filtered exosome-containing culture supernatant was stored at 4°C. Fresh culture supernatant collection medium was then added to the cells remaining in the dish, and the cells were cultured for 48 hours. The same procedure was repeated three times, and approximately 300 mL of filtered exosome-containing culture supernatant was collected. On day 41 after induction, the culture supernatant was collected, and the cells remaining on the dish were detached using cell detachment agent 1 and counted. The total number of cells obtained was 4.42 × 10 in the NHA-YA group. 7 cells, 1.51 × 10 in the NHA group 7 The survival rate of the total number of cells seeded was 94.1% in the NHA-YA group and 84.0% in the Normal group.

[0186] As a control medium (CM) for assessing exosome function, 20 mL of culture supernatant recovery medium was added to a 150 mm cell culture dish without cells seeded therein, and the culture supernatant was recovered under the same conditions as during cell culture.

[0187] 3. Concentration of exosomes in culture supernatant 180 mL of the culture supernatant from the NHA-YA group, NHA group, and CM collected above was transferred to a tube and centrifuged at 250,000 × g for 70 minutes at 4°C using the ultracentrifuge. After removing the supernatant, phosphate buffered saline was added to the resulting precipitate to wash it, and then centrifuged at 250,000 × g for 70 minutes at 4°C. The supernatant was then removed to obtain a washed precipitate. The washed precipitate was resuspended in phosphate buffered saline to a volume approximately 1 / 1000 of the culture supernatant volume.

[0188] 4. Measurement of particle count and protein content in 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 the number of particles in the diluted solution was measured using a ZetaView X20 (Particle Metrix). The total number of particles in the suspension was calculated by multiplying the measured particle number by the dilution factor, i.e., 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 assay medium was subtracted from the measured value for each culture supernatant to calculate the number of particles in the culture supernatant. In addition, the absorbance at 280 nm was measured using a NanoDrop (Thermo Scientific) to determine the protein concentration in the suspension. The total amount of protein in the suspension was calculated by multiplying the measured protein concentration by the volume of the suspension. The measurement results are shown in Table 10. The notation "aE+b" in the "Total Particle Number" column in Table 10 represents a × 10 b As shown in Table 10, the NHA-YA group contained the highest amount of total protein, and the number of particles was also significantly higher than that of the control medium (CM).

[0189] [Table 10]

[0190] <Evaluation experiment protocol> The frozen tube of PC-12 cells was thawed in a water bath at 37°C. The thawed cells were then transferred to a growth medium in a total volume of 10 mL, centrifuged at 180 × g for 5 minutes at room temperature, and the medium was then removed. The cells were then suspended in the growth medium and placed in a 100 mm cell culture dish at approximately 5,300 cells / cm. 2 The cells were cultured at 37°C with medium changes every 2 or 3 days, and the cells were propagated with intermittent passages. The obtained PC-12 cells were detached using cell detachment agent 2, washed with assay medium, and then cultured at a density of 3.96 × 10 4 The suspended cells were resuspended at a density of 1.98 × 10 cells / mL per well in a 96-well plate. 3 cells / 50 μL medium, i.e., 6,000 cells / cm 2 The cells were seeded at a seeding density of 100 μg / cm² and allowed to adhere for approximately 12 hours, after which the medium was replaced with each of the evaluation samples listed above. They were then cultured at 37°C, and the state of the PC-12 cells was observed under a microscope (BZ-X710, KEYENCE) on days 3, 4, and 5 of culture, and images were taken. The length of the neurites and the number of cells in the images were measured using the Multi-point tool (cell number) and the Straight tool (neurite length) of the image analysis software Fiji (ImageJ2 ver. 2.3.0). Statistical processing was performed using Microsoft Excel.

[0191] As a result, in the PC-12 cell line, a sympathetic neuron, no significant neurite outgrowth was observed in the above (2) and (3) containing exosomes compared to (1), indicating that neurite outgrowth was suppressed. This suggests that the exosomes of the present disclosure do not have the function of extending neurites in PC-12 cells, but rather have the function of inhibiting neurite extension, thereby enabling the exosomes of the present disclosure to suppress the sympathetic nervous system.

Claims

1. A method for producing hyperproliferative cells, comprising: (i) providing a starting ectodermal cell; (ii) contacting the source ectodermal cells with a small molecule signaling pathway inhibitor in a medium containing the inhibitor for a period of more than 28 days; and (iii) after the contact, further culturing in a medium containing the inhibitor to obtain a culture containing highly proliferative cells having enhanced cell proliferation ability compared to the starting ectodermal cells, the raw material ectodermal cells are astrocytes, the inhibitor comprises 10 μM to 30 μM of Y27632 and 0.5 μM to 3 μM of A-83-01; A method for producing highly proliferative cells.

2. The method of claim 1, wherein the number of cells after contact with the inhibitor for a culture period of more than 28 days is more than 1.0 times the number of ectodermal cells cultured under the same culture conditions but without contact with the inhibitor for the same culture period.

3. The method according to claim 1 or 2, wherein ectodermal cells of the central nervous system are cultured together with the raw material ectodermal cells.

4. Culturing cells of an ectodermal cell lineage related to astrocytes together with the starting ectodermal cells; The cells of the ectodermal cell lineage include radial glial cells, oligodendrocyte precursor cells (also called polydendrocytes), oligodendrocytes, and / or neuroepithelial cells. The method according to claim 1 or 2.

5. The method of claim 1 or 2, wherein the inhibitor further comprises at least one compound selected from the group consisting of a TGFβ receptor inhibitor and a ROCK inhibitor.

6. The method according to claim 5, wherein the concentration of the TGFβ receptor inhibitor is in the range of 0.001 μM to 100 μM.

7. The method according to claim 5, wherein the concentration of the ROCK inhibitor is in the range of 0.001 μM to 100 μM.

8. The method of claim 1 or 2, wherein the highly proliferative cells express at least one gene specific to mature cells at the same or higher level as the starting ectodermal cells, and express at least one gene specific to progenitor cells at the same or higher level as the starting ectodermal cells.

9. The method of claim 1 or 2, wherein the highly proliferative cells are negative for at least one selected from the group consisting of Musashi1, Notch1, Nestin, and SOX2.

10. Obtaining highly proliferative cells by the production method according to claim 1 or 2, obtaining a culture containing cell secretions secreted from said hyperproliferative cells; and separating the cell secretions from the culture. Method for producing cell secretions.

11. The method of claim 10, wherein the cell secretion is an exosome.

12. A method for producing ectodermal progenitor cells, comprising: the ectodermal progenitor cells are hyperproliferative cells; (i) providing a starting ectodermal cell; (ii) contacting the source ectodermal cells with a small molecule signaling pathway inhibitor in a medium containing the inhibitor for a period of more than 28 days; and (iii) after the contact, further culturing in a medium containing the inhibitor to obtain a culture containing highly proliferative cells having enhanced cell proliferation ability compared to the starting ectodermal cells, the raw material ectodermal cells are astrocytes, the inhibitor comprises 10 μM to 30 μM of Y27632 and 0.5 μM to 3 μM of A-83-01; A method for producing ectodermal progenitor cells.

13. The method of claim 12, further comprising isolating the hyperproliferative cells from the culture.

14. The cell secretion A cell secretion containing a protein and / or miRNA, the proteins include a combination of vinculin (P18206), integrin beta-1, CD29 (P05556), pyruvate kinase M1 / 2 (P14618), and ephrin type-A receptor 2 (P29317); The miRNAs include 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 (MIMAT T0000737), 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), The method for producing cell secretions according to claim 10.

15. The method for producing a cell secretion product according to claim 14, wherein the cell secretion product is an exosome.

16. A cell secretion product is produced by the production method according to claim 10, producing a pharmaceutical composition containing the cell secretion for use in the prevention or treatment of disorders related to peripheral nerve cells or central nerve cells; Method for producing a pharmaceutical composition.

17. The pharmaceutical composition comprises: The method includes contacting the cell secretion with a neuron, Uses include suppression of the sympathetic nervous system, A method for producing the pharmaceutical composition of claim 16.

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