Highly proliferative cell, method for producing same, and use thereof

Abstract

Provided are: a highly proliferative cell having improved cell proliferation ability; and a method for producing the highly proliferative cell. A highly proliferative cell derived from ectoderm cells, which has the characteristics of ectoderm cells and exhibits the expression behavior of A1 described below. (A1) In the highly proliferative cells, the expression level (E1) of at least one marker gene selected from the group consisting of GFAP, S100B, Musashhi1, CSPG4, Netin, and SLC1A3 exhibits the following relative value (E1 / EC) with respect to the expression level (EC) of a control gene. GFAP (E1 / EC) gt; 0.2 part; s100B (E1 / EC) gt; 0.017%, 0.017%; musashhi1 (E1 / EC) gt; 0.15, 0.15; cSPG4 (E1 / EC) lt; 0.015% of the total weight; the Nestin (E1 / EC) gt is obtained; 0.6 part; sLC1A3 (E1 / EC) gt; and 0.09%. A highly proliferative cell having the characteristics of ectoderm cells, the number of cells after a culture period of 28 days or less in contact with a low-molecular-weight signaling pathway inhibitor is more than 1.0 times the number of ectoderm cells after the same culture period under the same culture conditions except for the same culture conditions in which the cells are not in contact with the low-molecular-weight signaling pathway inhibitor.

Description

Technical Field

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

[0002] In the field of regenerative medicine, various cells in a living body, cells at various differentiation stages, or cells induced to differentiate in a specific direction are used. Considering use in a living body, it is desirable that these cells are not accompanied by genetic modification. To date, it has been reported that by contacting a specific low-molecular compound with mature cells of the endoderm, the above-mentioned mature cells can be reprogrammed into stem cells or progenitor cells without genetic modification (Patent Document 1 and Patent Document 2).

[0003] Prior Art Documents

[0004] Patent Documents

[0005] Patent Document 1: International Publication No. 2017 / 119512

[0006] Patent Document 2: International Publication No. 2020 / 080550 Summary of the Invention

[0007] On the other hand, cells obtained without genetic modification, especially cells relatively close to mature cells, mostly have limited proliferative ability and tend to be unable to be maintained for a long time in vivo or in vitro. Therefore, there is still a need for highly proliferative cells that are effectively used for long-term utilization of cell functions or mass production of useful substances produced by cells. In particular, for ectodermal cells containing nervous system cells, unlike endodermal cells, they have not been fully utilized.

[0008] Problems to be Solved by the Invention

[0009] An object of the present disclosure is to provide highly proliferative cells, a method for producing highly proliferative cells, and uses thereof.

[0010] Means for Solving the Problems

[0011] The present inventors conducted intensive studies and as a result, obtained highly proliferative cells derived from ectodermal cells having specific properties.

[0012] That is, the present disclosure includes the following aspects.

[0013] [1] A highly proliferative cell derived from an ectodermal cell, which has the characteristics of an ectodermal cell and shows the expression behavior of A1 below.

[0014] (A1) In the above-mentioned highly proliferative cells, the expression level (E1) of at least one marker gene selected from the group consisting of GFAP, S100B, Musashi1, CSPG4, Nestin, and SLC1A3 and the expression level (E C ) The relative value (E1 / E C ) satisfies the reference range shown in Table 1 below.

[0015] [Table 1]

[0016] Marker gene Reference range GFAP ＞0.2 S100B ＞0.017 Musashi1 ＞0.15 CSPG4 ＜0.015 Nestin ＞0.6 SLC1A 3 ＞0.09

[0017] [2] A highly proliferative cell having the characteristics of ectodermal cells and having the following proliferative ability: the number of cells after a culture period of 28 days or less in contact with a low-molecular-weight signal transduction pathway inhibitor relative to the number of ectodermal cells cultured under the same culture conditions for the same culture period except without contact with the above inhibitor exceeds 1.0 times.

[0018] [3] The highly proliferative cell according to [1] or [2], wherein the highly proliferative cell contains cells in which the expression level of at least one marker gene selected from GFAP, SOX2, Musashi1, and Nestin is 1.0 times or more that of ectodermal cells cultured under the same culture conditions for the same culture period except without contact with a low-molecular-weight signal transduction pathway inhibitor.

[0019] [4] The highly proliferative cell according to any one of [1] to [3], wherein the highly proliferative cell contains ectodermal progenitor cells.

[0020] [5] The highly proliferative cell according to any one of [1] to [4], wherein the ectodermal cells contain astrocytes.

[0021] [6] A method for producing a highly proliferative cell, comprising:

[0022] (i) Culturing ectodermal cells as a raw material in the presence of a low-molecular-weight signal transduction pathway inhibitor to obtain highly proliferative cells with a higher cell proliferation ability than the above-mentioned ectodermal cells as a raw material,

[0023] In the above (i), the culture period in the presence of the inhibitor is 28 days or less.

[0024] [7] A method for producing a highly proliferative cell, comprising:

[0025] (i) Culturing ectodermal cells as a raw material in the presence of a low-molecular-weight signal transduction pathway inhibitor to obtain highly proliferative cells with a higher cell proliferation ability than the above-mentioned ectodermal cells as a raw material,

[0026] In (i) above, the culturing in the presence of the above inhibitor is carried out until at least one condition selected from the group consisting of condition a1, condition b1, and condition c1 is satisfied.

[0027] (a1) In the cells cultured in the presence of the above inhibitor, the expression level (E1) of at least one marker gene selected from the group consisting of GFAP, S100B, Musashi1, CSPG4, Nestin, and SLC1A3 and the expression level (E C ) of the control gene, the determined relative value (E1 / E C ) satisfies the reference range shown in Table 2 below.

[0028] [Table 2]

[0029] Marker gene Reference range GFAP ＞0.2 S100B ＞0.017 Musashi1 ＞0.15 CSPG4 ＜0.015 Nestin ＞0.6 SLC1A3 ＞0.09

[0030] (b1) The number of cells of the cells cultured in the presence of the above inhibitor is more than 1.0 times the number of cells of the ectodermal cells cultured under the same culture conditions for the same culture period except in the absence of the above inhibitor.

[0031] (c1) In the cells cultured in the presence of the above inhibitor, the expression level of at least one marker gene selected from GFAP, SOX2, Musashi1, and Nestin is 1.0 times or more that of the ectodermal cells cultured under the same culture conditions for the same culture period except in the absence of the above inhibitor.

[0032] [8] The method for producing highly proliferative cells according to [6] or [7], wherein the above-mentioned ectodermal cells used as the raw material contain cells of the central nervous system.

[0033] [9] The method for producing highly proliferative cells according to any one of [6] to [8], wherein the above-mentioned ectodermal cells used as the raw material contain astrocytes.

[0034]

[10] The method for producing highly proliferative cells according to any one of [6] to [9], wherein the above inhibitor contains at least one compound selected from the group consisting of TGFβ receptor inhibitors and ROCK inhibitors.

[0035]

[11] The method for producing highly proliferative cells according to any one of [6] to

[10] , wherein in (i) above, the culturing in the presence of the above inhibitor is carried out using a medium containing the above inhibitor, and the concentration of the above inhibitor in the medium is in the range of 0.001 μM to 100 μM.

[0036]

[12] A method for producing a cell secretion from highly proliferative cells, which comprises:

[0037] (I) Obtain a culture of highly proliferative cells derived from ectodermal cells as described in any one of [1] to [5], or obtain a culture of highly proliferative cells by implementing the production method described in any one of [6] to

[11] ; and

[0038] (II) Isolate the cell secretion secreted by the above highly proliferative cells from the above culture.

[0039]

[13] The method for producing a cell secretion as described in

[12] , wherein the cell secretion contains exosomes.

[0040]

[14] A cell secretion, which is a cell secretion containing at least one of a protein and an miRNA, wherein

[0041] the above protein contains a combination of glycoprotein M6B (Q13491), HLA class II histocompatibility antigen DRα chain (P01903), tweety homolog 1 (Q9H313), fibrillin 2 (P35556), HLA class II histocompatibility antigen DRB1β chain (P01911), and S100 calcium-binding protein A8 (P05109),

[0042] the above miRNA contains a combination of hsa-miR-206 (MIMAT0000462), hsa-miR-204-5p (MIMAT0000265), hsa-miR-128-3p (MIMAT0000424), hsa-miR-363-3p (MIMAT0000707), and hsa-miR-323a-3p (MIMAT0000755).

[0043] Effects of the Invention

[0044] According to the present disclosure, highly proliferative cells, a method for producing the same, and uses thereof can be provided. Brief Description of the Drawings

[0045] Figure 1-1 and Figure 1-2 shows the morphology of ectodermal cells after culturing in Example 1, Figure 1-1 and is a magnified view at 40 times.

[0046] Figure 1-1 and Figure 1-2 shows the morphology of ectodermal cells after culturing in Example 1, Figure 1-2 and is a magnified view at 100 times.

[0047] Figure 2 shows the proteins selected by proteomic analysis of NHA-YA-derived exosomes in Example 5.

[0048] Figure 3 Shows the proteins selected by proteomic analysis of NHA-YA-derived exosomes in Example 5.

[0049] Figure 4 Shows the proteins selected by proteomic analysis of NHA-YA-derived exosomes in Example 5.

[0050] Figure 5 Shows the miRNAs selected as Parkinson's disease markers by miRNA analysis of NHA-YA-derived exosomes in Example 6.

[0051] Figure 6 Shows the miRNAs selected as Alzheimer's disease markers by miRNA analysis of NHA-YA-derived exosomes in Example 6.

[0052] Figure 7 Shows the miRNAs selected as Alzheimer's disease markers by miRNA analysis of NHA-YA-derived exosomes in Example 6.

[0053] Figure 8 Shows the miRNAs selected as Alzheimer's disease markers by miRNA analysis of NHA-YA-derived exosomes in Example 6.

[0054] Figure 9 Shows the miRNAs selected as Alzheimer's disease markers by miRNA analysis of NHA-YA-derived exosomes in Example 6.

[0055] Figure 10 Shows the miRNAs selected as depression markers by miRNA analysis of NHA-YA-derived exosomes in Example 6.

[0056] Figure 11-1 and Figure 11-2 Shows the cell morphology of the NHA-YA group in Example 8, Figure 11-1 Shows the cell morphology of N-D9 (P3), YA-D9 (P3), N-D28 (P5), and YA-D28 (P5).

[0057] Figure 11-1 and Figure 11-2 Shows the cell morphology of the NHA-YA group in Example 8, Figure 11-2 Shows the morphology of N-D9 (P6), YA-D9 (P6), N-D28 (P8), and YA-D28 (P8).

[0058] Figure 12 Shows examples of miRNAs contained in the cell secretions of the present disclosure. Detailed implementation mode

[0059] Examples are given below to illustrate the present invention. It should be noted that as long as there is no special statement, the descriptions related to each invention can be cited from their respective descriptions.

[0060] In this specification, unless otherwise specified, the term "cell" is interpreted as a term including at least one cell. Therefore, unless otherwise specified, the cells referred to by the term "cell" are not limited to the case of a single cell, and can also be used in the same meaning as a cell population.

[0061] In this specification, unless otherwise specified, the term "cell population" is interpreted as a term including at least one type of cell. Therefore, unless otherwise specified, the cells referred to by the term "cell population" are not limited to the case of one type of cell.

[0062] "Ectoderm" is one of the three germ layers generated during the development of metazoans. The ectoderm comes from the outer layer of the embryo and develops into the skin, nervous system, sensory organs, etc. In this specification, as the skin, for example, the epidermis, hair, nails, and skin glands can be cited. As the nervous system, for example, the cranial nerves, spinal cord, and peripheral nerves can be cited. As the sensory organs, for example, the visual organ, auditory organ, balance organ, taste organ, olfactory organ, and tactile organ can be cited.

[0063] In this specification, unless otherwise specified, the term "Progenitor cell" refers to a cell at a differentiation stage before reaching a mature cell, and is a cell produced by a stem cell and capable of differentiating into the final differentiated cells that make up the body.

[0064] In this specification, the term "low-molecular-weight signaling pathway inhibitor" is sometimes also simply referred to as "inhibitor".

[0065] In this specification, the "expression level of a gene" can, for example, directly be the expression level of the gene itself, or indirectly be the expression level of the protein encoded by the gene. Therefore, in this specification, the expression of a gene can, for example, be replaced by the expression of the protein encoded by the gene.

[0066] <1>High-proliferation cells

[0067] As the high-proliferation cells of the present disclosure, the first high-proliferation cells or the second high-proliferation cells can be cited.

[0068] (First high-proliferation cells)

[0069] First, as described above, the first high-proliferation cells of the present disclosure are high-proliferation cells derived from ectodermal cells that possess the characteristics of ectodermal cells and exhibit the expression behavior of the following A1.

[0070] (A1) In the above highly proliferating cells, the expression level (E1) of at least one marker gene selected from the group consisting of GFAP, S100B, Musashi1, CSPG4, Nestin, and SLC1A3 and the expression level (E C ) of the control gene to determine the relative value (E1 / E C ) satisfy the reference range shown in Table 1 below.

[0071] [Table 1]

[0072] Marker gene Reference range GFAP ＞0.2 S100B ＞0.017 Musashi1 ＞0.15 CSPG4 ＜0.015 Nestin ＞0.6 SLC1A3 ＞0.09

[0073] In the present disclosure, the expression level (E1) of the above marker gene can be represented, for example, by the expression level of the protein encoded by the above marker gene, and the expression level (E C ) of the control gene can be represented, for example, by the expression level of the protein encoded by the above control gene. In addition, for example, when using the marker gene and when using the protein encoded by the marker gene, the relative value (E1 / E C ) can be the same value or different values.

[0074] In the present disclosure, highly proliferating cells (in this specification, sometimes referred to as "HP cells") refer to cells with high proliferation ability. Cells with high proliferation ability refer to, for example, cells having the following properties: compared with control cells, they have higher cell proliferation activity and the property of proliferating in a shorter time (that is, the property of having a shorter cell doubling time; the property of proliferating for a longer period; or satisfying both of these properties.

[0075] The highly proliferating cells of the present disclosure are highly proliferating cells derived from ectodermal cells and exhibit high cell proliferation ability. Therefore, the highly proliferating cells of the present disclosure can, for example, be cultured for a long time and the number of cells can be effectively increased by culturing. In addition, the highly proliferating cells of the present disclosure can, for example, generate cell secretions as described later, so that, for example, the recovery amount of the above cell secretions can be increased by obtaining a large number of cells.

[0076] In the highly proliferating cells of the present disclosure, as described above, "high proliferation ability" means higher proliferation ability compared with the above control cells. The above control cells are, for example, ectodermal cells that have not been contacted with a low-molecular-weight signal transduction pathway inhibitor. Here, the low-molecular-weight signal transduction pathway inhibitor mentioned in relation to the highly proliferating cells corresponds to the substances described in the manufacturing method described later.

[0077] In this specification, the "ectodermal cells not in contact with the low-molecular-weight signal transduction pathway inhibitor" refers to, for example, ectodermal cells not treated with the above-mentioned inhibitor, specifically, ectodermal cells not cultured in the presence of the above-mentioned inhibitor. In the method for producing highly proliferative cells of the present disclosure, as described below, the proliferative ability of the above-mentioned ectodermal cells is improved by culturing in the presence of the above-mentioned inhibitor. Therefore, the ectodermal cells not in contact with the above-mentioned inhibitor, that is, the ectodermal cells not treated with the above-mentioned inhibitor, can be referred to as cells in which the improvement of proliferative ability has not been induced, and in this specification, they are sometimes referred to as "ectodermal cells in which proliferation has not been induced".

[0078] The highly proliferative cells of the present disclosure can be obtained, for example, using ectodermal cells as a raw material and culturing them in the presence of the above-mentioned low-molecular-weight signal transduction pathway inhibitor. As a specific example, they can be obtained by the first production method or the second production method of the highly proliferative cells of the present disclosure described below (hereinafter also collectively referred to as "the method for producing the highly proliferative cells of the present disclosure"). In the method for producing the highly proliferative cells of the present disclosure, the ectodermal cells used as a raw material are sometimes referred to as "raw material cells" in this specification. The above-mentioned raw material cells are, for example, ectodermal cells not in contact with the above-mentioned inhibitor, that is, the above-mentioned ectodermal cells in which proliferation has not been induced. For example, when the highly proliferative cells of the present disclosure are obtained by the method for producing the highly proliferative cells of the present disclosure, the highly proliferative cells of the present disclosure can be said to be cells with a proliferative ability higher than that of the above-mentioned raw material cells (the above-mentioned ectodermal cells in which proliferation has not been induced). It should be noted that as long as the highly proliferative cells of the present disclosure exhibit the characteristics of the highly proliferative cells of the present disclosure shown below, they are not limited to the cells produced by the method for producing the highly proliferative cells of the present disclosure and can also be produced by other production methods.

[0079] Regarding the highly proliferative cells of the present disclosure, unless otherwise specified, the descriptions of the first production method or the second production method of the highly proliferative cells of the present disclosure described below can be cited. Specifically, in the description of the highly proliferative cells of the present disclosure, for example, the above-mentioned low-molecular-weight signal transduction pathway inhibitor, the above-mentioned ectodermal cells (raw material cells), etc. can cite the descriptions in the method for producing the highly proliferative cells of the present disclosure.

[0080] The highly proliferative cells of the present disclosure can be, for example, a cell population. The above cell population can, for example, contain only HP cells, or can contain, in addition to HP cells, other cells. The above other cells can be, for example, cells with a lower proliferative ability than HP cells, that is, non-highly proliferating cells (in this specification, sometimes referred to as "non-HP cells"). For example, in the case of obtaining the highly proliferative cells of the present disclosure by the method for manufacturing highly proliferative cells of the present disclosure, in the above cell population, in addition to HP cells, as the above other cells, for example, cells with no increased proliferative ability compared to the above raw material cells (the above undifferentiated ectodermal cells) and having the same proliferative ability as the above raw material cells can also be contained. The proportion of HP cells in the above cell population can be, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more based on the number of cells.

[0081] In the highly proliferative cells of the present disclosure, its characteristics can be represented by the relative value of the expression level (E1) of a marker gene relative to the expression level (E C ) of a control gene. Specifically, in the highly proliferative cells of the present disclosure, the relative value (E1 / E C ) of at least one marker gene selected from the group consisting of GFAP, S100B, Musashi1, CSPG4, Nestin, and SLC1A3 needs to satisfy the reference range of Table 1 above. It should be noted that CSPG4 is sometimes also referred to as NG2. The marker genes that satisfy the reference range of Table 1 above can be, for example, 2 or more, 3 or more, 4 or more, 5 or more, or all.

[0082] The reference range of the relative value (E1 / E C ) of each marker gene can be set, for example, based on the following criteria. The culture days (more than 28 days or 28 days or less) in the following (1) or (2) refer to the culture days of the above raw material cells (the above ectodermal cells) in the presence of the above small molecule signal transduction pathway inhibitor.

[0083] (1) In the culture for more than 28 days, outside the expression range of the above marker gene; and

[0084] (2) In the culture for 28 days or less, within the expression range of the above marker gene.

[0085] However, in the culture for 28 days or less, it is not necessary to exclude the expression range of the above marker gene in the culture for more than 28 days throughout the entire period.

[0086] In the highly proliferative cells of the present disclosure, for example, it is preferred that at least the relative value (E1 / E C)Meet the reference ranges in Table 1 above. Additionally, in the highly proliferative cells of the present disclosure, combinations of marker genes that meet the reference ranges in Table 1 above can be exemplified as follows:

[0087] A combination of GFAP and CSPG4;

[0088] A combination of GFAP, Musashi1, CSPG4, and SLC1A3;

[0089] A combination of GFAP, Musashi1, Nestin, CSPG4, and SLC1A3;

[0090] A combination of GFAP, Musashi1, and CSPG4;

[0091] A combination of GFAP, Nestin, CSPG4, and SLC1A3; or

[0092] A combination of GFAP, Musashi1, Nestin, S100B, CSPG4, and SLC1A3; and so on.

[0093] In Table 1 above, the reference range for SLC1A3 is ">0.09", preferably ">0.090". Additionally, in Table 1 above, the reference range for CSPG4 is "<0.015", preferably "<0.0150".

[0094] In the highly proliferative cells of the present disclosure, the expression behavior of A1 above further includes, for example, OLIG2 as the above marker gene, and the relative value (E1 / E C ) can meet the reference range of "<0.0001", and the above reference range is preferably "<0.00010".

[0095] In the highly proliferative cells of the present disclosure, for example, the relative value (E1 / E C ) of at least one of the above marker genes can meet the reference range of A1 above, and the relative value (E1 / E C ) of at least one of the above marker genes can meet the range of the lower limit and the upper limit in Table 3 below.

[0096] [Table 3]

[0097]

[0098] The above control gene is preferably a gene with constitutive expression regardless of cell type, and can be exemplified by β-actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The control gene in Table 1 above is, for example, GAPDH or ACTB, preferably GAPDH.

[0099] The highly proliferative cells of the present disclosure are, for example, cells showing high undifferentiatedness. Regarding the highly proliferative cells of the present disclosure, high undifferentiatedness means, for example, higher undifferentiatedness compared to the above-mentioned control cells. The above-mentioned control cells are, for example, ectodermal cells that have not been contacted with the above-mentioned low-molecular signaling pathway inhibitor, i.e., the above-mentioned non-induced proliferative ectodermal cells, in the same manner as above. For example, in the case of obtaining the highly proliferative cells of the present disclosure by the method for producing the highly proliferative cells of the present disclosure, the highly proliferative cells of the present disclosure can be said to be cells with improved undifferentiatedness compared to the above-mentioned raw material cells. In addition, unless otherwise stated, the "highly proliferative cells" in the present disclosure includes the meaning of highly proliferative cells with high undifferentiatedness. That the highly proliferative cells of the present disclosure are cells with high undifferentiatedness can be determined, for example, by the expression behavior of markers related to undifferentiation. The above-mentioned markers can be, for example, gene markers or protein markers encoded by the above-mentioned gene markers.

[0100] In this specification, undifferentiatedness means, for example, having the characteristics of cells at an intermediate stage before reaching the final stage of differentiation (in other words, the stage where differentiation no longer occurs). Or, undifferentiatedness means, for example, maintaining an undifferentiated state, and can mean showing undifferentiated properties (such as showing the properties of stem cells) or showing properties of a differentiation stage closer to undifferentiated (such as showing properties closer to stem cells).

[0101] As described above, the characteristics of the highly proliferative cells of the present disclosure can be determined in the form of the relative value (E1 / E C ) of the expression level of the above-mentioned marker gene relative to the above-mentioned control gene. In addition to this, for example, it can also be determined by comparing the same items with the above-mentioned control cells. The above-mentioned items (hereinafter also referred to as determination items) are, for example, the properties of cells. Specifically, for example, the above-mentioned proliferative ability, undifferentiatedness, expression behavior of markers, etc. The above-mentioned markers can be, for example, marker genes or proteins (marker proteins) encoded by the above-mentioned marker genes. The above-mentioned marker genes and the above-mentioned marker proteins are not limited to the above-mentioned genes and proteins, and in addition, can also include the markers exemplified below.

[0102] The comparison between the above-mentioned highly proliferative cells and the above-mentioned control cells can be evaluated as follows, for example, using ectodermal cells that have not been contacted with the above-mentioned low-molecular signaling pathway inhibitor (the above-mentioned non-induced proliferative ectodermal cells) as the above-mentioned control cells.

[0103] In the first mode of the above-mentioned evaluation, for example, the above-mentioned highly proliferative cells and the above-mentioned control cells (the above-mentioned non-induced proliferative ectodermal cells) are cultured separately in the absence of the above-mentioned inhibitor, and the same items are compared.

[0104] In the first method described above, there are no particular restrictions on the other culture conditions except for the absence of the above-mentioned inhibitor. Examples of the other culture conditions may include an inoculation density of 4×10 3 cells / cm 2 , a culture temperature of 37°C, a CO2 concentration of 5%, etc. In addition, the culture period (hereinafter also referred to as culture period S) is not particularly limited and is, for example, 5 to 24 days. The above-mentioned culture period may be the same or different according to the above items, and the same period is preferred. In addition, the culture medium used in the culture is not particularly limited, and the culture medium exemplified in the method for producing highly proliferative cells of the present disclosure described below can be used. In the culture of each of the above-mentioned highly proliferative cells and the above-mentioned control cells (the above-mentioned uninduced proliferative ectodermal cells), for example, the same culture medium without the addition of the above-mentioned inhibitor can be used.

[0105] Next, in the second method of the above evaluation, for example, in the above-mentioned highly proliferative cells obtained by culturing the above-mentioned raw material cells (the above-mentioned uninduced proliferative ectodermal cells) for a culture period of 28 days or less (hereinafter also referred to as culture period T) in the presence of the above-mentioned inhibitor, and the above-mentioned control cells (the above-mentioned uninduced proliferative ectodermal cells) cultured for the above-mentioned culture period T in the absence of the above-mentioned inhibitor, the same items are compared. The above-mentioned culture period T can, for example, refer to the examples in the method for producing highly proliferative cells of the present disclosure described below.

[0106] In the second method described above, there are no particular restrictions on the culture conditions other than the presence or absence of the above-mentioned inhibitor and the above-mentioned culture period T. Examples of the other culture conditions may include an inoculation density of 4×10 3 cells / cm 2 , a culture temperature of 37°C, a CO2 concentration of 5%, etc. The culture medium used in the culture is not particularly limited, and the culture medium exemplified in the method for producing highly proliferative cells of the present disclosure described below can be used. In the culture of the above-mentioned raw material cells (the above-mentioned uninduced proliferative ectodermal cells) in the presence of the above-mentioned inhibitor and the culture of the above-mentioned control cells (the above-mentioned uninduced proliferative ectodermal cells) in the absence of the above-mentioned inhibitor, for example, except for the presence or absence of the above-mentioned inhibitor, the same culture medium can be used, and it can be carried out under the same culture conditions as those applicable to the production and maintenance of the above-mentioned highly proliferative cells.

[0107] As a specific example, the proliferation ability of the above-mentioned highly proliferative cells can be evaluated as follows, for example, by comparing the cell numbers of the above-mentioned highly proliferative cells with those of the above-mentioned control cells. In the evaluation of the above-mentioned proliferation ability, as the above-mentioned control cells, as described above, ectodermal cells that have not been contacted with the above-mentioned low-molecular-weight signal transduction pathway inhibitor, that is, the above-mentioned non-induced proliferative ectodermal cells, can be used. In addition, when the highly proliferative cells of the present disclosure are produced by the method for producing highly proliferative cells of the present disclosure, the above-mentioned control cells can be the above-mentioned raw material cells (the above-mentioned non-induced proliferative ectodermal cells).

[0108] The highly proliferative cells of the present disclosure, for example, exhibit the following proliferation ability N1 with respect to the above-mentioned control cells, which are ectodermal cells (the above-mentioned non-induced proliferative ectodermal cells) that have not been contacted with the above-mentioned low-molecular-weight signal transduction pathway inhibitor. In N1, the culture period is not particularly limited, and for example, the number of days exemplified in the above-mentioned culture period S can be cited.

[0109] (N1) When the above-mentioned highly proliferative cells and the above-mentioned control cells are respectively cultured under the same culture conditions for the same culture period in the absence of the above-mentioned inhibitor, the cell number (N1) of the obtained above-mentioned highly proliferative cells and the cell number (N1') of the obtained above-mentioned control cells satisfy (N1 / N1') > 1.0.

[0110] In this method, the ratio (N1 / N1') of the cell number (N1) of the obtained above-mentioned highly proliferative cells to the cell number (N1') of the obtained above-mentioned control cells, for example, exceeds 1.0 times as described above, and can be 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 above ratio (N1 / N1') is preferably 1.5 times or more.

[0111] In this method, the first method of the above-mentioned evaluation can be referred to. In this case, the above-mentioned highly proliferative cells and the above-mentioned control cells are respectively cultured under the same culture conditions for the same culture period in the absence of the above-mentioned inhibitor. In addition to the presence or absence of the above-mentioned inhibitor and the item of the above-mentioned culture period, the items of further identical culture conditions are, for example, the composition of the medium without the above-mentioned inhibitor, the inoculation density, the culture temperature, the CO2 concentration, etc. The above-mentioned culture period is not particularly limited, and the number of days exemplified in the above-mentioned culture period S can be cited.

[0112] In addition, the highly proliferative cells of the present disclosure, for example, have the following proliferation ability N2 with respect to the above-mentioned control cells, and the above-mentioned control cells are ectodermal cells (the above-mentioned non-induced proliferative ectodermal cells) that have not been contacted with the above-mentioned inhibitor.

[0113] (N2) The number of cells (N2) of the above-mentioned highly proliferative cells obtained by culturing the above-mentioned raw material cells (the above-mentioned uninduced and proliferating ectodermal cells) for a culture period T of less than 28 days in the presence of the above-mentioned inhibitor satisfies (N2 / N2') > 1.0 with the number of cells (N2') of the above-mentioned control cells obtained by culturing under the same culture conditions for the above-mentioned culture period T except that the above-mentioned inhibitor is absent.

[0114] In this method, the ratio (N2 / N2') of the number of cells (N2) of the above-mentioned highly proliferative cells obtained to the number of cells (N2') of the above-mentioned control cells obtained is, for example, more than 1.0 times as described above, and can be 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 above ratio (N2 / N2') is preferably 1.5 times or more.

[0115] In this method, the second method of the above-mentioned evaluation can be referred to. In this case, regarding the above-mentioned culture period T, as described above, its upper limit is 28 days or less, for example, 20 days, 18 days, 14 days, and its lower limit is, for example, 1 day, 4 days, 5 days, 7 days, and its range is, for example, 1 - 28 days, 4 - 28 days, 5 - 28 days, 7 - 28 days, 7 - 20 days, 7 - 18 days, 7 - 14 days. In this method, for the above-mentioned raw material cells and the above-mentioned control cells, except that the former is in the presence of the above-mentioned inhibitor and the latter is in the absence of the above-mentioned inhibitor, they can be cultured under the same conditions for the same culture period T. In addition, except for the item of the above-mentioned culture period T, the items of the same culture conditions are, for example, the composition of the culture medium (excluding the presence or absence of the above-mentioned inhibitor), the inoculation density, the culture temperature, the CO2 concentration, etc.

[0116] As an evaluation method, for example, the following method can be cited: For the above-mentioned raw material cells (the above-mentioned uninduced and proliferating ectodermal cells), except for using a culture medium containing the above-mentioned inhibitor and a culture medium not containing the above-mentioned inhibitor, they are cultured under the same conditions for the same period (for example, 5 - 14 days or 5 - 10 days), and the number of cells of the cells cultured with the culture medium containing the above-mentioned inhibitor (that is, highly proliferative cells) is compared with the number of cells of the cells cultured with the culture medium not containing the above-mentioned inhibitor (that is, raw material cells). In addition, for example, the following method can also be: For the highly proliferative cells obtained by culturing the above-mentioned raw material cells with a culture medium containing the above-mentioned inhibitor for 28 days or less and the above-mentioned raw material cells (the above-mentioned uninduced and proliferating ectodermal cells), they are respectively cultured with a culture medium not containing the above-mentioned inhibitor under the same conditions for the same period (for example, 5 - 14 days or 5 - 10 days), and the number of cells of the former is compared with the number of cells of the latter.

[0117] In addition, as described above, the highly proliferative cells can be obtained, for example, by culturing the ectodermal cells in the presence of the above inhibitor for a culture period of 28 days or less (referred to as "period T" in this specification). The high proliferative ability of the highly proliferative cells thus obtained can be confirmed as follows. That is, when the number of cells of the highly proliferative cells obtained from the above culture (referred to as "the number of cells N of the highly proliferative cells" in this specification) exceeds 1.0 times the number of cells of the ectodermal cells cultured under the same culture conditions for the same culture period except that the above inhibitor is absent (referred to as "the number of cells N' of the ectodermal cells" in this specification), it is evaluated as having high proliferative ability.

[0118] In one embodiment, there is no particular limitation as long as the number of cells N of the highly proliferative cells exceeds 1.0 times the number of cells N' of the ectodermal cells, and for example, it can be 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 of the number of cells N'. Preferably, the number of cells N of the highly proliferative cells is 1.5 times or more of the number of cells N' of the ectodermal cells.

[0119] As described above, the highly proliferative cells can be determined by the expression of cell-specific markers, that is, markers specific to the highly proliferative cells (such as genes or proteins). The expression of the markers of the highly proliferative cells can be confirmed based on either the above marker gene or the marker protein encoded thereby as described above. The method for confirming the expression of a gene or protein is not particularly limited, and for example, it can be carried out by the presence or absence of expression, the specific expression level or relative expression level depending on the measurement method, comparison with the expression level of the same gene or protein in specific cells different from the above highly proliferative cells, etc.

[0120] In this specification, regarding the expression of the above markers (the above marker gene or the above marker protein), it is sometimes expressed as "positive" or "negative". Regarding the expression of a gene or protein, generally, "positive" means that the expression of the gene or protein as the object is observed, while "negative" means that the expression of the gene or protein as the object is not observed, or it is below the detection limit. Regarding the expression of a gene or protein, when expressed as "high expression" or "positive", or "low expression" or "negative", specific cells different from the above highly proliferative cells can be used as a comparison control. The specific cells used as the comparison control, that is, the "control cells" for comparison, are not particularly limited, and can be cells with extremely low expression levels of the gene or protein as the object (hereinafter also referred to as low-expression cells), or cells with extremely high expression levels of the same (hereinafter also referred to as high-expression cells). The above low-expression cells can be, for example, cells in which the expression level of the marker as the object is below the detection limit and cannot be detected.

[0121] The above-mentioned control cells for comparison with the above-mentioned highly proliferating cells can be, for example, ectodermal cells used as raw materials in the manufacturing method of the present disclosure, that is, ectodermal cells not in contact with the above-mentioned inhibitor (the above-mentioned non-induced proliferating ectodermal cells). In addition, the above-mentioned control cells can be, for example, mature cells or their progenitor cells. The above-mentioned control cells can be, for example, ectodermal mature cells (hereinafter also referred to as mature cells), or can also be progenitor cells (hereinafter also referred to as ectodermal progenitor cells).

[0122] In addition, the expression of the above-mentioned marker being "positive" for the highly proliferating cells of the present disclosure can mean that the expression level of the above-mentioned marker in the above-mentioned highly proliferating cells is 1.0 times or more of the expression level of the above-mentioned marker in the ectodermal cells not in contact with the above-mentioned inhibitor (the above-mentioned non-induced proliferating ectodermal cells). In contrast, the expression of the above-mentioned marker being "negative" for the above-mentioned highly proliferating cells can mean that the expression level of the above-mentioned marker in the above-mentioned highly proliferating cells is less than 1.0 times of the expression level of the above-mentioned marker in the ectodermal cells not in contact with the above-mentioned inhibitor (the above-mentioned non-induced proliferating ectodermal cells).

[0123] In this specification, regarding the expression of the above-mentioned marker, unless otherwise specified, "the same or more" means an expression level that is 1.0 times or more of the expression level in the control cells for comparison.

[0124] When the ectodermal cells not in contact with the above-mentioned inhibitor (the above-mentioned non-induced proliferating ectodermal cells) are used as the above-mentioned control cells and the highly proliferating cells are determined by comparing the expression of the above-mentioned marker, the above-mentioned comparison can be performed, for example, by the first method or the second method of the above-mentioned evaluation as described above.

[0125] That is, in the case of the first method of the above-mentioned evaluation, the above-mentioned highly proliferating cells and the above-mentioned control cells are cultured, for example, under the same conditions for the same culture period S in the absence of the above-mentioned inhibitor, and the expression of the above-mentioned marker as an evaluation item is compared. In addition to the item of the above-mentioned inhibitor, the items of further identical culture conditions are, for example, the composition of the culture medium, the inoculation density, the culture temperature, the CO2 concentration, etc.

[0126] In addition, in the case of the second method of the above evaluation, for the above-mentioned raw material cells (the above-mentioned uninduced and proliferated ectodermal cells) and the above-mentioned control cells (the above-mentioned uninduced and proliferated ectodermal cells), except that the former is in the presence of the above-mentioned inhibitor and the latter is in the absence of the above-mentioned inhibitor, they are cultured under the same conditions for the same culture period T, and the expression of the above-mentioned marker as an evaluation item is compared. In addition to the item of the above-mentioned culture period T, the items of further identical culture conditions are, for example, the composition of the culture medium (excluding the presence or absence of the above-mentioned inhibitor), the seeding density, the culture temperature, the CO2 concentration, etc.

[0127] It should be noted that in the second method of the above evaluation, when comparing the expression of the above-mentioned marker, regarding the culture conditions of the highly proliferating cells and the ectodermal cells for the method of confirming the expression level, except for contact or non-contact with the inhibitor, they can be set to the same conditions. The above-mentioned culture conditions can be adjusted, for example, based on the cell state, proliferation ability, and cell number, etc., for the culture period, the number of cell passages, and the cell density, etc., so that the cells to be targeted reach the same or extremely similar state, etc. Those skilled in the art can select the conditions based on the cell state.

[0128] The expression level of the above-mentioned marker is, for example, in the form of a relative value (E1 / E C ) of the expression level relative to the control, that is, the expression level (E1) of the marker relative to the expression level (E C ) of the control. The above-mentioned control is, for example, a gene or a protein. As a specific example, when the above-mentioned marker is a marker gene, the above-mentioned control is a gene; when the above-mentioned marker is a marker protein, the above-mentioned control is a protein. The above-mentioned control is preferably, for example, a gene or a protein that is constitutively expressed regardless of the cell type. As the above-mentioned control gene, for example, ACTB and GAPDH can be used as described above, and as the above-mentioned control protein, for example, ACTB protein and GAPDH protein can be used.

[0129] As markers related to undifferentiatedness (undifferentiatedness markers), for example, SOX2, Nestin, CSPG4, Musashi1 (MSI1), Notch1, PAX6, etc. can be cited. The undifferentiatedness of cells can be evaluated based on the expression behavior of these undifferentiatedness markers, for example, according to whether the expression is positive or the expression level is relatively high. Here, being positive for the above undifferentiatedness marker means, for example, that the expression level is 1.0-fold or more compared to the expression level in ectodermal cells not in contact with the above inhibitor (the above non-induced proliferating ectodermal cells). In the highly proliferating cells of the present disclosure, for example, at least one marker gene selected from the group consisting of SOX2, Nestin, CSPG4, Musashi1 (MSI1), Notch1, and PAX6 is positive. In the highly proliferating cells of the present disclosure, for example, two or more of SOX2, Nestin, CSPG4, Musashi1, Notch1, and PAX6 can be positive, or all can be positive.

[0130] In the highly proliferating cells of the present disclosure, for example, regarding the above undifferentiatedness marker, it can include at least one cell selected from the group consisting of the following (1) to (6). In the following examples, as described above, being positive means, for example, that the expression level is the same or higher than the expression level in ectodermal cells not in contact with the above inhibitor (the above non-induced proliferating ectodermal cells), and as a specific example, it is 1.0-fold or more.

[0131] (1) SOX2-positive cells, preferably further at least one selected from the group consisting of Musashi1, Nestin, Notch1, and S100B is positive and CSPG4 is negative SOX2-positive cells

[0132] (2) Musashi1-positive cells, preferably further at least one selected from the group consisting of SOX2, Nestin, Notch1, and S100B is positive and CSPG4 is negative Musashi1-positive cells

[0133] (3) Nestin-positive cells, preferably further at least one selected from the group consisting of SOX2, Musashi1, Notch1, and S100B is positive and CSPG4 is negative Nestin-positive cells

[0134] (4) Notch1-positive cells, preferably further at least one selected from the group consisting of SOX2, Musashi1, Nestin, and S100B is positive and CSPG4 is negative Notch1-positive cells

[0135] (5) S100B-positive cells, preferably S100B-positive cells that are further positive for at least one selected from the group consisting of SOX2, Musashi1, Nestin, and Notch1 and negative for CSPG4

[0136] (6) CSPG4-negative cells that are positive for at least one selected from the group consisting of SOX2, Musashi1, Nestin, Notch1, and S100B and negative for CSPG4

[0137] As described above, the highly proliferative cells of the present disclosure may be any cells having high proliferative ability derived from ectodermal cells, and their differentiation stage is not limited. The differentiation stage of the highly proliferative cells of the present disclosure can be determined, for example, by detecting markers specific to progenitor cells, markers specific to mature cells, or markers specific to the differentiation stage from progenitor cells to mature cells.

[0138] In one aspect, the highly proliferative cells of the present disclosure are, for example, cells at an immature differentiation stage. When the highly proliferative cells of the present disclosure exhibit high proliferation and are at an immature differentiation stage, they can be referred to as, for example, ectodermal progenitor cells exhibiting high proliferation, and are sometimes hereinafter also referred to as "highly proliferative ectodermal progenitor cells or highly proliferative progenitor cells". In addition, unless otherwise specified, the "highly proliferative cells" in the present disclosure include highly proliferative progenitor cells.

[0139] In one aspect, the highly proliferative cells of the present disclosure can, for example, express at least one marker specific to mature cells at the same or higher level compared to ectodermal cells not contacted with the above inhibitor (the above non-induced proliferative ectodermal cells), and can express at least one marker specific to progenitor cells at the same or higher level compared to ectodermal cells not contacted with the above inhibitor (the above non-induced proliferative ectodermal cells).

[0140] Marker genes specific to mature cells can be exemplified by GFAP, S100B, and SLC1A3. They are, for example, genes that are highly expressed in glial cells classified as ectodermal cells (as a specific example, there are astrocytes, etc.). For mature cells represented by ectodermal mature cells, these genes and the proteins they encode are known as markers indicating that they are mature cells.

[0141] Marker genes specific to progenitor cells can be exemplified by CSPG4, Notch1, Nestin, and SOX2, etc. They are, for example, genes that are highly expressed in oligodendrocyte precursor cells, neuroepithelial cells, radial glial cells, etc. For progenitor cells represented by ectodermal progenitor cells, these genes and the proteins they encode are known as markers indicating that they are progenitor cells.

[0142] As described above, the highly proliferative cells of the present disclosure may be any cells having high proliferative ability and showing the expression behavior of A1 described above. Regarding other markers (genes or proteins), they may be cells showing any expression profile, and may be a cell population containing the above highly proliferative cells. The highly proliferative cells of the present disclosure may, for example, show the expression profiles of the markers listed below. In addition to the expression behavior of A1 described above, the highly proliferative cells of the present disclosure can be quickly identified and extracted based on the expression profiles of the markers exemplified below.

[0143] The highly proliferative cells of the present disclosure may, for example, contain cells that are positive for GFAP. In this case, the above highly proliferative cells may, for example, express GFAP at the same level as or higher than the above control cells (the above non-induced proliferating ectodermal cells). As a specific example, the lower limit of the expression level of GFAP relative to the above control cells is, for example, 1.0-fold or more, 4.0-fold or more, and the upper limit is, for example, 200-fold or less.

[0144] In another embodiment, the above highly proliferative cells may, for example, contain cells that are positive for GFAP and negative for CSPG4, or may contain cells that express GFAP at the same level as or higher than the above control cells (the above non-induced proliferating ectodermal cells) and the expression level of CSPG4 is lower than that of the above control cells (less than 1.0-fold). In another embodiment, the above highly proliferative cells may contain cells that are positive for GFAP and S100B and negative for CSPG4, or may contain cells that express GFAP and S100B at the same level as or higher than the above control cells and the expression level of CSPG4 is lower than that of the above control cells (less than 1.0-fold).

[0145] In yet another embodiment, the above highly proliferative cells may, for example, contain cells that express at least one gene specific to the above mature ectodermal cells at the same level as or higher than the above control cells (ectodermal cells not contacted with the above inhibitor) and express at least one gene specific to the above ectodermal progenitor cells at the same level as or higher than the above control cells.

[0146] In one embodiment, the above highly proliferative cells may, for example, contain cells that are negative for CSPG4, or may contain cells in which the expression level of CSPG4 is lower than that of CSPG4 in the above control cells (ectodermal cells not contacted with the above inhibitor). That is, in this embodiment, the above highly proliferative cells may, for example, be cells in which the expression level of CSPG4 is lower than that of CSPG4 in the above control cells.

[0147] In one mode, the above-mentioned highly proliferative cells may include, for example, cells that are positive for S100B and negative for CSPG4, or may express S100B at the same level as or higher than that of the above-mentioned control cells (ectodermal cells not in contact with the above-mentioned inhibitor), and express CSPG4 less than the above-mentioned control cells.

[0148] The highly proliferative cells of the present disclosure only need to be highly proliferative cells with high proliferative ability, especially highly proliferative ectodermal cells. As described above, their differentiation stage is not restricted. The cell population of the above-mentioned highly proliferative cells may be, for example, a heterogeneous cell population containing both the above-mentioned highly proliferative mature ectodermal cells and the above-mentioned highly proliferative ectodermal progenitor cells, or may be a cell population in which two or more highly proliferative ectodermal cells with relatively close differentiation stages account for the vast majority.

[0149] In one mode, the cell population of the highly proliferative cells of the present disclosure may be, for example, a cell population containing the above-mentioned highly proliferative ectodermal progenitor cells as the main component, or may be a cell population containing both the above-mentioned highly proliferative ectodermal progenitor cells and the above-mentioned highly proliferative mature ectodermal cells. Here, the "main component" in the cell population refers to, for example, cells that account for at least 50% based on the number of cells in the cell population. In this specification, the "vast majority" refers to, for example, the number of specific cells in the cell population being 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more based on the number of cells.

[0150] The proportion of the above-mentioned highly proliferative mature ectodermal cells in the cell population of the above-mentioned highly proliferative cells may be less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% based on the number of cells.

[0151] The cell population of the above-mentioned highly proliferative cells may include, for example, cells of the ectodermal cell system related to astrocytes. That is, the above-mentioned highly proliferative cells may be, for example, cells formed from ectodermal cells using astrocytes as a constituent raw material, in other words, cells induced by astrocytes. When manufactured by the manufacturing method of the present disclosure, it may include cells of the ectodermal cell system related to astrocytes. Examples of the cells of the ectodermal cell system related to astrocytes may include astrocytes, radial glial cells, oligodendrocyte precursor cells (also called multipolar glial cells), oligodendrocytes, and neuroepithelial cells. In addition, the above-mentioned cell population containing the cells of the ectodermal cell system related to astrocytes only needs to be a cell population showing high proliferative ability, and may include, for example, cells having some characteristics observed in ectodermal cells and not classifiable as any of the above-mentioned cells.

[0152] In addition, in the manufacturing method of the present disclosure, when the ectodermal cells used as raw materials contain astrocytes (mature astrocytes) that have reached the final differentiation stage, for example, the highly proliferative cells of the present disclosure may contain cells that exhibit characteristics of an immature differentiation stage compared to astrocytes. Such ectodermal cells at the immature differentiation stage have a higher proliferative ability than astrocytes, and in addition, may further have the characteristics of the following (a), (b), (c), or (d).

[0153] (a) Compared with astrocytes,

[0154] the combined expression level of GFAP and S100B, GFAP and Notch1, GFAP and Nestin, GFAP and SOX2,

[0155] the combined expression level of S100B and Notch1, S100B and Nestin, S100B and SOX2,

[0156] the combined expression level of Notch1 and Nestin, Notch1 and SOX2, or

[0157] the combined expression level of Nestin and SOX2

[0158] increases.

[0159] (b) Compared with astrocytes,

[0160] the combined expression level of GFAP, S100B, and Notch1; the combined expression level of GFAP, S100B, and Nestin; the combined expression level of GFAP, S100B, and SOX2;

[0161] the combined expression level of GFAP, Notch1, and Nestin; the combined expression level of GFAP, Notch1, and SOX2;

[0162] the combined expression level of GFAP, Nestin, and SOX2;

[0163] the combined expression level of S100B, Notch1, and Nestin; the combined expression level of S100B, Notch1, and SOX2;

[0164] or the combined expression level of Notch1, Nestin, and SOX2 increases.

[0165] (c) Compared with astrocytes,

[0166] Combinations of GFAP, S100B, Notch1, and Nestin; combinations of GFAP, S100B, Notch1, and SOX2; combinations of GFAP, S100B, Nestin, and SOX2; combinations of GFAP, Notch1, Nestin, and SOX2;

[0167] or combinations of S100B, Notch1, Nestin, and SOX2

[0168] show increased expression levels.

[0169] (d) Compared with astrocytes,

[0170] combinations of GFAP, S100B, Notch1, Nestin, and SOX2 show increased expression levels.

[0171] As described above, the highly proliferative cells of the present disclosure can, for example, include cells that are positive for at least one selected from the group consisting of GFAP, S100B, Notch1, Nestin, and SOX2. The above-mentioned highly proliferative cells can, for example, include at least one selected from the group consisting of the following cells (I) to (V). It should be noted that in the following examples, positive cells can also be replaced with cells that express a marker gene at the same level or higher (e.g., 1.0-fold or higher) compared to the ectodermal cells not in contact with the above inhibitor. In the case of obtaining the highly proliferative cells of the present disclosure by the method for producing highly proliferative cells of the present disclosure described below, the "ectodermal cells not in contact with the above inhibitor" can be, for example, "ectodermal cells as raw materials".

[0172] (I-1) GFAP-positive cells;

[0173] (I-2) S100B-positive cells;

[0174] (I-3) Notch1-positive cells;

[0175] (I-4) Nestin-positive cells;

[0176] (I-5) SOX2-positive cells;

[0177] (II-1) Cells that are positive for GFAP and positive for S100B;

[0178] (II-2) Cells that are positive for GFAP and positive for Notch1;

[0179] (II-3) Cells that are positive for GFAP and positive for Nestin;

[0180] (II-4) Cells that are positive for GFAP and positive for SOX2;

[0181] (II-5) S100B is positive and Notch1 is positive;

[0182] (II-6) S100B is positive and Nestin is positive;

[0183] (II-7) S100B is positive and SOX2 is positive;

[0184] (II-8) Notch1 is positive and Nestin is positive;

[0185] (II-9) Notch1 is positive and SOX2 is positive;

[0186] (II-10) Nestin is positive and SOX2 is positive;

[0187] (III-1) Cells that are positive for GFAP, positive for S100B, and positive for Notch1;

[0188] (III-2) Cells that are positive for GFAP, positive for S100B, and positive for Nestin;

[0189] (III-3) Cells that are positive for GFAP, positive for S100B, and positive for SOX2;

[0190] (III-4) Cells that are positive for GFAP, positive for Notch1, and positive for Nestin;

[0191] (III-5) Cells that are positive for GFAP, positive for Notch1, and positive for SOX2;

[0192] (III-6) Cells that are positive for GFAP, positive for Nestin, and positive for SOX2;

[0193] (III-7) Cells that are positive for S100B, positive for Notch1, and positive for Nestin;

[0194] (III-8) Cells that are positive for S100B, positive for Notch1, and positive for SOX2;

[0195] (III-9) Cells that are positive for S100B, positive for Nestin, and positive for SOX2;

[0196] (III-10) Cells that are positive for Notch1, positive for Nestin, and positive for SOX2;

[0197] (IV-1) Cells that are positive for GFAP, positive for S100B, positive for Notch1, and positive for Nestin;

[0198] (IV-2) Cells that are positive for GFAP, positive for S100B, positive for Notch1, and positive for SOX2;

[0199] (IV-3) Cells that are positive for GFAP, positive for S100B, positive for Nestin, and positive for SOX2;

[0200] (IV-4) Cells that are positive for GFAP, positive for Notch1, positive for Nestin, and positive for SOX2;

[0201] (IV-5) Cells that are positive for S100B, positive for Notch1, positive for Nestin, and positive for SOX2;

[0202] (V-1) Cells that are positive for GFAP, positive for S100B, positive for Notch1, positive for Nestin, and positive for SOX2.

[0203] The highly proliferative cells of the present disclosure may include, for example, cells that are positive for at least one selected from the group consisting of GFAP and S100B, positive for at least one selected from Musashi1, Notch1, Nestin, and SOX2, and negative for CSPG4. That is, in this manner, among the cells of the above (I) to (V), for example, CSPG4 is negative.

[0204] In the highly proliferative cells of the present disclosure, for example, SLC1A3, which is a marker of astrocytes, may be positive. That is, in this manner, among the cells of the above (I) to (V), for example, SLC1A3 is further positive. In the highly proliferative cells of the present disclosure, for example, OLIG2, which is a marker of oligodendrocytes, may be negative. That is, in this manner, among the cells of the above (I) to (V), for example, OLIG2 is further negative.

[0205] In the cell population of highly proliferative cells of the present disclosure, the proportion of GFAP-positive cells is, for example, 10% or more, preferably 40% or more, 80% or more; the proportion of Musashi1-positive cells is, for example, 10% or more, preferably 40% or more, 80% or more; the proportion of SOX2-positive cells is, for example, 10% or more, 10% or more and less than 80%, preferably 40% or more, 40% or more and less than 80%; the proportion of Nestin-positive cells is, for example, 10% or more, 10% or more and less than 80%, preferably 40% or more, 40% or more and less than 80%. It should be noted that Musashi1-positive cells can be, for example, SOX2-positive, can be Nestin-positive; in addition, SOX2-positive cells can be, for example, Musashi1-positive, can be Nestin-positive; SOX2-positive cells can be, for example, Musashi1-positive, can be Nestin-positive.

[0206] (Second highly proliferative cell)

[0207] Next, the second highly proliferative cell of the present disclosure is the following highly proliferative cell, which has the characteristics of ectodermal cells and has the following proliferative ability: the number of cells after a culture period of 28 days or less when the ectodermal cells are brought into contact with the above-mentioned low-molecular signal transduction pathway inhibitor exceeds 1.0 times the number of ectodermal cells cultured under the same culture conditions for the same culture period except for not being brought into contact with the above-mentioned inhibitor.

[0208] The second highly proliferative cell of the present disclosure can be obtained in the same manner as the above-mentioned first highly proliferative cell, for example, by the method for producing the highly proliferative cell of the present disclosure. That is, for example, by culturing the ectodermal cells (the above-mentioned raw material cells) in contact with the above-mentioned low-molecular signal transduction pathway inhibitor for 28 days or less, the highly proliferative cell of the present disclosure can be obtained. And the highly proliferative cell obtained by the above-mentioned culture for 28 days or less in the presence of the above-mentioned inhibitor has the following proliferative ability: exceeding 1.0 times the number of ectodermal cells cultured under the same culture conditions for the same culture period except for not being brought into contact with the above-mentioned inhibitor.

[0209] All the descriptions in the above-mentioned first highly proliferative cell of the present disclosure can be cited as examples in the second highly proliferative cell of the present disclosure, for example.

[0210] The second highly proliferative cell of the present disclosure, for example, contains cells in which the expression level of at least one marker gene selected from GFAP, SOX2, Musashi1, and Nestin is 1.0 times or more that of the ectodermal cells cultured under the same culture conditions for the same culture period except for not being brought into contact with the above-mentioned inhibitor.

[0211] <2>First production method of highly proliferative cells

[0212] The first method for manufacturing highly proliferative cells according to the present disclosure is a method for manufacturing highly proliferative cells, which includes:

[0213] (i) Culturing ectodermal cells as a raw material in the presence of a low-molecular-weight signaling pathway inhibitor to obtain highly proliferative cells with a higher cell proliferation ability than the above-mentioned ectodermal cells as the raw material,

[0214] In the above (i), the culturing period in the presence of the inhibitor is 28 days or less.

[0215] According to the manufacturing method of the present disclosure, through the culturing in the presence of the above-mentioned low-molecular-weight signaling pathway inhibitor in the above (i), highly proliferative cells (the above-mentioned HP cells) with a higher cell proliferation ability than the above-mentioned ectodermal cells as the raw material can be obtained. That is, according to the manufacturing method of the present disclosure, the highly proliferative cells of the present disclosure described in the above <1> can be manufactured. It should be noted that the description of the manufacturing method of the present disclosure does not limit the manufacturing method of the highly proliferative cells of the present disclosure described in the above <1>.

[0216] The manufacturing method of the highly proliferative cells of the present disclosure can also be replaced, for example, with a method for manufacturing highly proliferative ectodermal cells or a method for enriching ectodermal cells with improved cell proliferation ability.

[0217] According to the manufacturing method of the present disclosure, as described above, by culturing ectodermal cells as a raw material in the presence of the above-mentioned low-molecular-weight signaling pathway inhibitor for a culturing period of 28 days or less, highly proliferative cells with improved cell proliferation ability can be obtained, for example, without gene introduction or genetic modification of the above-mentioned ectodermal cells. Compared with ectodermal cells cultured in the absence of the above-mentioned inhibitor, the highly proliferative cells obtained in this way can, for example, be cultured for a longer period and a larger number of cells can be obtained.

[0218] In addition, according to the manufacturing method of the present disclosure, through the culturing in the presence of the above-mentioned low-molecular-weight signaling pathway inhibitor in the above (i), highly proliferative cells with properties showing a higher cell proliferation ability than the above-mentioned ectodermal cells as the raw material and a higher undifferentiated state than the above-mentioned ectodermal cells as the raw material can be obtained, for example. Therefore, the manufacturing method of the highly proliferative cells of the present disclosure can also be replaced, for example, with a method for manufacturing highly proliferative cells with a high undifferentiated state. In addition, unless otherwise specified, the "highly proliferative cells" in the present disclosure include highly proliferative cells with a high undifferentiated state.

[0219] In addition, according to the manufacturing method of the present disclosure, through the cultivation in the presence of the above-mentioned low-molecular-weight signal transduction pathway inhibitor in (i) above, for example, highly proliferative cells with a cell proliferation ability higher than that of the above-mentioned ectodermal cells used as raw materials and showing characteristics of an immature differentiation stage can be obtained. Highly proliferative cells such as these that show high proliferative ability and are in an immature differentiation stage are, for example, the above-mentioned highly proliferative ectodermal progenitor cells (highly proliferative progenitor cells) exemplified in <1> above. Therefore, the method for manufacturing highly proliferative cells of the present disclosure can also be replaced with, for example, a method for manufacturing highly proliferative progenitor cells. In addition, unless otherwise specified, "highly proliferative cells" in the present disclosure includes highly proliferative progenitor cells.

[0220] The above-mentioned ectodermal cells used in the present disclosure, for example, contain cells of the nervous system. As a specific example, for example, they can contain cells of the central nervous system, can contain cells of the peripheral nervous system, or can also contain cells of the central nervous system and the peripheral nervous system. Therefore, in the present disclosure, the ectodermal cells can, for example, contain cells of the central nervous system. In the present disclosure, the ectodermal cells can, for example, contain glial cells. As a specific example, as described below, they can contain astrocytes.

[0221] The above-mentioned ectodermal cells used in the present disclosure can be provided from any source, for example, from a mammal. As a mammal, for example, a human or a non-human animal can be cited. Examples of the above-mentioned non-human animals include rats, mice, cats, dogs, guinea pigs, rabbits, sheep, horses, pigs, cows, and monkeys, etc. The mammal is preferably a human, a rat, a mouse, a cat, or a dog, and more preferably a human, a rat, or a mouse.

[0222] The above-mentioned ectodermal cells used in the present disclosure are, for example, cells that make up ectodermal tissues and ectodermal organs. As ectodermal tissues and ectodermal organs, for example, the central nervous system (such as the central nervous system of the brain or the central nervous system of the spinal cord), the pituitary gland, the peripheral nerves, the enteric nerves, the adrenal medulla, melanocytes, facial cartilage, dentin of teeth, the epidermis, hair, nails, skin, sebaceous glands, salivary glands, sweat glands, mammary glands, the nasal cavity, nasal mucosa, oral epithelium, the oral cavity, eyes, the bladder, the anus, etc. can be cited. The brain can be roughly divided into the cerebrum, cerebellum, and brainstem. The cerebrum can be further divided into the telencephalon and diencephalon, and the brainstem can be further divided into the midbrain, pons, and medulla oblongata. The central nervous system is composed of nerve cells (neurons) and glial cells. In the manufacturing method of the present disclosure, for example, these cells, that is, nerve cells, glial cells, or both, can be selected as raw material cells (also referred to as the starting material for cultivation). A large number of mature cells that have completed the final differentiation stage exist in the tissues or organs exemplified above.

[0223] The above-mentioned ectodermal cells used in the present disclosure are not particularly limited and are, for example, glial cells. As the above-mentioned glial cells, for example, at least one selected from the group consisting of astrocytes, microglia, oligodendrocytes, oligodendrocyte precursor cells (or also referred to as polydendrocytes), ependymal cells, Schwann cells, and satellite cells can be selected. The above-mentioned glial cells can be, for example, one type or a mixture of two or more types. These cells can be, for example, primary cells obtained from an organism, established cell lines, or ectodermal cells induced from pluripotent stem cells such as ES cells or induced pluripotent stem cells (iPS cells).

[0224] The above-mentioned ectodermal cells used in the present disclosure can be, for example, cells prepared from a brain sample collected from a mammal through separation and purification, etc. The collection of the brain sample from a mammal can be, for example, the removal of the whole brain or the excision of a brain tissue slice depending on the state of the mammal.

[0225] As a specific example, when the above-mentioned mammal is a rat, for example, it is preferable to use a brain sample removed from an adult rat aged 10 weeks to 20 weeks, and this is not limited thereto. For example, a brain sample from a juvenile rat less than 10 weeks old can also be used. The collection of the brain sample can be carried out, for example, by biopsy or by surgery. When the mammal is a human, the brain sample can be obtained, for example, by biopsy or surgery. For example, both nerve cells and glial cells can be obtained from a brain tissue slice collected by biopsy. In the case of surgery, the preparation of the above-mentioned ectodermal cells can use, for example, an excised adult brain tissue slice or a brain removed from a dead fetus. In addition, for example, nerve cells or glial cells can be isolated and purified from these collected brain samples and then frozen, and the frozen cells can be used as the above-mentioned ectodermal cells.

[0226] The above-mentioned ectodermal cells used in the present disclosure can be defined, for example, as cells showing the characteristics of ectodermal cells. As the characteristics of ectodermal cells, for example, the morphology of the cells, the expression of genes or proteins specific to the cells (sometimes referred to as "marker genes or marker proteins"), etc. can be cited. That the cell is the above-mentioned ectodermal cell can be confirmed by the presence of the expression of at least one marker gene selected from the group consisting of GFAP, S100B, and CSPG4, for example, at any stage of the differentiation stage. The expression of the marker gene of the above-mentioned ectodermal cells can be confirmed based on either genes or proteins. The confirmation of the expression of genes or proteins can be carried out by methods well known in the art, such as the presence or absence of expression, the specific expression level depending on the measurement method, and the comparison with the expression level of the same gene or protein in a specific cell.

[0227] It should be noted that in this specification, the expression of the above-mentioned markers can be confirmed using techniques well-known in the art. In the case of the above-mentioned marker genes, for example, it can be confirmed by measuring the gene expression level using quantitative PCR (sometimes referred to as "qPCR") and the like. In the case of the above-mentioned marker proteins, for example, it can be confirmed by measuring the protein amount using immunoassays such as ELISA and flow cytometry methods.

[0228] In the production method of the present disclosure, the above-mentioned ectodermal cells (raw material cells) used as raw materials in the production of the above-mentioned highly proliferative cells are as described above. The above-mentioned raw material cells are, for example, ectodermal cells that have not been contacted with the above-mentioned small molecule signal transduction pathway inhibitor before performing the above-mentioned (i) culture, that is, the above-mentioned uninduced proliferative ectodermal cells.

[0229] In the production method of the present disclosure, the prepared raw material cells (the above-mentioned uninduced proliferative ectodermal cells) are contacted with the above-mentioned small molecule signal transduction pathway inhibitor. The contact between the above-mentioned raw material cells and the above-mentioned inhibitor can be an in vitro contact. There is no particular limitation on the above-mentioned inhibitor that can be used in the production method of the present disclosure, as long as it is a small molecule inhibitor that inhibits the signal transduction pathway. As the above-mentioned inhibitor, for example, a transforming growth factor (TGF) β receptor inhibitor, a ROCK [Rho-associated protein kinase] inhibitor, and a glycogen synthase kinase 3 (GSK3) inhibitor can be cited. In the production method of the present disclosure, the above-mentioned inhibitor can, for example, use any one kind, or two or more kinds can be used in combination, and their combination has no particular limitation.

[0230] In the production method of the present disclosure, the above-mentioned inhibitor can, for example, contain 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 of the present disclosure, the above-mentioned inhibitor can, for example, contain at least one compound selected from the group consisting of a TGFβ receptor inhibitor and a ROCK inhibitor.

[0231] The TGFβ receptor inhibitor is not particularly limited as long as it has the function of inhibiting the TGFβ receptor. Examples of the above-mentioned TGFβ receptor inhibitor 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-phenylthio carbamoyl-1H-pyrazole (A-83-01), [2-(5-chloro-2-fluorophenyl)-4-(4-pyridylamino)]pteridine (SD-208), 3-(pyridin-2-yl)-4-(4-quinolyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (all of the above are purchased from Merck), SB431542 (Sigma Aldrich), and CultureSure (registered trademark) A-83-01 (FUJIFILM Wako Pure Chemical Corporation). The TGFβ receptor inhibitor is preferably CultureSure (registered trademark) A-83-01. The TGFβ receptor inhibitor also includes, for example, a TGFβ receptor antagonist. The TGFβ receptor inhibitor can be used alone or in combination of two or more.

[0232] The ROCK inhibitor is not particularly limited as long as it has the function of inhibiting Rho-associated protein kinase. Examples of the above-mentioned ROCK inhibitor include GSK269962A (Axon medchem), Fasudil hydrochloride (Tocris Bioscience), CultureSure (registered trademark) Y-27632 (FUJIFILM Wako Pure Chemical Corporation), and H-1152 dihydrochloride (FUJIFILM Wako Pure Chemical Corporation). The ROCK inhibitor is preferably CultureSure (registered trademark) Y-27632. The ROCK inhibitor can be used alone or in combination of two or more.

[0233] The GSK3 inhibitor is not particularly limited as long as it has the function of inhibiting glycogen synthase kinase (GSK) 3. Examples of the above-mentioned GSK3 inhibitor include SB216763 (Selleck), CHIR 98014 (Axon medchem), CHIR 99021 (Axon medchem), SB415286 (Tocris Bioscience), and Kenpaullone (Cosmo Bio). The GSK3 inhibitor is preferably CHIR 99021. The GSK3 inhibitor can be used alone or in combination of two or more.

[0234] The above-mentioned low-molecular-weight signaling pathway inhibitor used in the manufacturing method of the present disclosure can be, for example, at least one selected from the group consisting of a TGFβ receptor inhibitor and a ROCK inhibitor. That is, the above-mentioned inhibitor used in the manufacturing method of the present disclosure can be, for example:

[0235] · A combination of a TGFβ receptor inhibitor and a ROCK inhibitor;

[0236] · A TGFβ receptor inhibitor; or

[0237] · A ROCK inhibitor.

[0238] In one embodiment of the present disclosure, the above-mentioned inhibitor used is a combination of a TGFβ receptor inhibitor and a ROCK inhibitor.

[0239] In one embodiment of the present disclosure, for example, as the above-mentioned inhibitor, the following inhibitors are included:

[0240] (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-quinolyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, SB431542, and CultureSure (registered trademark) A-83-01;

[0241] (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-quinolyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, SB431542, and CultureSure (registered trademark) A-83-01 and

[0242] A combination of at least one compound selected from the group consisting of GSK269962A, Fasudil hydrochloride, CultureSure® Y-27632, and H-1152 dihydrochloride; and

[0243] (3) at least one compound selected from the group consisting of GSK269962A, Fasudil hydrochloride, CultureSure® Y-27632, and H-1152 dihydrochloride.

[0244] In the production method of the present disclosure, the above-mentioned raw material cells (the above-mentioned uninduced proliferating ectodermal cells) are cultured in the presence of the above-mentioned inhibitor. For example, the above-mentioned culture can be carried out using a culture medium containing the above-mentioned inhibitor, and the above-mentioned inhibitor can be contacted with the above-mentioned raw material cells in the above-mentioned culture medium for culturing. The concentration of the above-mentioned inhibitor in the above-mentioned culture medium is not particularly limited, and the following concentrations can be exemplified, for example. That is, the lower limit of the concentration of the above-mentioned inhibitor in the above-mentioned culture medium is, for example, 0.001 μM, 0.05 μM, and the upper limit is, for example, 30 μM, 50 μM, 80 μM, 100 μM, and the range thereof is, for example, 0.001 μM to 100 μM, 0.01 μM to 80 μM, 0.01 μM to 50 μM, 0.01 to 30 μM, or 0.05 μM to 30 μM. The concentration of the above-mentioned inhibitor can be appropriately adjusted according to the type of the above-mentioned inhibitor used. In this specification, "M" of the concentration can be replaced with "mol / L".

[0245] The exemplified concentration range of the above-mentioned inhibitor is applicable, for example, when using one inhibitor alone or when using two or more inhibitors in combination. The above-mentioned inhibitor can be directly added to the culture medium, or can be added to the culture medium after being dissolved in a solvent to reach the above-mentioned final concentration. Examples of the above-mentioned solvent include aqueous solvents and organic solvents. Examples of the aqueous solvent include water, buffer solutions, physiological saline, etc. When the above-mentioned inhibitor is water-insoluble or hardly water-soluble, for example, an organic solvent is used. The above-mentioned organic solvent is preferably low-toxic, and as a specific example, DMSO, etc. can be used, and the above-mentioned inhibitor is preferably dissolved in a small amount of the above-mentioned organic solvent.

[0246] When the above-mentioned inhibitor is a TGFβ receptor inhibitor, the concentration of the TGFβ receptor inhibitor in the culture medium is, for example, in the range of 0.001 μM to 100 μM, 0.01 μM to 50 μM, or 0.05 μM to 30 μM, and can be appropriately adjusted according to the type of the TGFβ receptor used. In one embodiment, the concentration of the TGFβ receptor inhibitor in the culture medium, for example, CultureSure® A-83-01, is 0.1 μM to 3 μM.

[0247] When the above-mentioned inhibitor is a ROCK inhibitor, the concentration of the ROCK inhibitor in the medium is, for example, in the range of 0.001 μM to 100 μM, 0.01 μM to 80 μM, or 0.1 μM to 50 μM, and can be appropriately adjusted according to the type of the ROCK inhibitor used. In one embodiment, the concentration of the ROCK inhibitor in the medium, such as CultureSure (registered trademark) Y-27632, is 1 μM to 30 μM.

[0248] When the above-mentioned inhibitor is a GSK3 inhibitor, the concentration of the GSK3 inhibitor in the medium is, for example, in the range of 0.001 μM to 100 μM, 0.01 μM to 80 μM, or 0.1 μM to 50 μM, and can be appropriately adjusted according to the type of the GSK3 inhibitor used.

[0249] The above-exemplified concentration ranges are applicable, for example, when the TGFβ receptor inhibitor, the ROCK inhibitor, and the GSK3 inhibitor are used alone, and when these inhibitors are used in combination. In addition, when the above-mentioned inhibitor is water-insoluble or hardly water-soluble, as described above, it can be dissolved in a small amount of a low-toxic organic solvent (such as DMSO, etc.) and then added to the medium to achieve the above-mentioned final concentration.

[0250] As described above, the production method of the present disclosure performs the culture of the above-mentioned starting material cells (the above-mentioned uninduced and proliferating ectodermal cells) in the presence of the above-mentioned inhibitor. In the above-mentioned culture, a medium containing the above-mentioned inhibitor can be used, and the above-mentioned starting material cells can be cultured in a state of being in contact with the above-mentioned inhibitor in the above-mentioned medium. Specifically, it is preferable to add the above-mentioned inhibitor to the medium at the exemplified concentration and perform the culture of the above-mentioned starting material cells. By culturing the above-mentioned starting material cells in the presence of the above-mentioned inhibitor, highly proliferative cells derived from the above-mentioned ectodermal cells with improved cell proliferation ability compared to the above-mentioned starting material cells (the above-mentioned uninduced and proliferating ectodermal cells) at the start stage of the culture can be obtained.

[0251] In the production method of the present disclosure, the above-mentioned raw material cells (the above-mentioned uninduced and proliferated ectodermal cells) can be prepared and directly cultured in the presence of the above-mentioned inhibitor, or can be cultured first in the absence of the above-mentioned inhibitor and then in the presence of the above-mentioned inhibitor. In the latter case, the culture of the above-mentioned raw material cells in the absence of the above-mentioned inhibitor, that is, the culture before contacting with the above-mentioned inhibitor, is also called "pre-culture" or "first culture". And, the culture of the above-mentioned raw material cells in the presence of the above-mentioned inhibitor, that is, the culture in the state of contacting with the above-mentioned inhibitor, is also called "main culture" or "second culture". It should be noted that the above-mentioned main culture is a culture for a specified time in the presence of the above-mentioned inhibitor. Since the above-mentioned culture medium contains the above-mentioned inhibitor, during the above-mentioned main culture, the cells are continuously in contact with the inhibitor.

[0252] In the present disclosure, the culture medium used in the pre-culture and the main culture is not particularly limited. A culture medium widely used for culturing animal cells can be used as the basal medium, or a commercially available basal medium can be used. As the commercially available basal medium, for example, Astrocyte Growth Medium (AGM), Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), RPMI1640 medium, Medium 199, Ham's F12 medium, William's E medium, and NS basal medium (FUJIFILM Wako Pure Chemical Corporation) etc. can be cited, but it is not particularly limited to these. As the culture medium, the above-mentioned culture medium can be used alone, or two or more of them can be used in combination. For the pre-culture and the main culture, for example, the same culture medium can be used, or different culture media can be used.

[0253] The above-mentioned culture medium can further contain additives, for example. As the additives added to the culture medium, for example, cytokines, growth factors (such as epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2)), hormones (such as insulin, estradiol, progesterone, testosterone, and thyroxine), steroids (such as dexamethasone (Dex)), plasma-derived proteins (such as transferrin), various amino acids (such as L-glutamine and L-proline), various inorganic salts (such as selenite and NaHCO3), various vitamins (such as nicotinamide and ascorbic acid derivatives), N2 supplement (FUJIFILM Wako Pure Chemical Corporation), NS supplement (FUJIFILM Wako Pure Chemical Corporation), B-27Plus supplement (ThermoFisher Scientific), various antibiotics (such as penicillin and streptomycin), antifungal agents (such as amphotericin), and buffers (such as Good's buffers such as HEPES) can be cited.

[0254] For the pre-culture (first culture) medium and the main culture (second culture) medium, for example, either a serum-supplemented medium or a serum-free medium can be used respectively.

[0255] In the case of using a serum-supplemented medium, as the serum, for example, fetal bovine serum (FBS) can be used. In addition, as described below, in the case of isolating extracellular vesicles (such as exosomes) as cell secretions from cultured cells, for example, to facilitate exosome isolation, FBS from which exosomes have been removed can also be used. As a commercially available exosome-depleted medium, for example, disposable format exosome-depleted FBS (FBS exosome-depleted, OneShot format) (Gibco (registered trademark), Thermo Fisher Scientific) can be cited. The serum concentration in the medium is, for example, 0.5 to 25% (v / v), 1 to 25% (v / v), 1 to 20% (v / v), 1 to 15% (v / v), 2 to 15% (v / v), 2 to 10% (v / v), 3 to 10% (v / v), 3 to 8% (v / v), and 3 to 5% (v / v). In a specific embodiment, the serum concentration in the medium is, for example, 3% (v / v).

[0256] In the case of using a serum-free medium, a serum substitute can be added to the above medium. As the serum substitute, for example, bovine serum albumin (BSA), KnockOut Serum Replacement (KSR), human serum albumin (HAS or Human albumin serum; HAS), etc. can be cited. As human serum albumin, for example, human serum albumin isolated from human plasma or human serum albumin purified from rice expressing the human serum albumin gene (FUJIFILM Wako Pure Chemical Corporation) can be used. In the case of a serum-free medium, factors such as growth factors (hEGH, etc.), cytokines, and hormones are usually further added. As these added factors, for example, epidermal growth factor (EGF), insulin, transferrin, hydrocortisone 21-succinate or its salt, and dexamethasone (Dex), N2 supplement (FUJIFILM Wako Pure Chemical Corporation), NS supplement (FUJIFILM Wako Pure Chemical Corporation), and B-27 serum-free supplement (Thermo Fisher Scientific) can be cited, but are not limited to these.

[0257] There are no particular limitations on the culture vessel used in the culture. For example, a vessel suitable for adherent culture can be used, such as a petri dish, a tissue culture dish, a multi-well culture dish, a microtiter plate, a microwell plate, a multi-purpose plate, a multi-well plate, a chamber culture slide, a flat dish, a culture tube, a culture tray, and a culture bag. In the case of adherent culture, for example, a vessel whose inner surface is coated with a cell-supporting matrix to improve cell adhesion can also be used. As the above-mentioned cell-supporting matrix, for example, collagen, gelatin, Matrigel, poly-L-lysine, laminin, and fibronectin can be cited. It is preferred that the cell-supporting matrix be collagen or Matrigel. In addition, for example, in the case of suspension culture of cells, as the culture vessel, a culture vessel whose surface is processed so that cells cannot adhere can also be used.

[0258] In the manufacturing method of the present disclosure, there are no particular limitations on the inoculation conditions of the above-mentioned raw material cells (the above-mentioned uninduced and proliferated ectodermal cells), such as the cell density to be inoculated. As a specific example, the above-mentioned raw material cells can be inoculated on the culture vessel at a cell density of, 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 .

[0259] The culture conditions of the above-mentioned raw material cells can be directly applied to the conditions generally used for culturing ectodermal cells. The culture can be carried out using a CO2 incubator. There are no particular limitations on the culture temperature and CO2 concentration, as long as they are the generally used culture temperature and CO2 concentration. For example, they can be 37°C and 5% (v / v).

[0260] In the production method of the present disclosure, the culture period (culture period T) of the above-mentioned main culture is a period during which the culture is carried out in the presence of the above-mentioned inhibitor, that is, together with the above-mentioned inhibitor. The above-mentioned culture period T can be, for example, a continuous period or a discontinuous period, that is, it can be a period composed of the sum of a plurality of discontinuous periods. The upper limit of the culture period T of the above-mentioned main culture is 28 days, and can be, for example, 20 days, 18 days, or 14 days. The lower limit of the culture period T of the above-mentioned main culture can be, for example, 1 day, 4 days, 5 days, or 7 days. The range of the culture period T of the above-mentioned main culture can be, for example, 1 to 28 days, 4 to 28 days, 5 to 28 days, 7 to 28 days, 7 to 20 days, 7 to 18 days, or 7 to 14 days. In the above-mentioned main culture, the passage of the cells being cultured is not particularly limited, and can be appropriately carried out, for example, according to the cell density in the culture vessel, the state of the culture medium, or the state of the cells, etc. The passage interval can be, for example, about 2 to 20 days.

[0261] In the production method of the present disclosure, when pre-culture is carried out before the main culture, the culture period in the absence of the above-mentioned inhibitor is not particularly limited.

[0262] The production method of the highly proliferative cells of the present disclosure can further include the following (ii) after the main culture of the above-mentioned (i), for example.

[0263] (ii) Separating the above-mentioned highly proliferative cells from the culture obtained by the culture in the presence of the low molecular weight signal transduction pathway inhibitor of the above-mentioned (i)

[0264] According to the production method of the present disclosure, the above-mentioned highly proliferative cells are obtained through the above-mentioned main culture. And, the culture of the above-mentioned main culture contains a cell population containing the above-mentioned highly proliferative cells. Therefore, in the production method of the present disclosure, for example, the above-mentioned highly proliferative cells can be further separated from the above-mentioned culture. The separation of the above-mentioned highly proliferative cells can be, for example, a separation in the form of a cell population containing the above-mentioned highly proliferative cells, or a separation and purification of the above-mentioned highly proliferative cells.

[0265] In the separation of the above-mentioned (ii), the separation of the above-mentioned highly proliferative cells can be, for example, the application of known means for separating specific cells. The above-mentioned separation method can be, for example, the fluorescence-activated cell sorting (FACS) method based on the expression of proteins from the above-mentioned marker genes specific to the above-mentioned highly proliferative cells, and the magnetic cell separation method using magnetic beads (Dynabeads), etc.

[0266] According to the manufacturing method of the present disclosure, as described above, the highly proliferative cells of the present disclosure can be obtained. Moreover, the characteristics of the highly proliferative cells obtained by the manufacturing method of the present disclosure can refer to the characteristics of the highly proliferative cells described in <1> above. It should be noted that the characteristics of the highly proliferative cells obtained by the manufacturing method of the present disclosure can be represented, for example, by comparison with the above-mentioned ectodermal cells used as raw materials. The "ectodermal cells used as raw materials" for comparison with the above-mentioned highly proliferative cells can be, for example, the above-mentioned control cells described in <1> above, specifically, the ectodermal cells that have not been contacted with the above-mentioned inhibitor, that is, the above-mentioned non-induced proliferative ectodermal cells. In addition, the "ectodermal cells used as raw materials" for comparison with the above-mentioned highly proliferative cells are, for example, the ectodermal cells that have not undergone the above-mentioned main culture in the manufacturing method of the present disclosure, that is, the ectodermal cells that have not been cultured in the presence of the above-mentioned inhibitor, and are equivalent to the above-mentioned non-induced proliferative ectodermal cells. The "ectodermal cells used as raw materials" for comparison with the above-mentioned highly proliferative cells are not limited to the ectodermal cells actually used as raw materials in the production of the above-mentioned highly proliferative cells, as long as they are ectodermal cells that can be used as raw materials in the manufacturing method of the present disclosure and have not been contacted with the above-mentioned inhibitor (the above-mentioned non-induced proliferative ectodermal cells). Therefore, in this specification, unless otherwise specified, the "ectodermal cells used as raw materials" in the comparison with the above-mentioned highly proliferative cells do not limit whether they have actually been used as raw materials in the production of the above-mentioned highly proliferative cells, but refer to ectodermal cells that can be used as raw materials (the above-mentioned non-induced proliferative ectodermal cells).

[0267] As described above, the evaluation of the proliferation ability of the above-mentioned highly proliferative cells obtained by the manufacturing method of the present disclosure can refer to the method described in the highly proliferative cells of the present disclosure above. In addition, regarding the proliferation ability of the above-mentioned highly proliferative cells obtained by the above-mentioned manufacturing method of the present disclosure, as described above, for example, regarding the cell number of the above-mentioned highly proliferative cells (the above-mentioned N1, N2, or N), the ratio (the above-mentioned N1 / N1', N2 / N2', or N / N') of the cell number of the above-mentioned highly proliferative cells (the above-mentioned N1, N2, or N) to the cell number of the above-mentioned control cells (the above-mentioned N1', N2', or N') is greater than 1.0 times, and can 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 above-mentioned control cells are, for example, the above-mentioned non-induced proliferative ectodermal cells as described above.

[0268] The highly proliferative cells obtained by the manufacturing method of the present disclosure can be determined, for example, by markers specific to the cells as described above. In the present disclosure, the types of markers of the above-mentioned highly proliferative cells, the method for confirming the expression of the markers, the expression behavior, etc. can refer to the description in the highly proliferative cells of the present disclosure in <1> above.

[0269] In the manufacturing method of the present disclosure, when the ectodermal cells as raw materials are astrocytes, for example, the cell population of the above-mentioned highly proliferative cells may include cells of the ectodermal cell system related to astrocytes. Examples of the cells of the above-mentioned ectodermal cell system related to astrocytes may include astrocytes, radial glial cells, oligodendrocyte precursor cells (also referred to as polydendrocytes), oligodendrocytes, and neuroepithelial cells. In addition, the cell population of the above-mentioned ectodermal cell system related to astrocytes may be, for example, a cell population showing improved cell proliferation ability, and may also include cells having some characteristics observed in ectodermal cells and not classifiable into any of the above-mentioned cells.

[0270] In one mode, the manufacturing method of the present disclosure uses, for example, primary human mature astrocytes as the above-mentioned raw material cells, and uses a combination of a TGFβ receptor inhibitor and a ROCK inhibitor as the above-mentioned inhibitor. Compared with the cells obtained by culturing the above-mentioned raw material cells in the absence of the above-mentioned inhibitor (also referred to as the inhibitor-untreated cell group), the above-mentioned highly proliferative cells (also referred to as the inhibitor-treated group) obtained in this mode can be cultured for a longer period, for example. That is, in the highly proliferative cells (inhibitor-treated group) obtained in this mode, for example, cell proliferation can be continuously carried out for a longer period compared with the above-mentioned inhibitor-untreated group. In addition, regarding the number of cells finally obtained by proliferation, the number of cells in the above-mentioned inhibitor-treated group is, for example, 2 times or more, 5 times or more, 10 times or more, 50 times or more, 100 times or more, 200 times or more, or 400 times the number of cells in the above-mentioned inhibitor-untreated group.

[0271] <3>Second manufacturing method of highly proliferative cells

[0272] The second manufacturing method of the highly proliferative cells of the present disclosure is a manufacturing method of highly proliferative cells, which includes:

[0273] (i) Culturing ectodermal cells as raw materials in the presence of a low-molecular-weight signal transduction pathway inhibitor, thereby obtaining highly proliferative cells with a cell proliferation ability higher than that of the above-mentioned ectodermal cells as raw materials,

[0274] In the above (i), the culturing in the presence of the above-mentioned inhibitor is carried out until at least one condition selected from the group consisting of condition a1, condition b1, and condition c1 is satisfied.

[0275] (a1) In the cells cultured in the presence of the above-mentioned inhibitor, the relative value (E1 / E determined by the expression level (E1) of at least one marker gene selected from the group consisting of GFAP, S100B, Musashi1, CSPG4, Nestin, and SLC1A3 and the expression level (E C ) of the control geneC )Meet the reference ranges shown in Table 2 below.

[0276] [Table 2]

[0277] Marker gene Reference range GFAP ＞0.2 S100B ＞0.017 Musashi1 ＞0.15 CSPG4 ＜0.015 Nestin ＞0.6 SLC1A3 ＞0.09

[0278] (b1) The number of cells of the cells cultured in the presence of the above inhibitor is more than 1.0 times the number of cells of the ectodermal cells cultured under the same culture conditions for the same culture period except in the absence of the above inhibitor.

[0279] (c1) In the cells cultured in the presence of the above inhibitor, the expression level of at least one marker gene selected from GFAP, SOX2, Musashi1, and Nestin is 1.0 times or more that of the ectodermal cells cultured under the same culture conditions for the same culture period except in the absence of the above inhibitor.

[0280] According to the second production method of the present disclosure, through the culture in the presence of the above-mentioned low-molecular-weight signaling pathway inhibitor in (i) above, highly proliferative cells (the above-mentioned HP cells) with a cell proliferation ability higher than that of the above-mentioned ectodermal cells used as raw materials can be obtained in the same manner as the first production method of the present disclosure. That is, according to the production method of the present disclosure, the highly proliferative cells of the present disclosure described in <1> above can be produced. It should be noted that the description of the production method of the present disclosure does not limit the production method of the highly proliferative cells of the present disclosure described in <1> above.

[0281] In the second production method of the present disclosure, it is preferable that the culture in the presence of the inhibitor in (i) above is carried out until at least condition a1 and condition b1 are satisfied, or at least condition b1 and condition c1 are satisfied.

[0282] The culture of the above-mentioned raw material cells (ectodermal cells) in (i) above in the presence of the inhibitor is carried out until the cells in the culture satisfy condition b1 or condition c1. When judging condition b1 or condition c1, the number of cells of the above-mentioned raw material cells (ectodermal cells) cultured in the absence of the above inhibitor or the expression level of the above marker gene can be obtained, for example, by culturing the above-mentioned raw material cells in the absence of the above inhibitor in parallel with the culture of the above-mentioned raw material cells in the presence of the inhibitor in (i). In addition, the raw material cells (ectodermal cells) used in (i) can be cultured separately in the absence of the above inhibitor to obtain information on the number of cells or the expression level of the above marker gene.

[0283] In the second production method of the present disclosure, unless otherwise specified, for example, the descriptions in the production methods of the highly proliferative cells of the present disclosure in <1> above and the second highly proliferative cells of the present disclosure in <2> above can be cited.

[0284] The present disclosure may include, for example, a method for evaluating highly proliferative cells. According to the evaluation method of the present disclosure, it is possible to evaluate whether the cells to be evaluated are the highly proliferative cells of the present disclosure. Each of the following steps in the evaluation method of the present disclosure may refer to the description in the method for producing highly proliferative cells of the present disclosure, for example.

[0285] That is, the method for evaluating highly proliferative cells of the present disclosure includes:

[0286] Preparing ectodermal cells as raw materials;

[0287] Culturing the above-mentioned ectodermal cells as raw materials in the presence of the above-mentioned low-molecular-weight signal transduction pathway inhibitor;

[0288] When the cells in the culture satisfy at least one of the conditions selected from the group consisting of condition a1, condition b1, and condition c1, the above-mentioned cells are evaluated as highly proliferative cells with a cell proliferation ability higher than that of the above-mentioned ectodermal cells used as raw materials.

[0289] (a1) In the cells cultured in the presence of the above-mentioned inhibitor, the relative value (E1 / E C ) determined by the expression level (E1) of at least one marker gene selected from the group consisting of GFAP, S100B, Musashi1, CSPG4, Nestin, and SLC1A3 and the expression level of the control gene (E C ) satisfies the reference range shown in Table 2 above.

[0290] (b1) The number of cells of the cells cultured in the presence of the above-mentioned inhibitor exceeds 1.0 times the number of cells of the ectodermal cells cultured under the same culture conditions for the same culture period except in the absence of the above-mentioned inhibitor.

[0291] (c1) In the cells cultured in the presence of the above-mentioned inhibitor, the expression level of at least one marker gene selected from GFAP, SOX2, Musashi1, and Nestin is 1.0 times or more that of the ectodermal cells cultured under the same culture conditions for the same culture period except in the absence of the above-mentioned inhibitor.

[0292] In the method for evaluating highly proliferative cells of the present disclosure, for example, when the cells in the above-mentioned culture further satisfy the expression of various markers exemplified in the highly proliferative cells of the present disclosure in <1> above, they can be evaluated as the above-mentioned highly proliferative cells.

[0293] <4> Uses of highly proliferative ectodermal progenitor cells

[0294] As the highly proliferative cells of the present disclosure, for example, as shown in <1> above, the highly proliferative ectodermal precursor (the highly proliferative progenitor cell) can be cited. The highly proliferative progenitor cell can be used for the following uses, for example. It should be noted that the present disclosure is not restricted by any of these uses.

[0295] The ectodermal progenitor cells of the present disclosure can be used, for example, in an evaluation method of a therapeutic agent for a nerve disorder. Specifically, for example, the usefulness of a candidate agent for a therapeutic agent for a nerve disorder can be evaluated by bringing the candidate agent into contact with the above-mentioned ectodermal progenitor cells. Therefore, the present disclosure relates to an evaluation method of a candidate agent for a therapeutic agent for a nerve disorder, which includes bringing a candidate agent for a therapeutic agent for a nerve disorder into contact with the above-mentioned ectodermal progenitor cells of the present disclosure. According to the evaluation method of the present disclosure, for example, if the function reduced due to damage of a damaged nerve (for example, cells damaged by corticosterone) is restored by the contact with the above-mentioned candidate agent, the above-mentioned candidate agent can be evaluated as useful.

[0296] The present disclosure further relates to an evaluation method of a nerve disease model, which includes the use of the above-mentioned highly proliferative ectodermal progenitor cells. For example, in the case of identifying a gene related to a nerve disease, it includes: artificially creating a mouse with a mutation in the gene, administering the above-mentioned highly proliferative ectodermal progenitor cells to the gene-mutated mouse, and evaluating nerve functions corresponding to the nerve disease, behaviors of the mouse, etc.

[0297] The present disclosure further relates to a method for producing ectodermal mature cells, which includes using the above-mentioned highly proliferative ectodermal progenitor cells and obtaining ectodermal mature cells by inducing the differentiation of the above-mentioned highly proliferative ectodermal progenitor cells. The conditions for the differentiation induction are not particularly limited, and common differentiation induction methods can be adopted. For example, the above-mentioned highly proliferative ectodermal progenitor cells can be cultured under maturation conditions that induce their differentiation into mature cells.

[0298] Here, "under maturation conditions" is not particularly limited and refers to culture conditions using known differentiation-inducing factors. As the above-mentioned differentiation-inducing factors, for example, fibroblast growth factor 2 (FGF-2); leukemia inhibitory factor (LIF); or IL-6 family cytokines such as ciliary neurotrophic factor (CNTF); bone morphogenetic protein (BMP) family cytokines such as BMP2 or BMP4, Notch family proteins; Wnt gene family proteins; sonic hedgehog protein; and retinoic acid can be cited. In addition, as factors required for the proliferation or maintenance of mature cells, for example, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 / 5 (NT-4 / 5), and platelet-derived growth factor (PDGF) can be cited.

[0299] As the culture conditions using the above-mentioned differentiation-inducing factors, for example, known differentiation-inducing conditions applied in the differentiation-inducing method based on the differentiation-inducing factors can be selected, and the above-mentioned differentiation-inducing conditions can be appropriately selected according to the type of differentiation-inducing factor used.

[0300] <5>Use of inhibitors

[0301] The low-molecular-weight signaling pathway inhibitor used in the manufacturing method of the present disclosure can be used as described above to maintain or promote the proliferation ability of ectodermal cells. Therefore, another aspect of the present disclosure relates to a proliferation regulator for ectodermal cells, which contains the above-mentioned low-molecular-weight signaling pathway inhibitor. Specifically, the present disclosure also relates to a proliferation regulator that contains the above-mentioned low-molecular-weight signaling pathway inhibitor and is used to maintain or promote the proliferation ability of the above-mentioned highly proliferative cells or ectodermal cells. The above inhibitor can, for example, be the inhibitor exemplified in the first manufacturing method of the present disclosure in <2> above. In addition, the combination of the above inhibitors can also be the exemplified combination. Regarding the above inhibitor, for example, as an example, it contains at least one inhibitor selected from the group consisting of TGFβ receptor inhibitors and ROCK inhibitors. Here, in this specification, the term "proliferation regulation" can refer to achieving at least one of cell proliferation maintenance and proliferation promotion. Regarding the terms "proliferation maintenance" and "proliferation promotion", for example, as long as the number of cells is maintained or increased during the culture period, there is no special distinction. There is no particular limitation on the use concentration of the above proliferation regulator for cell proliferation regulation. For example, the concentration of the above-mentioned low-molecular-weight signaling pathway inhibitor described in the first manufacturing method of the present disclosure can be directly applied.

[0302] <6>Cell Secretions and Their Manufacturing Methods

[0303] Another aspect of the present disclosure relates to a method for manufacturing cell secretions, which includes: obtaining a culture of the above-mentioned highly proliferative cells of the present disclosure, or obtaining a culture of highly proliferative cells by implementing the manufacturing method of the above-mentioned highly proliferative cells of the present disclosure; and separating the cell secretions from the above-mentioned highly proliferative cells from the above culture. The manufacturing method of the present disclosure can, for example, further include optional steps as needed. The above-mentioned highly proliferative cells of the present disclosure are highly proliferative cells with improved cell proliferation ability. Therefore, for example, compared with ectodermal cells not in contact with the above inhibitor, they can proliferate in a shorter time, can proliferate for a longer period, or can proliferate in a shorter time and proliferate for a longer period. Therefore, in the manufacturing method of the cell secretions of the present disclosure, for example, compared with control cells, the cell secretions secreted by the above-mentioned highly proliferative cells can be obtained in a larger amount in a shorter time. The above control cells are, for example, the above-mentioned control cells, which are ectodermal cells not in contact with the above inhibitor.

[0304] Cell secretions are usually also referred to as the "secretome". The cell secretions of the present disclosure are not particularly limited as long as they are substances secreted by the highly proliferating cells of the present disclosure. For example, extracellular vesicles such as exosomes, microvesicles, and apoptotic bodies can be mentioned; functional proteins such as cytokines, hormones, and antibodies, etc. In one aspect of the present disclosure, the cell secretions can be, for example, the above-mentioned extracellular vesicles, and particularly exosomes.

[0305] In the method for producing cell secretions of the present disclosure, the prepared culture is a culture of the highly proliferating cells of the present disclosure, and the culture contains cell secretions secreted by the highly proliferating cells. The culture containing the cell secretions can be produced, for example, by implementing the method for producing the highly proliferating cells of the present disclosure, can be produced by obtaining the highly proliferating cells of the present disclosure and culturing them, can be separately obtained by implementing the method for producing the highly proliferating cells of the present disclosure to obtain a culture, or can be separately obtained as a culture of the highly proliferating cells of the present disclosure. The "culture" in this specification refers to, for example, a combination of cultured cells and culture supernatant (conditioned medium) obtained by culturing cells.

[0306] As described above, the method for producing cell secretions of the present disclosure includes separating the cell secretions from the above-mentioned culture. The separation method for separating the cell secretions from the above-mentioned culture is not particularly limited. For example, known separation methods such as recovering the supernatant from the above-mentioned culture and centrifugation can be used. The cell secretions separated from the above-mentioned culture can be obtained, for example, in the form of a composition containing the cell secretions. When the cell secretions are a composition containing the cell secretions, i.e., a cell secretion-containing composition, the cell secretion-containing composition can contain, for example, the cell secretions and an aqueous medium. The aqueous medium is not particularly limited. For example, it is a known aqueous medium selected according to the type of the cell secretions, etc. Specific examples include the culture supernatant in the above-mentioned culture, water, buffers (such as phosphate buffer, Good's buffer), physiological saline, and culture media.

[0307] The method for producing cell secretions of the present disclosure can further include purifying or separating the cell secretions separated from the above-mentioned culture. The purification method or separation method for the cell secretions is not particularly limited, and known methods can be applied according to the type of the cell secretions.

[0308] As the method for separating or purifying the above-mentioned cell secretion, for example, filtration and concentration can be cited. For example, the above-mentioned composition containing the cell secretion separated from the above-mentioned culture can be subjected to separation or purification. In the case of the above-mentioned filtration, for example, a membrane having a size or molecular weight cut-off value corresponding to the size of the target cell secretion can be used to filter the above-mentioned composition containing the cell secretion, and the above-mentioned cell secretion can be separated or purified. As another method, for example, tangential flow filtration or ultrafiltration can be used to filter or concentrate the above-mentioned composition containing the cell secretion, and the above-mentioned cell secretion can be separated or purified. The above-mentioned cell secretion after separation or purification is also referred to as, for example, separated cell secretion.

[0309] The above-mentioned cell secretion can be used, for example, in the form of a composition containing the above-mentioned cell secretion or in the form of the above-mentioned separated cell secretion obtained after separation or purification. The above-mentioned cell secretion can be directly stored in the form of the above-mentioned composition or the above-mentioned separated cell secretion. In addition, the composition of the above-mentioned cell secretion or the above-mentioned separated cell secretion can be made into a dry body by known treatments such as spray drying and freeze drying, can be used in the form of the above-mentioned dry body, and can also be stored in the form of the above-mentioned dry body. The storage method is not particularly limited, and examples include storage at room temperature, refrigerated storage, or frozen storage.

[0310] Specifically, the cell secretion from the above-mentioned highly proliferative cells of the present disclosure is a cell secretion containing at least one of the following proteins and miRNAs, or a cell secretion containing both of the following proteins and miRNAs. The above-mentioned proteins are selected from, for example, Figures 2 to 4 as shown, and the above-mentioned miRNAs are selected from, for example, Figures 5 to 10 and Figure 12 as shown. Figures 2 to 4 The symbols in are the abbreviations of the proteins, the names are the names of the proteins, and the accession numbers are the accession numbers in UniProtKB / Swiss-Prot (https: / / www.uniprot.org / ). Figures 5 to 10 and Figure 12 In, the miRNA column is the miRNA name, and the accession number is the accession number in miRBase (Release 21) (https: / / www.mirbase.org / ). The cell secretion of the present disclosure only needs to contain at least one selected from these proteins, or at least one selected from these miRNAs, and can also contain at least one selected from these proteins and at least one selected from these miRNAs.

[0311] In one embodiment of the cell secretion of the present disclosure, as the protein,

[0312] · Group PA: It can contain at least a combination of glycoprotein M6B (Q13491), HLA class II histocompatibility antigen DR alpha chain (P01903), tweety homolog 1 (Q9H313), profilin 2 (P35556), HLA class II histocompatibility antigen DRB1 beta chain (P01911), and S100 calcium-binding protein A8 (P05109);

[0313] · Group PB: It can contain a combination of glycoprotein M6B (Q13491), HLA class II histocompatibility antigen DR alpha chain (P01903), tweety homolog 1 (Q9H313), profilin 2 (P35556), HLA class II histocompatibility antigen DRB1 beta chain (P01911), and S100 calcium-binding protein A8 (P05109); and at least one selected from the group consisting of endophilin A2 (Q99961), NEDD4-like E3 ubiquitin ligase (Q96PU5), integrin subunit beta 8 (P26012), intercellular adhesion molecule 1 (P05362), ephrin B2 (P52799), DnaJ homolog subfamily A member 2 (O60884), protein containing coiled-coil region 50 (Q8IVM0), latent transforming growth factor-beta binding protein 2 (Q14767), midkine (P21741), and basal cell adhesion molecule (P50895);

[0314] · PC group: may include a combination of glycoprotein M6B (Q13491), HLA class II histocompatibility antigen DRα chain (P01903), tweety homolog 1 (Q9H313), fibrillin 2 (P35556), HLA class II histocompatibility antigen DRB1β chain (P01911), and S100 calcium-binding protein A8 (P05109); a combination of endophilin A2 (Q99961), NEDD4-like E3 ubiquitin ligase (Q96PU5), integrin subunit β8 (P26012), intercellular adhesion molecule 1 (P05362), ephrin B2 (P52799), DnaJ homolog subfamily A member 2 (O60884), protein containing coiled-coil region 50 (Q8IVM0), latent transforming growth factor β-binding protein 2 (Q14767), midkine (P21741), and basal cell adhesion molecule (P50895); and at least one selected from the group consisting of tetraspanin 3 (O60637), radixin (P35241), G protein-coupled receptor family C member 5B (Q9NZH0), tetraspanin 6 (O43657), galectin-3-binding protein (Q08380), phospholipid scramblase 1 (O15162), ephrin B1 (P98172), gliomedin (P07093), fibrillin 1 (P35555), Ras-related protein Rab-5A (P20339), galectin-7 (P47929), Toll-interacting protein (Q9H0E2), clusterin (P10909), CD47 (Q08722), cadherin 13 (P55290), ezrin (P15311), and vacuolar protein sorting protein 37C (A5D8V6); or

[0315] · PD group: may include Figure 2 , Figure 3 or Figure 4 all the proteins described in

[0316] The miRNAs contained in the cell secretions of the present disclosure may include, for example, Figure 12 the miRNAs listed in Figure 12 Among the miRNAs listed in Figure 12 , the miRNAs involved in the treatment of Parkinson's disease may be exemplified by Figure 5 the miRNAs listed in Figures 6 to 9 Among the miRNAs listed in Figures 6 to 9 , the miRNAs involved in the treatment of Alzheimer's disease may be exemplified by Figure 10 the miRNAs listed in

[0317] In one embodiment of the cell secretions of the present disclosure, as the miRNA,

[0318] · Group RA: may comprise a combination of at least hsa-miR-206 (MIMAT0000462), hsa-miR-204-5p (MIMAT0000265), hsa-miR-128-3p (MIMAT0000424), hsa-miR-363-3p (MIMAT0000707) and hsa-miR-323a-3p (MIMAT0000755);

[0319] · Group RB: may comprise a combination of hsa-miR-206 (MIMAT0000462), hsa-miR-204-5p (MIMAT0000265), hsa-miR-128-3p (MIMAT0000424), hsa-miR-363-3p (MIMAT0000707) and hsa-miR-323a-3p (MIMAT0000755); and at least one selected from the group consisting of hsa-miR-29c-3p (MIMAT0000681), hsa-miR-708-5p (MIMAT0004926), hsa-miR-218-5p (MIMAT0000275), hsa-miR-4484 (MIMAT0019018), hsa-miR-183-5p (MIMAT0000261), hsa-miR-186-5p (MIMAT0000456), hsa-miR-484 (MIMAT0002174), hsa-miR-374b-5p (MIMAT0004955) and hsa-miR-93-5p (MIMAT0000093);

[0320] · RC group: It may include the combination of hsa-miR-206 (MIMAT0000462), hsa-miR-204-5p (MIMAT0000265), hsa-miR-128-3p (MIMAT0000424), hsa-miR-363-3p (MIMAT0000707), and hsa-miR-323a-3p (MIMAT0000755); the combination of hsa-miR-29c-3p (MIMAT0000681), hsa-miR-708-5p (MIMAT0004926), hsa-miR-218-5p (MIMAT0000275), hsa-miR-4484 (MIMAT0019018), hsa-miR-183-5p (MIMAT0000261), hsa-miR-186-5p (MIMAT0000456), hsa-miR-484 (MIMAT0002174), hsa-miR-374b-5p (MIMAT0004955), and hsa-miR-93-5p (MIMAT0000093); and at least one selected from the group consisting of hsa-miR-222-3p (MIMAT0000279), hsa-miR-25-3p (MIMAT0000081), hsa-miR-425-5p (MIMAT0003393), hsa-miR-328-3p (MIMAT0000752), hsa-miR-485-5p (MIMAT0002175), hsa-miR-151a-3p (MIMAT0000757), hsa-miR-16-5p (MIMAT0000069), hsa-miR-4534 (MIMAT0019073), and hsa-miR-320a (MIMAT0000510); or

[0321] · RD group: It may include Figures 5 to 10 all miRNAs recorded in any of the figures in Figure 12 or all miRNAs recorded in

[0322] In another embodiment of the cell secretion of the present disclosure, it may include the proteome of the above PA group, PB group, PC group, or PD group as proteins, and the miRNA group of the above RA group, RB group, RC group, or RD group as miRNAs. Thus, the above cell secretion in one embodiment can be used, for example, as a pharmaceutical composition having known effects brought by these proteins and miRNAs.

[0323] In one aspect, the cell secretion of the present disclosure is the above-mentioned extracellular vesicles, specifically vesicles containing a lipid bilayer. The diameter of the above-mentioned extracellular vesicles is, for example, 50 nm to 5 μm or 50 nm to 1000 nm. The diameter of exosomes, which are one of the above-mentioned extracellular vesicles, is, for example, 50 nm to 200 nm. Exosomes contain various bioactive substances such as proteins, nucleic acids, carbohydrates, and lipids, and thus their utilization in the treatment and diagnosis of diseases, pharmaceuticals, cosmetics, etc. can be expected.

[0324] When the cell as the source is an adherent cell, exosomes are secreted from the above-mentioned adherent cell into the culture, particularly the culture supernatant. In addition, when the cell as the source is a non-adherent cell and the above-mentioned non-adherent cell is present in a cell suspension, exosomes are secreted from the above-mentioned non-adherent cell into the culture supernatant or the cell suspension. When the cell secretion of the present disclosure contains extracellular vesicles (such as exosomes) from the above-mentioned adherent cell or the above-mentioned non-adherent cell, for example, as the above-mentioned highly proliferative cells of the present disclosure, adherent cells (also called highly proliferative ectodermal adherent cells) or non-adherent cells (also called highly proliferative ectodermal non-adherent cells) can be prepared, and the above-mentioned extracellular vesicles can be obtained from the above-mentioned adherent cells or the above-mentioned non-adherent cells. As the above-mentioned exosomes, for example, the exosomes described in Journal of Controlled Release, Volume 323, Pages 225 - 239 (2020) can be cited.

[0325] Exosomes can be separated based on, for example, molecular weight, size, shape, composition, or biological activity. Specifically, they can be separated by the following methods: fractionation of sediment based on ultracentrifugation, fractionation of fractions based on density gradient ultracentrifugation, fractionation using size exclusion chromatography, fractionation using ion exchange chromatography (such as CIMmultus TM EV (manufactured by BIA separations)), fractionation based on capture using a protein (such as, MagCapture TM Exosome Isolation Kit PS (manufactured by Fujifilm Wako Pure Chemical Corporation)), fractionation based on capture using an antibody, fractionation of precipitates using a polymer such as polyethylene glycol, etc. These methods can be implemented, for example, singly or in combination of multiple.

[0326] In the method for manufacturing the cell secretion of the present disclosure, when manufacturing exosomes as the above-mentioned cell secretion, the properties of exosomes can be used to trace the activity of exosomes. The activity of exosomes can be confirmed using, for example, static light scattering, dynamic light scattering, ultraviolet-visible light detector, fluorescence detector, or differential refractive index detector.

[0327] In the method for producing the cell secretion of the present disclosure, for example, the cell secretion can be separated from the culture after the main culture in the method for producing the highly proliferative cells of the present disclosure described above. Alternatively, the obtained highly proliferative cells can be further subcultured, and the cell secretion can be separated from the culture. From the aspect of obtaining exosomes, which are the cell secretion, with good purity, when separating or purifying exosomes, for example, for the culture containing the highly proliferative cells, the culture medium for culturing the culture (for example, the medium in the main culture) can be further replaced with a recovery medium, and subculture can be performed. Specifically, the recovery medium is a culture supernatant recovery medium. The subculture is, for example, the culture using the recovery medium after the culture using the culture medium. The period of the subculture is not particularly limited, and it is a culture for a shorter time than the main culture. For example, it can be 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 for example, it can be 96 hours or less or 72 hours or less. By performing the above subculture, for example, the contamination rate of exosomes derived from a different origin from the highly proliferative cells, that is, exosomes that may be contained in the main culture medium used in the main culture, can be reduced, and the purity of exosomes derived from the target highly proliferative cells can be increased.

[0328] Examples of the recovery medium include a medium without serum addition and a medium that has been subjected to exosome removal treatment. Examples of commercially available media that have been subjected to exosome removal treatment include a disposable type exosome-depleted FBS (FBS exosome-depleted, OneShot format) (Gibco (registered trademark), Thermo Fisher Scientific). The recovery medium preferably does not contain the low molecular weight signaling pathway inhibitor described above.

[0329] The medium without serum addition preferably uses a medium that does not contain serum from non-human mammals (as a specific example, bovine serum such as fetal bovine serum). Generally, fetal bovine serum or the like containing factors for promoting cell proliferation is added to the medium used for cell culture, so bovine-derived exosomes may be mixed in the medium. Moreover, it is difficult to separate human-derived exosomes from bovine-derived exosomes. Therefore, for example, in the case of separating exosomes from human cells, by performing a short-term subculture again using a recovery medium that does not contain fetal bovine serum after the main culture, exosomes derived from human cells can be separated with further good purity.

[0330] When using the above-described culture medium for recovery, for example, after the above-described additional culture, centrifugation can be performed on the cultured product of the additional culture, and the culture supernatant can be separated and taken as a composition containing exosomes. If necessary, the separated culture supernatant can be subjected to the above-described method for separating or purifying exosomes, whereby exosomes can be obtained.

[0331] <7>Use of highly proliferative cells and cell secretions

[0332] The cell secretions derived from the highly proliferative cells of the present disclosure described above can be expected to have various effects on various cell types, such as a neuroprotective effect, a neurite outgrowth inhibitory effect, a neurite outgrowth effect, a neurite network formation effect, a neurocyte death prevention effect, and a neurocyte proliferation promoting effect. Based on these functions, the cell secretions derived from the highly proliferative cells described above can be expected to be useful as a pharmaceutical composition for preventing or treating disorders related to peripheral nerve cells or central nerve cells. The cell secretions derived from the highly proliferative cells are not particularly limited, and examples thereof include the cell secretions listed in the above <6>, and exosomes are preferred among them.

[0333] The above-described neuroprotective effect is, for example, an effect in which nerve cells are not easily damaged or the development of nerve disorders is delayed. For example, it is known that some hormones such as corticosterone are locally excessive in the central nervous system, and thus may cause central diseases such as depression due to nerve disorders and dysfunctions caused by overstimulation of hormone receptors. When the cell secretions of the present disclosure have an effect of inhibiting neurocyte death, nerve cells are not easily damaged or the development of nerve disorders can be delayed, and thus protection of nerves can be expected.

[0334] The above-described neurite outgrowth inhibitory effect is involved in the differentiation of peripheral nerve cells, and is an effect of inhibiting the differentiation from less differentiated cells into peripheral nerve cells, particularly sympathetic nerve cells. Therefore, by using the cell secretions of the present disclosure for, for example, peripheral nerve cells, inhibition of the peripheral nervous system, particularly the sympathetic nervous system, can be expected. In addition, for example, by using the cell secretions of the present disclosure for, for example, central nerve cells, activation of the central nervous system can be expected based on its neurite outgrowth effect, neurite network formation effect, neurocyte death prevention effect, and neurocyte proliferation promoting effect.

[0335] Therefore, the present disclosure relates to a pharmaceutical composition comprising a pharmaceutical composition containing a therapeutically or prophylactically effective amount of the cell secretions of the present disclosure and a pharmaceutically acceptable carrier, and further, the present disclosure relates to a method for treatment or prophylaxis, which includes administering the above-described pharmaceutical composition of the present disclosure to a subject. The term "treatment" in this specification includes not only the radical cure of disorders, but also the improvement of disorder symptoms such as alleviation and remission.

[0336] The above-mentioned disorders to be treated or prevented are not particularly limited and are disorders related to peripheral nerve cells or central nerve cells. For example, disorders selected from the group consisting of cancer, pain, Alzheimer's disease, Parkinson's disease, depression, schizophrenia, and dementia can be cited. As disorders related to peripheral nerve cells, particularly disorders related to sympathetic nerve cells, for example, cancers, pain, etc. related to sympathetic nerve cells can be cited. Disorders related to central nerve cells are, for example, selected from the group consisting of Alzheimer's disease, Parkinson's disease, depression, schizophrenia, and dementia.

[0337] There is no particular limitation on the above-mentioned pharmaceutically acceptable carrier. For example, carriers well-known in the art can be used, such as physiological saline and the like. In addition, the present disclosure relates, for example, to a method for inhibiting the sympathetic nerve or a method for inhibiting neurite outgrowth related to the above-mentioned action, including bringing the cell secretion (specifically, exosomes) derived from the above-mentioned highly proliferative cells into contact with nerve cells (such as sympathetic nerve cells).

[0338] In the treatment or prevention method of the present disclosure, the administration subject is a human or a non-human animal, and for the above-mentioned non-human animal, the above-mentioned examples can be cited. In the treatment or prevention method of the present disclosure, the administration method is not particularly limited. For example, there are oral administration or parenteral administration. The above-mentioned parenteral administration can include, for example, injection into the affected part, intravenous injection, subcutaneous injection, intradermal injection, drip injection, transdermal administration, etc.

[0339] In this specification, the numerical range represented by "~" means a range that includes the numerical values described before and after "~" as the minimum value and the maximum value respectively. In the numerical ranges described step by step in this specification, the upper limit value or the lower limit value of a certain step's numerical range can be arbitrarily combined with the upper limit value or the lower limit value of other step's numerical ranges.

[0340] All the documents, patent applications, and technical standards described in the present disclosure are incorporated into the present disclosure in their entirety by reference.

[0341] Examples

[0342] [Example 1] Cultivation of human astrocytes using a medium supplemented with an inhibitor

[0343] The following materials, reagents, and culture products were used to cultivate human astrocytes. Unless otherwise specified, the same materials, reagents, culture products, devices, etc. were also used in the examples after Example 1.

[0344] <Materials>

[0345] · Normal human astrocytes (NHA) (CC-2565, Lonza)

[0346] <Reagents and culture products>

[0347] · Culture medium for culturing: AGM astrocyte growth medium bullet kit (CC-3186, Lonza) (Composition of AGM: basal medium, FBS (3% (v / v)), L-glutamine, ascorbic acid, hEGF, insulin, antibiotics)

[0348] · Inhibitor Y: CultureSure (registered trademark) Y-27632 (034-24024, FUJIFILM Wako Pure Chemical Corporation), final concentration: 10 μM

[0349] · Inhibitor A: CultureSure (registered trademark) A-83-01 (035-24113, FUJIFILM Wako Pure Chemical Corporation), final concentration: 0.5 μM

[0350] · Cell detachment agent: Accutase (AT104, ICT)

[0351] · Cell buffer: phosphate buffered saline solution (PBS(-); Dulbecco; calcium- and magnesium-free) (14190250, Thermo Fisher Scientific)

[0352] · Cell cryopreservation medium: CELLBANKER 1 (CB011 TaKaRa, Nippon Zenyaku Kogyo Co., Ltd.)

[0353] · Cell culture dish 60 mm (150462, Thermo Fisher Scientific)

[0354] · Cell culture dish 100 mm (150466, Thermo Fisher Scientific)

[0355] · Cell culture dish 150 mm (150468, Thermo Fisher Scientific)

[0356] · Cell culture flask T-75 (3290, Corning)

[0357] · Stericup Quick Release-GP sterile vacuum filtration system (registered trademark, S2GPU02RE, MerckMillipore)

[0358] · Medium without inhibitors for collecting culture supernatant: AGM Astrocyte Growth Medium BulletKit (CC-3186, Lonza) (Composition of AGM: basal medium, L-glutamine, ascorbic acid, hEGF, insulin, antibiotics, supplemented with exosome-depleted FBS [3% (v / v)] (A2720803, Gibco) instead of the attached FBS)

[0359] · Centrifuge: Optima XE-90, Beckman Coulter

[0360] <Culture device>

[0361] · CO2 incubator: MCO-170AICUVD-PJ, PHC Corporation

[0362] <Culture conditions>

[0363] Temperature 37°C, CO2 concentration 5%

[0364] <Cell observation and imaging device>

[0365] · Fluorescence microscope BZ-X810, CKX53, Olympus Corporation

[0366] <Culture process>

[0367] The culture is carried out according to the following process. Thaw the cryotube of human astrocytes (NHA) in a 37°C water bath. Then transfer the thawed cells to the culture medium to a total volume of 10 mL, and subject them to centrifugation at 180×g for 5 minutes at room temperature, and remove the above medium. Resuspend the cells in a new culture medium, and inoculate them into a 150 mm cell culture dish at an inoculation density of 5.8×10 3 cells / cm 2 . On the 10th day of culture, detach the cells from the above dish using a cell detachment agent and resuspend them in a new culture medium. For the analysis of gene expression and protein expression, inoculate this cell suspension into a 60 mm cell culture dish at an inoculation density of 4.8×10 3 ~1.4×10 4 cells / cm 2 . Additionally, for subculture purposes, inoculate the above cell suspension at an inoculation density of 5.5×10 3 / cm 2The cells were inoculated into a 100-mm cell culture dish at the inoculation density. The next day, the culture dishes for analysis and subculture were divided into a normal (Normal) group (also referred to as the NHA group), a YA group (also referred to as the NHA-YA group), a Y group (also referred to as the NHA-Y group), and an A group (also referred to as the NHA-A group), and the main culture media corresponding to each group were replaced (culture day 0), and the main culture was carried out. As the main culture medium, the above-mentioned normal group used a culture medium without an inhibitor, the above-mentioned YA group used a YA main culture medium supplemented with the above-mentioned inhibitor Y and the above-mentioned inhibitor A, the above-mentioned Y group used a culture medium supplemented with the above-mentioned inhibitor Y, and the above-mentioned A group used a culture medium supplemented with the above-mentioned inhibitor A. The concentration of the inhibitor in the above-mentioned main culture medium was as described above. Hereinafter, the description of inhibitor YA refers to the addition of both the above-mentioned inhibitor Y and the above-mentioned inhibitor A. During the culture period, the culture medium was changed every 2 or 3 days. At this time, it was replaced with a fresh main culture medium (with or without an inhibitor), and the cells were observed and photographed using a microscope (KEYENCE, BZ-X810 or Tomy Seiko, MX-307).

[0368] The subculture cells were detached from the above-mentioned culture dish every 7 days using a cell detachment agent and suspended in the above-mentioned main culture medium (with or without an inhibitor). After that, again for the analysis of gene expression and protein expression, the above-mentioned cell suspension was inoculated into a 60-mm cell culture dish at an inoculation density of 4.8×10 3 ~1.4×10 4 cells / cm 2 ; again for subculture purposes, the above-mentioned cell suspension was inoculated into a 100-mm cell culture dish at an inoculation density of 5.5×10 3 / cm 2 to perform subculture. While repeating the above steps, the main culture was carried out for up to 28 days in the presence or absence of the above-mentioned inhibitor.

[0369] The cells for gene expression and protein expression analysis were cultured in the above-mentioned 60-mm cell culture dish for up to 14 days, and then, as described above, the subculture cells were re-inoculated and the culture was continued. After that, in the main culture using the above-mentioned main culture medium (with or without an inhibitor), on the 1st, 3rd, 5th, 7th, 9th, 11th, 14th, 21st, and 28th days, the cells were detached from the culture dish for analysis, and the cell count, RNA recovery, and protein immunostaining were performed. A part of the sampled cells was cryopreserved for subsequent analysis.

[0370] The relationship between the number of days of main culture and the total number of cells in each group is shown in Table 4. The total cell number is expressed as the cumulative value obtained by assuming that all cells were passaged at the time of passage (the same applies hereinafter).

[0371] [Table 4]

[0372]

[0373] As can be seen from Table 4 above, in the YA group, after 5 days of main culture in the presence of the above inhibitor, the proliferation rate began to increase, and thereafter, until 28 days of main culture, a high cell proliferation rate continued. In addition, on the 7th day of main culture, the number of cells in the YA group was 1.2 times the proliferation rate of the normal group, 24 times on the 14th day, 92 times on the 21st day, and 326 times on the 28th day. From these results, it can be seen that the cell population obtained by main culture in the presence of the above inhibitor contains highly proliferative cells with increased proliferation. In addition, an increase in proliferation was also confirmed by main culture in the Y group and the A group.

[0374] For all experimental groups, cells were observed from the start of main culture until the 28th day. Figure 1 ( Figure 1-1 and Figure 1-2 ) shows micrographs of cells at specified days (the 3rd day, the 7th day, the 14th day, the 28th day) from the start of main culture. Figure 1-1 In, each photograph is a magnified photograph at 40 times, and the scale bar in the figure is 200 μm; Figure 1-2 In, each photograph is a magnified photograph at 100 times, and the scale bar in the figure is 100 μm. In Figure 1, AGM is the photograph of the normal group, YA is the photograph of the YA group, and Y is the photograph of the Y group. According to Figure 1, compared with the normal group cultured in the absence of the above inhibitor, the following characteristics can be confirmed in the YA group, Y group, and A group cultured in the presence of the above inhibitor.

[0375] In the Y group and the YA group, changes in cell morphology began to appear from the day after the addition of the above-mentioned inhibitor (day 1), and images of cells extending thin neurite-like structures were observed. In Figure 1, this feature was significant on day 3. In addition, in the cells of the A group, some cells underwent atrophy. Subsequently, on days 5 to 7 after the addition, in the Y group, A group, and YA group, the appearance of small and dense cell clusters and the colony-like proliferation of multiple cell clusters with different morphologies were observed in the cells. In Figure 1, this feature was observed on days 7 and 14. In addition, in the Y group and the YA group, cell clusters with different cell morphologies continued to proliferate, and cell proliferation was significantly promoted compared with the normal group. In contrast, in the normal group, the cells were slightly enlarged, and cell clusters with neurite-like structures continued to proliferate three-dimensionally above the small and dense cell layer even when the cell density reached the inhibitory proliferation level. In addition, a small and dense cell layer was also formed in the A group, but the proliferation of cell clusters with neurite-like structures was limited compared with the Y group or the YA group. In Figure 1, the above-mentioned feature was confirmed in the cell images on day 28.

[0376] [Example 2] Confirmation of ectodermal cell markers based on quantitative PCR

[0377] For the cell population containing highly proliferating cells (YA group) obtained by main culture in the presence of the above-mentioned inhibitor and the cell population cultured in the absence of the above-mentioned inhibitor (normal group) in the same manner as in Example 1 above, the expression levels of ectodermal cell marker genes were studied.

[0378] RNA was extracted from the following cell samples according to a conventional method, and cDNA was synthesized using this RNA sample. The preparation of the RNA sample and the setting of the reaction conditions were carried out according to the operating procedures attached to the product.

[0379] Cell samples:

[0380] · Normal human astrocytes (NHA)

[0381] The cell population (normal group) obtained by main culture in the absence of the above-mentioned inhibitor in Example 1 above was used. It should be noted that two passages were carried out during the 28-day main culture period.

[0382] · NHA-YA

[0383] The cell population (YA group) obtained by main culture in the presence of the above-mentioned inhibitor YA in Example 1 above was used. It should be noted that two passages were carried out during the 28-day main culture period.

[0384] <Recovery of total RNA>

[0385] Reagents: PureLink RNA Mini Kit (registered trademark, product number: 12183018A, ThermoFisher), PureLink DNase Set (registered trademark, product number: 12185010, Thermo Fisher)

[0386] Equipment: Cooling centrifuge MX-307, Bio·Rad; Spectrophotometer Nanodrop (trademark), ThermoFisher

[0387] <Synthesis of cDNA>

[0388] Reagents: PrimeScript RT Premix (registered trademark, product number: RR037A, Takara Bio)

[0389] Equipment: CFX96 Touch Real-Time PCR Detection System (Bio-Rad)

[0390] <Quantitative PCR>

[0391] Using the cDNA synthesized from the above total RNA as a template, the expression of ectodermal cell marker genes was confirmed by quantitative PCR using the following primers and probes. The preparation of the above total RNA and the setting of reaction conditions were carried out according to the operating procedures attached to the product.

[0392] Reagents: TaqMan Fast Advanced Premix (product number: 4444557, Thermo Fisher)

[0393] Equipment: CFX96 Touch Real-Time PCR Detection System (Bio-Rad)

[0394] Primer / Probe Set:

[0395] · TaqMan (registered trademark, the same below) Gene Expression Assay GAPDH: Hs02786624_g1 (product number: 4331182, Thermo Fisher)

[0396] · TaqMan Gene Expression Assay GFAP: Hs00909233_m1 (product number: the same above, Thermo Fisher)

[0397] · TaqMan Gene Expression Assay SOX2: Hs01053049_s1 (product number: the same above, Thermo Fisher)

[0398] · TaqMan Gene Expression Assay for NES: Hs04187831_g1 (Product number: same as above, Thermo Fisher)

[0399] · TaqMan Gene Expression Assay for MSI1: Hs01045894_m1 (Product number: same as above, Thermo Fisher)

[0400] · TaqMan Gene Expression Assay for Notch1: Hs01062014_m1 (Product number: same as above, Thermo Fisher)

[0401] · TaqMan Gene Expression Assay for S100b: Hs00902901_m1 (Product number: same as above, Thermo Fisher)

[0402] · TaqMan Gene Expression Assay for SLC1A3: Hs00904823_g1 (Product number: same as above, Thermo Fisher)

[0403] · TaqMan Gene Expression Assay for CSPG4: Hs00361541_g1 (Product number: same as above, Thermo Fisher)

[0404] · TaqMan Gene Expression Assay for PAX6: Hs01088114_m1 (Product number: same as above, Thermo Fisher)

[0405] · TaqMan Gene Expression Assay for Olig2: Hs00377820_m1 (Product number: same as above, Thermo Fisher)

[0406] Statistical analysis: The calculation of gene expression levels was performed using Excel (Microsoft).

[0407] (2-1) The relative value (E1 / E C ) of the expression level (E1) of the marker gene relative to the expression level of the control gene C )

[0408] The expression levels (E1) of various marker genes were calculated as relative values (E1 / E C ) relative to the expression level of the internal control gene (GAPDH). The results are shown in Table 5 (5-1, 5-2). In Table 5, the bold values are the highest and lowest values shown in Table 6 below. C )

[0409] [Table 5-1]

[0410] Expression level of marker gene (E1) / Expression level of control gene (Ec)

[0411]

[0412] [Table 5-2]

[0413] Expression level of marker gene (E1) / Expression level of control gene (Ec)

[0414]

[0415] Extract the maximum and minimum relative values (E1 / E C ) of the expression levels of each marker gene according to the results of Table 5-2 for culture days exceeding 28 days (35 days to 63 days). On the other hand, for the results of Table 5-1 for culture days less than 28 days (1 day to 28 days), extract the maximum and minimum relative values (E1 / E C ) of the expression levels of each marker gene according to the following criteria (1) and (2), and determine the cut-off values that are different from the results for culture days exceeding 28 days for each marker gene. These results are shown in Table 6 below. In the following table, the cut-off value can be, for example, the value in parentheses.

[0416] (1) In the culture exceeding 28 days, outside the expression range of the above-mentioned marker gene.

[0417] (2) In the culture less than 28 days, within the expression range of the above-mentioned marker gene.

[0418] However, in the culture less than 28 days, it is not necessary to exclude the expression range of the above-mentioned marker gene in the culture exceeding 28 days throughout the period.

[0419] [Table 6]

[0420] Expression level of marker gene (E1) / Expression level of control gene (Ec)

[0421]

[0422] The above cut-off value can be a condition for distinguishing the cells obtained from the culture less than 28 days in the presence of the above inhibitor from the cells obtained from the culture exceeding 28 days in the presence of the above inhibitor. Therefore, the above cut-off value can be set as a reference range for determining the expression behavior of the marker gene for the highly proliferative cells of the present disclosure.

[0423] (2-2) Relative value (E YA ) of the expression level of the marker gene in the YA group relative to the expression level of the marker gene in the normal group (E N ) (E YA / E N )

[0424] The expression confirmation results of Musashi1 and Notch1, which are markers of neural stem cells, Notch1 and Nestin, which are markers of neuroepithelial cells, SOX2, and Nestin, which is a marker of radial glial cells, obtained by quantitative PCR, as well as the expression confirmation results of GFAP and S100B, SLC1A3, which are markers of astrocytes, and NG2, which is a marker of oligodendrocyte progenitor cells, are shown in Table 7.

[0425] The expression levels of each marker gene on the 5th, 7th, 9th, 14th, and 28th days of primary culture are shown in Table 7. The expression level of the marker gene is a value obtained by correcting the measured value of the expression level of the marker gene using the measured value of the expression level of the control gene (GAPDH). And, the corrected expression level (E N ) of the marker gene in the normal group on the same culture day is set to "1", and is expressed in the form of the relative value (E YA ) of the corrected expression level (E Y A / E N ) of the marker gene in the YA group.

[0426] [Table 7]

[0427] Expression level in YA group / Expression level in normal group

[0428]

[0429] As shown in Table 7, in the cell population (YA group) obtained in the presence of the above inhibitor, the expressions of GFAP, S100B, and SLC1A3, which are markers of astrocytes, are high, while the expression of NG2, which is a marker of oligodendrocyte progenitor cells, is low, compared with astrocytes (normal group) cultured in the absence of the above inhibitor. In addition, regarding the generation of nerve cells, high expressions of Nestin, which is a marker of radial glial cells and neuroepithelial cells existing upstream of astrocytes, Notch1 and SOX2, which are also markers of neuroepithelial cells, and Musashi1, which is a marker of neural stem cells, were confirmed.

[0430] From these results, it can be seen that the cell population (YA group) obtained in the presence of the above inhibitor is closer to progenitor cells and has a high proliferation ability compared with astrocytes (normal group), which are mature cells. Since this cell population (YA group) has a high proliferation ability, it can proliferate more in a short time compared with astrocytes. In addition, it can be seen that it is advantageous to use the above YA group in order to obtain more exosomes in a short time.

[0431] [Example 3] Confirmation of ectodermal cell markers based on immunostaining

[0432] By the same method as in Example 1 above, a cell population (YA group) containing highly proliferative cells obtained by main culture in the presence of the above inhibitor and a cell population cultured in the absence of the above inhibitor (normal group) were prepared. For each cell population, the expression of marker proteins of ectodermal cells was confirmed using the immunostaining method.

[0433] [Reagents and culture products used]

[0434] Reagents:

[0435] · Cell fixative and cell membrane treatment solution: eBioscience Foxp3 / Transcription Factor Staining Buffer Set (trademark, product number: 00-5523-00, Invitrogen), 4% paraformaldehyde·phosphate buffer (PFA) (product number: 161-20141, Fujifilm Wako Pure Chemical), Triton X-100 (product number: 93443, Sigma Aldrich), Otsuka distilled water (product number: 05206903, Otsuka Pharmaceutical)

[0436] · Cell washing solution: phosphate buffered saline solution (PBS(-): calcium and magnesium free, the same below) (product number: 166-23555, Fujifilm Wako Pure Chemical), phosphate buffered saline containing Tween 20 (PBS-T) (product number: T9183, Fujifilm Wako Pure Chemical)

[0437] · Primary antibody:

[0438] · Rabbit monoclonal anti-human GFAP (product number: ab68428, Abcam)

[0439] · Rabbit monoclonal anti-human SOX2 (product number: ab92494, Abcam)

[0440] · Rabbit monoclonal anti-human NOTCH1 (product number: ab52627, Abcam)

[0441] · Rabbit monoclonal anti-human Musashi1 (product number: ab52865, Abcam)

[0442] · Rabbit monoclonal anti-human neural / glial antigen 2 (NG2) (product number: ab275024, Abcam)

[0443] · Rabbit IgG monoclonal isotype control (product number: ab172730, Abcam)

[0444] · Mouse IgG1 Isotype Control (Product No.: 401401, Biolegend)

[0445] · Alexa Flour 488-conjugated anti-human GFAP (Product No.: 53-9792-82, Invitrogen)

[0446] · Alexa Flour 488-conjugated anti-SOX2 (Product No.: 656109, Biolegend)

[0447] · Alexa Flour 488-conjugated anti-human Musashi1 (Product No.: ab199781, Abcam)

[0448] · Alexa Flour 488-conjugated anti-human neural / glial antigen 2 (NG2) (Product No.: 53-4504-80, Biolegend)

[0449] · PE-conjugated anti-Nestin (Product No.: 656805, Biolegend)

[0450] · FITC-conjugated mouse IgG1 Isotype Control (Product No.: 400107, Biolegend)

[0451] · PE-conjugated mouse IgG1 Isotype Control (Product No.: 400111, Biolegend)

[0452] · APC-conjugated mouse IgG1 Isotype Control (Product No.: 400119, Biolegend)

[0453] · Alexa Flour 488-conjugated mouse IgG1 Isotype Control (Product No.: 50-6714-80, Invitrogen)

[0454] · PE-conjugated mouse IgG2a Isotype Control (Product No.: 400211, Biolegend)

[0455] · Alexa Fluor plus 488-conjugated donkey anti-mouse IgG (Product No.: A32796, Invitrogen)

[0456] · Secondary Antibodies:

[0457] · Alexa Fluor plus 488-conjugated donkey anti-mouse IgG (Product No.: A32766, Invitrogen)

[0458] · Alexa Fluor plus 488-conjugated donkey anti-rabbit IgG (Product No.: A32790, Invitrogen)

[0459] · Nuclear staining solution: 7-AAD viability dye (Product No.: A07704, Beckman Coulter)

[0460] · FACS solution: A solution obtained by adding FBS [2% (v / v)] and 2 mM EDTA to phosphate-buffered saline solution (PBS(-)) (Product No.: 311-90075, Nippon Gene) and filtering with a 0.22-μm ASFIL syringe filter (2-856-01AZONE).

[0461] · Cell culture dish 100 mm (150466, Thermo Fisher Scientific)

[0462] · Proteosave SS 1.5-mL microtube (MS-4265M, SUMITOMO BAKELITE)

[0463] Equipment:

[0464] · Centrifuge: MX-307, Tomy Seiko

[0465] · Cell analysis device: Flow cytometer BD FACSVerse, trademark, Nippon Becton Dickinson

[0466] · Analysis software: BD FACSiute version 1.3, FlowJo version 10.8.1 (trademark, Nippon Becton Dickinson)

[0467] <Immunostaining>

[0468] · For human astrocytes (NHA), primary culture was performed for 7 days in the presence and absence of the above inhibitor YA as in Example 1 above (preparation of the normal group and the YA group). After that, the cells cultured for 7 days in primary culture were detached from the culture dish using a cell detachment agent, and a part of the cells was collected into a 15-mL centrifuge tube, suspended in 13 mL of proliferation medium (the inhibitor-free culture medium in Example 1 above), and then subjected to centrifugation at 1,500 rpm for 5 minutes at 4°C to wash the cells.

[0469] · The cells in the above centrifuge tube were suspended using the above FACS solution and adjusted to a cell concentration of 1.0×10 7 cells / mL.

[0470] · Pipette 100 μL (1.0×10 6 cells) of the cell suspension with adjusted concentration into a 1.5 mL microtube, and further add the above primary antibody in a volume ratio of 1 / 100 to 1 / 250, and react at 4°C for 1 hour. It should be noted that when the above primary antibody is directly labeled with a fluorescent dye, the reaction is carried out in the dark.

[0471] · Add 1 mL of PBS to the above tube, subject it to centrifugation at 1,200 rpm for 3 minutes at 4°C, and wash the cells.

[0472] · Suspend the cells in the above tube in 100 μL of PBS(-), further add 900 μL of the above 4% PFA, fix the above cells by culturing at room temperature for 30 minutes, and then subject it to centrifugation at 1,200 rpm for 3 minutes at 4°C. After centrifugation, remove the supernatant, add 1 mL of PBS(-) to the above tube, and wash the cells.

[0473] · Gently suspend the cells in the above tube in 1 mL of 0.2% Triton-X solution, and perform cell membrane permeabilization treatment at room temperature for 15 minutes. After the treatment, add 1 mL of FACS solution to the above tube, subject it to centrifugation at 1,500 rpm for 5 minutes at 4°C, and wash the cells. It should be noted that when using a commercially available reagent (eBioscience Foxp3 / Transcription Factor Staining Buffer Set) for cell fixation and cell membrane permeabilization treatment, follow the operating procedures attached to the product.

[0474] · Suspend the cells in the above tube in 100 μL of FACS solution, and further add the above secondary antibody in a volume ratio of 1 / 50 to 1 / 175, and react at 4°C for 30 minutes to 1 hour. It should be noted that at this time, when the above primary antibody is directly labeled with a fluorescent dye, the reaction is also carried out in the dark.

[0475] · Add 1 mL of PBS(-) to the above tube, subject it to centrifugation at 1,500 rpm for 5 minutes at 4°C, and wash the cells twice.

[0476] · Suspend the cells in the above tube in 500 μL of FACS solution. It should be noted that when live cell determination is required, instead of the above FACS solution, add a suspension obtained by suspending a 7-AAD active dye in a volume ratio of 1 / 50 in FACS solution.

[0477] · Then, for the suspended cells, use the above flow cytometer to measure the proportion of marker-positive cells, and use the above analysis software to analyze the composition of the cell population.

[0478] In Table 8, for the cell populations of the above normal group and the above YA group on the 7th day of main culture, the proportion of cells with positive expression of the marker protein (positive cell rate) and the proportion of the positive cell rate relative to that of the above normal group (ratio relative to normal) are shown respectively.

[0479] [Table 8]

[0480]

[0481] As shown in Table 8, in the YA group, GFAP as an astrocyte marker, Nestin as a radial glial cell marker and a neuroepithelial cell marker, Notch1 and SOX2 which are also neuroepithelial cell markers, and Musashi1 as a neural stem cell marker all showed a higher positive cell rate than the normal group. In contrast, the positive cell rate of NG2 as an oligodendrocyte progenitor cell marker was lower than that of the normal group. From these results, it was confirmed that the ratio of the expression levels of various ectodermal cell marker proteins in the YA group on the 7th day of culture relative to the normal group was consistent with the relative value (E YA / E N ) in Example 2 (2-2) above.

[0482] [Example 4] Recovery and Confirmation of Exosomes in Culture Supernatant

[0483] [Recovery of Culture Supernatant of Human Astrocytes]

[0484] According to the following procedure, human astrocytes were cultured using the above main culture medium containing the above inhibitor YA or not containing the above inhibitor, and their culture supernatants were recovered respectively.

[0485] First, the cryotube of NHA was thawed in a 37°C water bath in the same manner as in Example 1 above. The thawed cells were suspended in 30 mL of culture medium and inoculated into a T-75 cell culture flask at an inoculation density of 1.3×10 4 cells / cm 2 , and cultured. The next day, the medium was replaced with the above main culture medium (containing or not containing the above inhibitor YA) at a volume of 15 mL / flask, and the same medium replacement was performed every 2-3 days and the culture was continued. After culturing for 7 days using the above main culture medium, the cells were detached using a cell detachment agent, the cells were suspended in the above main culture medium, and at 6.6×10 3 cells / cm 2And the inoculation density with a volume of 20 mL / dish was passaged into a 150-mm cell culture dish (second passage). Five dishes were used under each condition of containing the above inhibitor YA and not containing the above inhibitor, so the total number of cells inoculated was 5 × 10 6 cells. Similar to Example 1 above, the cell group cultured with the main culture medium containing the above inhibitor YA was called the YA group, and the cell group cultured with the main culture medium without the above inhibitor YA was called the normal group.

[0486] Three days after the second passage above (main culture: 10 days in total), the medium in the above dish was replaced from the above main culture medium with a volume of 20 mL / dish to the inhibitor-free medium for recovering the above culture supernatant (hereinafter referred to as the recovery medium), and the additional culture for exosome recovery was started.

[0487] Forty-eight hours after the start of the above additional culture, the culture supernatant was recovered from the above dish and filtered using a 0.22-μm filtration system (Stericup, Merck Millipore), and the filtrate was stored at 4°C. Further, fresh above recovery medium was added to the above dish after the above culture supernatant was recovered with a volume of 20 mL / dish, and the additional culture was continued. The above culture supernatant was recovered three times in total for 6 days in this way, and a total of 300 mL of culture supernatant P2 (solution containing exosomes) was obtained under each condition. Here, P2 means that the subculture was carried out twice. Similarly, PN (here, N represents a positive integer) means that the subculture was carried out N times, and the same applies hereinafter. Therefore, the "culture supernatant P2" here refers to the culture supernatant obtained by carrying out the subculture twice.

[0488] [Example 5] Proteomic analysis of NHA-YA-derived exosomes

[0489] Using the cell supernatant P2 (solution containing exosomes) of Example 4 above, proteomic analysis of the proteins contained in the exosomes was carried out.

[0490] <Purification of exosomes in the supernatant>

[0491] Exosomes were purified from the respective culture supernatants P2 (solutions containing exosomes) of the above-mentioned YA group and the above-mentioned normal group obtained in Example 4 above using a commercially available kit (MagCapture Exosome Isolation Kit PS: trademark, 293-7760, Fujifilm Wako Pure Chemical Corporation). The particle size and particle concentration of the purified exosome samples were measured using a Nanosight NS300 (Malvern Panalytical). It should be noted that the particle concentration of each culture supernatant P2 was obtained by subtracting the particle concentration of the medium without inhibitors used for recovering the culture supernatant. Table 9 shows the measurement results of the particle size and particle concentration of the exosome samples of each group. In addition, a BCA assay was performed using a part of the above-mentioned exosome samples to identify the mass of the proteins contained in the exosomes.

[0492] [Table 9]

[0493]

[0494] As shown in Table 9, any of the exosome samples of the above-mentioned YA group and the above-mentioned normal group contained particles having a mean and a mode at a particle size of 100 nm to 200 nm, and the presence of exosomes was confirmed. In addition, compared with the exosome samples of the above-mentioned normal group, the content of the particles of the above-mentioned particle size in the exosome samples of the above-mentioned YA group was shown to be about 2.2 times. From this, it can be seen that exosomes can be efficiently prepared using the cell population of the above-mentioned YA group obtained by culturing in the presence of the above-mentioned inhibitor YA.

[0495] <Proteomic analysis of exosomes>

[0496] A reduction treatment solution was added to the culture supernatant P2 (solution containing exosomes) obtained in Example 4 above, and a reduction treatment was performed at 57 °C for 30 minutes. The above reduction treatment solution was prepared by dissolving 1.5 mg of DTT in 1 mL of 100 mM ammonium bicarbonate. An alkylation treatment solution was added to the reduced sample, and the reaction was carried out at 25 °C for 30 minutes. The above alkylation treatment solution was prepared by dissolving 10 mg of iodoacetamide in 1 mL of 100 mM ammonium bicarbonate. Then, 100 μL of trypsin digestion solution and 100 μL of 50 mM ammonium bicarbonate were sequentially added to the above reaction solution, and the treatment was carried out at 30 °C for 16 hours to decompose the exosomes. The resulting decomposition product of the exosomes was dried using a centrifugal concentrator, 30 μL of 0.1% formic acid was added and stirred, and then centrifuged (20,000×g, 10 minutes), and the supernatant was recovered and used as an analysis sample for nanoLC-MS / MS analysis.

[0497] In nanoLC-MS / MS analysis, for liquid chromatography (LC), UltiMate (registered trademark)3000 , the mass spectrometry device (MS) used Q-Exactive Plus, and Xcalibur (Thermo Fisher Scientific) was used to control LC and MS to carry out the determination. Database search was carried out based on the analysis conditions of nanoLC-MS / MS and Proteome Discoverer Ver2.5 (Thermo Fisher Scientific). Then the search results were output, and quantitative comparative analysis was carried out using Scaffold Ver5.2.0 (Proteome Sciences). For the MS / MS data of the analysis sample, Proteome Discoverer was used to search three databases: SwissProt_Homo sapiens (ID: 9606), UniProtKB_Bos taurus (ID: 9913), and SwissProt_Homo sapiens (ID: 9606)+UniProtKB_Bos taurus (ID: 9913) which is a mixture of them.

[0498] · Human proteins: SwissProt (number of sequences 20376)

[0499] · Bovine proteins: UniProtKB (number of sequences 47043)

[0500] The quantitative method for analysis used the iBAQ (intensity-based absolute quantification) value. As the search result, all protein fragments of the exosome fraction from the normal group source, the exosome fraction from the NHA-YA group source, and the culture medium components were identified. Proteins that were detected more in the exosome fraction from the YA group source compared to the normal group were selected from all the proteins detected in the above databases, and proteins with a quantitative value (number of detected spectra; corrected) of 5.0 or more were selected in descending order. Furthermore, human proteins were further selected from the selected proteins. In the case of bovine proteins, the identification probability in human proteins was studied, and proteins with a high identification probability were selected. Hereinafter, the steps for selecting characteristic proteins to be carried out later will be described.

[0501] a: Steps for selecting characteristic proteins such as membrane proteins and extracellular regions

[0502] As a retrieval result on SwissProt, a total of 1,966 protein fragments were detected, which were the exosome fractions from the normal group, the exosome fractions from the YA group, and the culture medium components combined. From all 1,966 detected proteins, proteins that were detected more in the exosome fractions from the YA group compared to the normal group and had a quantitative value (number of detected spectra; corrected) of 5.0 or more were selected, and 41 proteins were extracted.

[0503] (a1) Among the above-extracted 41 proteins, 29 proteins were selected as those classified as "membrane" by the GO of Scaffold Proteome viewer;

[0504] (a2) Among the above-extracted 41 proteins, 35 proteins were selected as those classified as "extracellular region" by the GO of Scaffold Proteome viewer;

[0505] (a3) Among the above-extracted 41 proteins, 1 protein was selected as a protein that was neither classified as "membrane" nor "extracellular region" by the GO of Scaffold Proteome viewer;

[0506] (a4) Among the proteins extracted through the above steps (a1), (a2), and (a3), 12 proteins were selected as proteins not included in the above culture medium. 8 of them were duplicates of the proteins selected through the following steps (b1) - (b4).

[0507] The 12 proteins selected through the above steps (a1) - (a5) were listed in Figure 2 in.

[0508] Characteristic proteins were selected using a selection step b different from the above selection step a.

[0509] b: Selection steps for characteristic proteins that can be expected to have a pharmacological effect on central nervous system diseases

[0510] (b1) As a combined retrieval result of SwissProt_Homo sapiens and UniProtKB_Bos taurus, a total of 1,642 protein fragments were detected, which were the exosome fractions from the normal group, the exosome fractions from the YA group, and the culture medium components combined. From all 1,642 detected sequences, proteins that were detected more in the exosome fractions from the YA group compared to the normal group and had a quantitative value (number of detected spectra; corrected) of 5.0 or more were selected, and 35 proteins were extracted.

[0511] (b2) For the 35 sequences selected in the above (b1), 30 proteins related to central nervous system diseases or proteins presumably related to the proliferation, development, differentiation, morphogenesis, migration, metabolism, etc. of the brain and nerve cells are selected through the paper retrieval website PubMed (https: / / pubmed.ncbi.nlm.nih.gov / ). Nine of the 30 selected proteins are duplicates of the proteins selected through the above steps (a1) to (a5).

[0512] Record the 30 proteins selected through the above steps (b1) to (b3) in Figure 3 .

[0513] Combining the above selection steps a and b can select a total of 33 proteins. They are shown in Figure 4 .

[0514] [Example 6] miRNA analysis of NHA-YA-derived exosomes

[0515] Using the culture supernatant P2 (solution P2 containing exosomes) of the above Example 4, analyze the small RNAs (Small-RNA: including miRNA, piRNA, tRNA, other RNAs) contained in the exosomes.

[0516] <Extract RNA from the exosomes derived from the above YA group>

[0517] Use the exoRNeasy Serum / Plasma Kit (77144, Qiagen) to extract total RNA from the solution P2 containing exosomes in the above Example 4.

[0518] <RNA analysis>

[0519] Use the Agilent RNA 6000 Picogram Kit (Agilent Technologies) and the Agilent Small RNA Kit (Agilent Technologies), and use the Agilent 2100 Bioanalyzer to perform quality identification on the obtained total RNA. After quality identification, use the Stranded RNA Library Preparation Kit and the NEBNextUltra II Directional RNA Library Preparation Kit for Illumina to prepare an RNA library from the above total RNA. Perform mRNA-Seq analysis using the prepared RNA library.

[0520] Similarly, a miRNA library was prepared using a miRNA library preparation kit, QIAseq miRNA Library Kit (Qiagen), and QIAseq miRNA NSG 96 Index IL (Qiagen) from the total RNA described above. The prepared miRNA library was used for miRNA-Seq analysis.

[0521] The quality of the sequencing library was identified using a high-sensitivity DNA kit (Agilent Technologies) with an Agilent 2100 Bioanalyzer.

[0522] NGS was performed using NextSeq500 and Illumina (single-end, 75 bp, with an average read count of approximately 10 million reads).

[0523] After quality assessment (FastQC) of the sequenced data, the reads were aligned (mapping) to the reference genome (Human hg38) using the GeneGlobe Data Analysis Center (QIAGEN), and the expression levels were normalized using the trimmed mean of M-values (TMM). Subsequently, an Excel file containing the annotation information of each miRNA and the classification statistics of the small RNA composition was created (analysis tools: StrandNGS v4.0, R v3.6.2; annotation information: based on miRBase Release 21). After that, as data analysis, after extracting genes with altered expression, GO analysis and Pathway analysis based on target gene prediction were performed.

[0524] The number of genes detected from the above solution P2 containing exosomes was 1350 in the normal group and 1346 in the above YA group. Next, when comparing the above normal group and the above YA group, 377 genes with a fold change of 1.1 or more were detected. Among them, in the above YA group, 121 genes with a fold change of 2 or more and overexpressed were detected.

[0525] <Selection of functional marker miRNAs>

[0526] 1. miRNAs as Parkinson's disease markers

[0527] Referring to the following literature, 39 miRNAs with decreased expression in the human brain tissue of Parkinson's disease were selected. By administering exosomes containing the above miRNAs to the cells of Parkinson's disease patients, the function of the above miRNAs can be exogenously supplemented in Parkinson's disease patients, and a therapeutic effect for Parkinson's disease can be expected.

[0528] References:

[0529] ·MicroRNAs in Parkinson’s disease and emerging therapeutictargets.Neural Regeneration Research,12(12),pp.1945-1959(2017)

[0530] ·"Lei Zhou et al.MicroRNA―128Protects Dopamine Neurons fromApoptosis and Upregulates the Expression of Excitatory Amino Acid Transporter4in Parkinson’s Disease by Binding to AXIN1.Cell Physiol Biochem.2018;51(5):2275-2289.

[0531] ·Eashita Das et al.MicroRNA-432contributes to dopamine cocktail andretinoic acid induced differentiation of human neuroblastoma cells bytargeting NESTIN and RCOR1genes.FEBS Lett.2014May 2;588(9):1706-14.

[0532] ·Anna-Elisa Roser et al.miR-182-5p and miR-183-5p Act as GDNFMimics.Molecular Therapy.Mol Ther Nucleic Acids.2018Jun 1;11:9-22.

[0533] ·Sifan Sun et al.MicroRNA-212-5p Prevents Dopaminergic Neuron Deathby Inhibiting SIRT2 in MPTP-Induced Mouse Model of Parkinson’s Disease.FrontMol Neurosci.2018Oct 11;11:381.

[0534] Next, three of them were selected as the miRNAs detected in the exosomal miRNAs derived from the above YA group. The three selected miRNAs are shown in Figure 5 .

[0535] 2. miRNAs as Alzheimer's disease markers

[0536] Using IMOTA (https: / / ccb-web.cs.uni-saarland.de / imota / ), miRNAs associated with the following proteins were screened. The proteins are proteins associated with the pathological conditions of Alzheimer's disease, particularly the following proteins associated with multiple proteins that contribute to the pharmacological effects of Alzheimer's disease therapeutics.

[0537] 2-1. miRNAs as Alzheimer's disease markers (1)

[0538] Using IMOTA, 70 miRNAs associated with amyloid precursor protein (APP: Amyloid Precursor Protein) in the cerebral cortex were selected. APP is the main component of senile plaques, which are deposits of amyloid-β protein, and is listed as one of the causes of Alzheimer's disease.

[0539] Next, five of them were selected as the miRNAs detected in the exosomal miRNAs derived from NHA-YA. The five selected miRNAs are shown in Figure 6 .

[0540] 2-2. miRNAs as Alzheimer's disease markers (2)

[0541] Using IMOTA, 42 miRNAs associated with BACE1 (β-site APP cleaving enzyme) in the cerebral cortex were selected. When Alzheimer's disease develops, BACE1 cleaves the N-terminal part of APP, thereby producing abnormal amyloid-β protein.

[0542] Next, two of them were selected as the miRNAs detected in the exosomal miRNAs derived from NHA-YA. The two selected miRNAs are shown in Figure 7 .

[0543] 2-3. miRNAs as Alzheimer's disease markers (3)

[0544] Using IMOTA (https: / / ccb-web.cs.uni-saarland.de / imota / ), 66 miRNAs related to NMDA receptors (N-methyl-D-aspartic acid receptors) in the cerebral cortex were selected. When Alzheimer's disease develops, abnormal proteins accumulate in the brain, thereby excessively releasing substances that excite nerves. Through this excitatory substance, NMDA receptors are over-activated, thereby causing disorders in nerve transmission and memory.

[0545] Next, 3 miRNAs detected as miRNAs derived from NHA-YA exosomes were selected from among them. The 3 selected miRNAs are shown in Figure 8 .

[0546] 2-4. miRNAs as Alzheimer's disease markers (4)

[0547] Using IMOTA (https: / / ccb-web.cs.uni-saarland.de / imota / ), 58 miRNAs related to glycogen synthase kinase 3β (GSK-3β: Glycogen synthase kinase-3β) in the cerebral cortex were selected. GSK-3β is an enzyme that phosphorylates various proteins and plays diverse roles in the maintenance of cell life and the regulation of physiological functions via multiple pathways. In Alzheimer's disease, it promotes the deposition of amyloid-β protein in the brain and the accumulation of Tau protein in nerve cells. As a result, it induces apoptosis of nerve cells.

[0548] Next, 2 miRNAs detected as miRNAs derived from NHA-YA exosomes were selected from among them. The 2 selected miRNAs are shown in Figure 9 .

[0549] 3. miRNAs as depression markers

[0550] Referring to the following literature, 24 miRNAs with reduced expression in the plasma of individuals with depression were selected. By administering exosomes containing this miRNA to the cells of patients with depression, the function of this miRNA is exogenously supplemented in patients with depression, and a therapeutic effect on depression can be expected.

[0551] Literature: MicroRNAs expressed in depression and their associated pathways: A systematic review and a bioinformatics analysis (Journal of Chemical Neuroanatomy 100(2019)101650)

[0552] Next, one of them was selected as the miRNA in which exosomal miRNA derived from NHA-YA was detected. The selected one miRNA is shown in Figure 10 .

[0553] [Example 7] Verification of the neuroprotective effect of NHA-YA-derived exosomes against corticosterone injury

[0554] As a functional evaluation test of NHA-YA-derived exosomes, a neurite outgrowth inhibition test was performed on a rat adrenal pheochromocytoma-derived cell line, PC-12 cells (RCB0009, RIKEN Cell Bank). Corticosterone is an adrenocortical hormone (glucocorticoid) synthesized by the adrenal cortex, which shows toxicity to PC-12 cells and induces apoptosis of the cells by adding a specified amount to the culture medium. The quantification of apoptosis induction was performed by measuring the activities of caspase 3 and caspase 7.

[0555] [Reagents and culture products]

[0556] The following reagents and culture products were used.

[0557] Culture of PC-12 cells

[0558] · Composition of the proliferation medium: basal medium, 10% (v / v) FBS, 10% (v / v) HS, antibiotics

[0559] Basal medium: DMEM, high glucose, pyruvate (11995-073; Gibco)

[0560] FBS: fetal bovine serum, premium grade, Brazil (10270-106; Gibco)

[0561] HS: horse serum, heat-inactivated, New Zealand blood source (26050-088; Gibco)

[0562] Antibiotics: Antibiotic-Antimycotic (100X) (15240-062; Gibco)

[0563] · Cell detachment agent 1: Accutase (AT104, ICT)

[0564] · Cell detachment agent 2: TrypLE Express Enzyme (1X), phenol red - free (12604 - 013, Gibco)

[0565] · Cell buffer: Phosphate - Buffered Saline Solution (PBS(-), Dulbecco; calcium - and magnesium - free) (BNDSBN200, KAC)

[0566] · Cell cryopreservation medium: CELLBANKER 1 (CB011 Takara Bio (Nippon Zenyaku Kogyo))

[0567] · Composition of the analysis medium: basal medium, additives, antibiotics

[0568] Basal medium: Advanced DMEM (12491 - 015, Gibco)

[0569] Additives: GlutaMAX Supplement (100X) (35050 - 061, Gibco)

[0570] Antibiotics: Antibiotic - Antimycotic (100X) (15240 - 062, Gibco)

[0571] · Apoptosis - inducing reagent: Corticosterone - 100MG (C0388 - 100MG Tokyo Chemical Industry)

[0572] · Dissolving solvent: Dimethyl Sulfoxide (DMSO) (472301 - 100ML SIGMA - ALDRICH)

[0573] · Apoptosis assay reagent: Caspase GlO 3 / 7 Assay System (G8091 Promega)

[0574] Equipment

[0575] · For proliferation: Cell culture dish 100mm (150466 THERMO Scientific)

[0576] · For analysis: Collagen I - coated 96 - well plate (4860 - 010 IWAKI)

[0577] · Luminescence measurement equipment: Synergy H4 (BioTeck)

[0578] Culture of astrocytes for exosome recovery

[0579] The culture media, reagents, and equipment used are in accordance with Example 4 above.

[0580] · Culture medium for cultivation: AGM astrocyte growth medium bullet kit (CC-3186, Lonza) (Composition of AGM: basal medium, FBS (3% (v / v)), L-glutamine, ascorbic acid, hEGF, insulin, antibiotics) (same as the medium used in Example 1)

[0581] · Inhibitor Y: CultureSure® Y-27632 (034-24024, Fujifilm Wako Pure Chemical Corporation), final concentration: 10 μM (same as the Inhibitor Y used in Example 1)

[0582] · Inhibitor A: CultureSure® A-83-01 (035-24113, Fujifilm Wako Pure Chemical Corporation), final concentration: 0.5 μM (same as the Inhibitor A used in Example 1)

[0583] · Exosome-depleted bovine serum: Disposable type premium exosome-depleted fetal bovine serum (A2720803, Gibco)

[0584] · 0.22 μm filtration system: Stericup Quick Release-GP sterile vacuum filtration system (S2GPU02RE, Merck Millipore)

[0585] · Culture medium for collecting culture supernatant: Medium obtained by adding 3% (v / v) of exosome-depleted bovine serum (A2720803; Gibco) to replace the bovine serum (FBS) which is a component of the AGM astrocyte growth medium bullet kit (CC-3186, Lonza)

[0586] · Washing buffer: Dulbecco PBS(-) powder "Nissui" (05913, Nissui Pharmaceutical)

[0587] · 96-well plate: Collagen I-coated 96-well plate (4860-010, IWAKI)

[0588] · T-75 flask for cell culture (430641, Corning)

[0589] · 100 mm Petri dish for cell culture: 150466, Thermo Fisher Scientific (same as the Petri dish used in Example 1)

[0590] · 150 mm Petri dish for cell culture: 150468, Thermo Fisher Scientific (same as the Petri dish used in Example 1)

[0591] Equipment

[0592] · Ultracentrifuge: Optima XE-90 (Beckman Coulter)

[0593] · Rotor: SW-41Ti 6×13.2 mL (Beckman Coulter)

[0594] · Centrifugation tube: Ultra-Clear Tube (344059 Beckman Coulter)

[0595] · Storage tube: Prokeep Protein Low Adsorption Tube 1.5 mL (PK-15C-500N Watson)

[0596] <Recovery and Confirmation of Exosomes in Culture Supernatant>

[0597] Using the same materials, reagents, culture products, and operating procedures as those used in Example 4, the culture supernatants during the culture of human astrocytes were recovered using the above-mentioned main culture medium containing inhibitor YA or the main culture medium without inhibitor, respectively. The specific method is as follows.

[0598] 1. Materials

[0599] As NHA, the same materials as those used in Example 1 were used.

[0600] 2. Recovery of Culture Supernatant of Human Astrocytes

[0601] The cryotube storing the cell culture solution containing NHA was thawed in a 37°C water bath. The thawed cells were resuspended in 30 mL of culture medium and seeded in a T-75 cell culture flask at an inoculation density of 1.4×10 4 cells / cm 2 for culture. The next day, the medium was replaced with the above-mentioned main culture medium containing inhibitor YA and the above-mentioned main culture medium without inhibitor (hereinafter referred to as the main culture medium) at a volume of 15 mL / flask, and the medium was replaced every 2 or 3 days and the main culture was continued.

[0602] After culturing for 7 days using the above-mentioned main culture medium, the cells were detached using a cell detachment agent, resuspended in fresh above-mentioned main culture medium, and seeded at 3.3×10 3 cells / cm 2The inoculation density with a volume of 20 mL / dish was passaged into a 150-mm cell culture dish (second passage). Five dishes were used under each condition with and without the above inhibitor YA, so the total number of cells inoculated was 5×10 6 cells. Similar to Example 1 above, the cell group cultured with the above main culture medium containing YA was called the YA group, and the cell group cultured with the above main culture medium without YA was called the normal group.

[0603] Three days after the second passage (main culture: 10 days in total), the medium in the above culture dish was changed from the above main culture medium with a volume of 20 mL / dish to the recovery medium without inhibitor for recovering the above culture supernatant (hereinafter referred to as the recovery medium), and additional culture for exosome recovery was started. It should be noted that the same medium as in Example 1 above was used for the above recovery medium.

[0604] Forty-eight hours after the start of the above additional culture, the culture supernatant was recovered from the above culture dish and filtered using a 0.22-μm filtration system (Stericup, Merck Millipore), and the filtrate was stored at 4°C. Further, fresh above recovery medium was added to the above culture dish after the above culture supernatant was recovered with a volume of 20 mL / dish, and the additional culture was continued. The above culture supernatant was recovered three times in total for 6 days in this way, and 305 mL of culture supernatant P2 (solution containing exosomes) was obtained under each condition.

[0605] The cells remaining on the above culture dish after recovering the culture supernatant three times were detached using a cell detachment agent, and the number of cells was counted. As a result, the total number of cells obtained as the normal group was 6.4×10 6 cells, and the residual rate relative to the total number of inoculated cells was 73.6%. In contrast, the total number of cells obtained as the above YA group was 7.1×10 7 cells, and the residual rate relative to the total number of inoculated cells was 96.7%, obtaining a result higher than the above normal group.

[0606] 3. Concentration of exosomes in the culture supernatant

[0607] Transfer 305 mL of the culture supernatant P2 (solution containing exosomes) of the above-mentioned recycled YA group and the above-mentioned normal group to a tube, and use an ultracentrifuge (main body: Optima XE-90, rotor: SW41 Ti; Beckman Coulter) to perform centrifugation at 250,000×g for 70 minutes at 4°C. After removing the supernatant and recovering the precipitate, in order to wash the obtained precipitate, add the above-mentioned washing buffer (PBS(-)) to the above-mentioned precipitate, and then perform centrifugation at 250,000×g for 70 minutes at 4°C. Remove the supernatant to obtain the washed precipitate. The washed precipitate is resuspended with PBS(-) to make it reach about 1 / 1000 of the volume of the above-mentioned culture supernatant P2. The resuspended solution is used as the evaluation sample. PBS(-) is used as the control for the above-mentioned evaluation sample.

[0608] <Evaluation experimental protocol>

[0609] Thaw the cryotube of the above-mentioned PC-12 cells in a 37°C water bath. Transfer the thawed cells to the above-mentioned proliferation medium in a total volume of 10 mL, and after subjecting them to centrifugation at 180×g for 5 minutes at room temperature, remove the above-mentioned medium. Resuspend the cells again in fresh proliferation medium and inoculate them in a 100 mm cell culture dish at an inoculation density of about 5,300 cells / cm 2 . Replace the medium every 2 or 3 days and culture at 37°C, and proliferate the cells while performing passage. Detach the obtained PC-12 cells using the above-mentioned cell detachment agent 2, wash them with the above-mentioned analysis medium, and then resuspend them in fresh analysis medium. Inoculate the suspended cells into a 96-well plate (96-well plate coated with collagen I) at an inoculation density of 2.0×10 4 cells / 100 μL medium, that is, 61,000 cells / cm 2 . After culturing for about 24 hours to allow the cells to adhere, replace the medium in the above-mentioned wells with 50 μL of analysis medium (protein mass: 10 μg / mL) containing 100 μM corticosterone and each of the above-mentioned evaluation samples. Thereafter, culture for 2 days at 37°C in the analysis medium containing the above-mentioned evaluation samples, and evaluate the damage status of the cells in the obtained culture by measuring caspase 3 / 7 activity.

[0610] Caspase 3 / 7 activity was measured using a commercially available apoptosis assay reagent based on the operating procedures attached to the above assay reagent. The above assay reagent uses a homogeneous luminescence analysis reagent for measuring caspase 3 / 7 activity. Activation of caspase 3 / 7 activity is an indicator of apoptosis induction. Therefore, the measurement results (luminescence amount) of caspase 3 / 7 activity are shown in Table 10 as an indirect result of the degree of apoptosis. In Table 10, YA is the result using the evaluation sample from the YA group, Normal is the result using the evaluation sample from the normal group, and PBS is the result using the control (PBS(-)). In Table 10, the luminescence amount is expressed as a relative luminescence value with the average luminescence amount of the control set to 1.

[0611] [Table 10]

[0612] Relative value of apoptosis amount

[0613] Group Relative luminescence value Normal 0.75 YA 0.73 Control (PBS) 1

[0614] As a result, as shown in Table 10, in the evaluation exosome samples from the above normal group and the evaluation exosome samples from the above YA group, the relative luminescence value was significantly reduced to a 25% decrease in the normal group and a 27% decrease in the NHA-YA group compared to the control (PBS(-)). In addition, compared with the evaluation exosome samples from the above normal group, a further decrease in the luminescence amount was confirmed in the evaluation exosome samples from the above YA group.

[0615] When cells (PC-12) are damaged by corticosterone, the caspase 3 / 7 pathway is activated and the cells tend to die. That is, apoptosis is induced. When caspase 3 / 7 is further activated, the luminescence amount also further increases in the above activity evaluation. That is, a relatively large luminescence amount means a strong degree of damage to the target cells and that they are heading towards apoptosis. Therefore, in the case of using the above evaluation sample from the YA group containing the exosomes of the present disclosure, compared with the cases of using the control (PBS(-)) and the evaluation sample from the above normal group, the target cells PC-12 showed the smallest degree of damage. Therefore, it can be known that the exosomes of the present disclosure secreted by the cells of the above YA group have the function of inhibiting cell damage by corticosterone and protecting cells.

[0616] [Example 8] Confirmation of the trait stability of YA-induced cells

[0617] It was confirmed whether the traits of the ectodermal cell population cultured using the above main culture medium containing the inhibitor YA could be maintained in the absence of the above inhibitor.

[0618] [Sample]

[0619] · Normal

[0620] The cell population (normal group) obtained by main culture in the absence of the above inhibitors in Example 1 above. Cells stored in cryotubes from the cells on the 9th day (second passage, N-D9) and the 28th day (fourth passage, N-D28) after the start of main culture were used. Hereinafter, the former is denoted as N-D9 (P2), and the latter is denoted as N-D28 (P4).

[0621] ·NHA-YA

[0622] The cell population (normal group) obtained by main culture in the presence of the above inhibitor Y and the above inhibitor A in Example 1 above. Cells stored in cryotubes from the cells on the 9th day (second passage, YA-D9) and the 28th day (fourth passage, YA-D28)) after the start of main culture were used.

[0623] <Reagents and culture products>

[0624] ·Culture medium for culture: AGM astrocyte growth medium bullet kit (CC-3186, Lonza) (Composition of AGM: basal medium, FBS (3% (v / v)), L-glutamine, ascorbic acid, hEGF, insulin, antibiotics)

[0625] ·Cell detachment agent: Accutase (AT104, ICT)

[0626] ·Cell buffer: Phosphate buffered saline solution (PBS(-), Dulbecco; calcium and magnesium free) (14190250, Thermo Fisher Scientific)

[0627] ·Cell cryopreservation medium: CELLBANKER 1 (CB011 TaKaRa (Nippon Zenyaku Kogyo Co., Ltd.))

[0628] ·Culture dish for cell culture 100 mm (150466, Thermo Fisher Scientific)

[0629] ·Centrifuge: Optima XE-90, Beckman Coulter

[0630] <Culture apparatus>

[0631] ·CO2 incubator: MCO-170AICUVD-PJ, PHC Corporation

[0632] <Culture conditions>

[0633] Temperature 37 °C, CO2 concentration 5%

[0634] <Cell Observation and Imaging Device>

[0635] ·Fluorescence microscope CKX53, Olympus Corporation

[0636] <Cultivation Process>

[0637] Unless otherwise specified, the cultivation process follows that of Example 1. Thaw the cryotubes of the above-mentioned N-D9 (P2), N-D28 (P2), YA-D9 (P4), and YA-D28 (P4) in a 37°C water bath. Then, transfer the thawed cells to the culture medium to a total volume of 10 mL, and subject them to centrifugation at 180×g for 5 minutes at room temperature, followed by removing the medium. Resuspend the cells in fresh culture medium and inoculate them into 100-mm cell culture dishes. At this time, for YA-D9 and YA-D28, divide the same cells into two portions and inoculate them into 100-mm cell culture dishes respectively. The next day, replace the medium in the above-mentioned dishes with the above-mentioned inhibitor-free culture medium (day 0 of cultivation start), and continue the cultivation. Thereafter, for N-D9, N-D28, YA-D9, and YA-D28, also cultivate them using the above-mentioned inhibitor-free culture medium. During the cultivation period, replace the medium with the above-mentioned inhibitor-free culture medium every 2 or 3 days. After 5 to 7 days of cultivation, repeat the subculture and perform cultivation with 4 subcultures in 24 days. During the cultivation period, replace the medium and observe and photograph the cells using a microscope (Tomy Seiko, MX-307).

[0638] After that, for the N-D9 and YA-D9 that have been subcultured twice, calculate the proliferation rates of each generation (P3, P4, P5, P6) during the cultivation period of further 4 subcultures in the absence of the above-mentioned inhibitor. For the N-D28 and YA-D28 that have been subcultured 4 times, calculate the proliferation rates of each generation (P5, P6, P7, P8) during the cultivation period of further 4 subcultures in the absence of the above-mentioned inhibitor. After that, for the proliferation rates of each generation of YA-D9 and YA-D28 in the absence of the inhibitor (YA(-)), set the proliferation rate of the same generation in the normal group to 1 and calculate the relative values. These results are shown in Table 11.

[0639] [Table 11]

[0640]

[0641] As shown in Table 11, in the above-mentioned Example 1, the above-mentioned YA group (YA-D9 and YA-D28) obtained by culturing in the presence of the above-mentioned inhibitor can maintain a high proliferation rate even when further cultured in the absence of the above-mentioned inhibitor YA, as compared with the above-mentioned normal group (N-D9 and N-D28).

[0642] As a representative example, in Fig. 11, Figure 11-1 shows the cell morphology of the 3rd passage (P3) of N-D9 and YA-D9 and the 5th passage (P5) of N-D28 and YA-D28, and Figure 11-2 shows the cell morphology of the 6th passage (P6) of N-D9 and YA-D9 and the 8th passage (P8) of N-D28 and YA-D28. In any passage of YA-D9 and YA-D28, in addition to small and dense cell clusters, images of cells with a protruded morphology proliferating three-dimensionally were confirmed. On the other hand, in any passage of N-D9 (P4) and N-D28 (P6), the cells increased in size and the cells with a protruded morphology decreased. In addition, in any passage of YA-D9 and YA-D28, although the small cells decreased, the cells with a protruded morphology were maintained more than N-D9 and N-D28. Since the morphology of YA-D9 and YA-D28 is similar to the morphology confirmed in the above-mentioned Example 1, it can be seen that even when the high-proliferation cells of the present disclosure are further cultured in the absence of the above-mentioned inhibitor, the proliferation and morphology of the above-mentioned high-proliferation cells can be maintained.

[0643] <Confirmation of ectodermal cell markers based on quantitative PCR>

[0644] In this example, RNA samples were extracted from the cells of the 4th passage (P4) of N-D9 and the 4th passage (P4) of YA-D9 cultured in the absence of the above-mentioned inhibitor, and the 6th passage (P6) of N-D28 and the 6th passage (P6) of YA-D28 cultured in the absence of the above-mentioned inhibitor, and the expression levels of the following ectodermal cell marker genes were studied.

[0645] The preparation of the above-mentioned RNA samples and the setting of the reaction conditions for quantitative PCR were carried out according to the above-mentioned Example 2. For the above-mentioned RNA samples, the expression levels of Musashi1 and Notch1 as neural stem cell markers, Notch1, Nestin and SOX2 as neuroepithelial cell markers, Nestin as radial glial cell marker, GFAP, S100B and SLC1A3 as astrocyte markers, and NG2 as oligodendrocyte progenitor cell marker were measured, and the expression level of the internal standard gene GAPDH was used for calibration (E1 / E C)。In addition, similarly, for the above-mentioned N-D9(P2), N-D28(P2), YA-D9(P4), and YA-D28(P4) before the start of subculture, RNA samples were similarly prepared from the cells on day 0 of the culture starting from the replacement with the above-mentioned inhibitor-free culture medium, and the expression levels of the above-mentioned marker genes were measured and corrected (E1 / E C )。Next, the increase or decrease was calculated based on the corrected relative values in the cells on day 0 of the culture and the corrected relative values in the cells of N-D9(P4), YA-D9(P4), N-D28(P6), and YA-D28(P6) after subculture. After that, the relative value of the increase or decrease in the expression of YA-D9 relative to the increase or decrease in the expression of N-D9 and the relative value of the increase or decrease in the expression of YA-D28 relative to the increase or decrease in the expression of N-D28 were calculated in the form of relative values (E YA / E N ). These results are shown in Table 12.

[0646] [Table 12]

[0647]

[0648] As shown in Table 12, in YA-D9(P4), the expression levels of GFAP, Musashi1, Notch1, Nestin, S100B, and SLC1A3 were higher than those in N-D9(P4), which was consistent with the results of Example 2 above. On the other hand, in YA-D9(P4), the expression level of SOX2 was slightly lower than normal and the expression level of NG2 was slightly higher. In addition, in YA-D28(P6), the expression levels of GFAP, Musashi1, Notch1, SOX2, Nestin, S100B, and SLC1A3 were all higher than those in N-28(P6), and the expression level of NG2 was lower than that in N-28(P6), which was consistent with the gene expression profile of the cells cultured in the presence of the YA inhibitor confirmed in Example 2 above.

[0649] Based on these results, it was confirmed that the characteristics of the ectodermal cell population cultured using the above-mentioned main culture medium containing the inhibitor YA were maintained even after subsequent culture in the absence of the above-mentioned inhibitor.

[0650] The present invention has been described with reference to the embodiments, but the present invention is not limited to the above-mentioned embodiments. Various modifications that can be understood by those skilled in the art can be made to the constitution and details of the present invention within the scope of the present invention.

[0651] This application claims priority based on Japanese Patent Application No. 2022-192595 filed on December 1, 2022, and incorporates the entire contents disclosed therein into this application.

[0652] Industrial applicability

[0653] According to the present disclosure, highly proliferative cells, a method for producing the same, and uses thereof can be provided.

Claims

1. A highly proliferative cell derived from ectodermal cells, which has the characteristics of ectodermal cells and shows the expression behavior of A1 below, (Al) Among the highly proliferating cells, the expression level E1 of at least one marker gene selected from the group consisting of GFAP, S100B, Musashi1, CSPG4, Nestin, and SLC1A3 and the expression level E of the control gene C The determined relative value E1 / E C Meets the reference range shown in Table 1 below, [Table 1] 。 2. A highly proliferative cell, which has the characteristics of ectodermal cells and has the following proliferative ability: the number of cells after a culture period of 28 days or less in contact with a low-molecular-weight signal transduction pathway inhibitor is more than 1.0 times the number of ectodermal cells cultured under the same culture conditions for the same culture period except without contact with the inhibitor.

3. The highly proliferative cell according to claim 1 or 2, wherein The highly proliferative cell includes the following cells: the expression level of at least one marker gene selected from GFAP, SOX2, Musashi1, and Nestin is more than 1.0 times that of ectodermal cells cultured under the same culture conditions for the same culture period except without contact with a low-molecular-weight signal transduction pathway inhibitor.

4. The highly proliferative cell according to any one of claims 1 to 3, wherein, The highly proliferative cell includes ectodermal progenitor cells.

5. The highly proliferating cell according to any one of claims 1 to 4, wherein, The ectodermal cells include astrocytes.

6. A method for producing a highly proliferative cell, which includes: (i) Culturing ectodermal cells as a raw material in the presence of a low-molecular-weight signal transduction pathway inhibitor to obtain a highly proliferative cell with a higher cell proliferation ability than the ectodermal cells as the raw material, In (i) above, the culture period in the presence of the inhibitor is 28 days or less.

7. A method for producing a highly proliferative cell, which includes: (i) Culturing ectodermal cells as a raw material in the presence of a low-molecular-weight signal transduction pathway inhibitor to obtain a highly proliferative cell with a higher cell proliferation ability than the ectodermal cells as the raw material, In (i) above, the culture in the presence of the inhibitor is carried out until at least one condition selected from the group consisting of condition a1, condition b1, and condition c1 is satisfied, (a1) In the cells cultured in the presence of the inhibitor, the expression level E1 of at least one marker gene selected from the group consisting of GFAP, S100B, Musashi1, CSPG4, Nestin, and SLC1A3 and the expression level E of the control gene C The determined relative value E1 / E C Satisfies the reference range shown in Table 2 below; [Table 2] (b1) The number of cells cultured in the presence of the inhibitor is more than 1.0 times the number of ectodermal cells cultured under the same culture conditions for the same culture period except without the inhibitor; (c1) In the cells cultured in the presence of the inhibitor, the expression level of at least one marker gene selected from GFAP, SOX2, Musashi1, and Nestin is more than 1.0 times that of ectodermal cells cultured under the same culture conditions for the same culture period except without the inhibitor.

8. The method for producing highly proliferative cells according to claim 6 or 7, wherein, The ectodermal cells as the raw material include cells of the central nervous system.

9. The method for producing highly proliferative cells according to any one of claims 6 to 8, wherein, The ectodermal cells as the raw material include astrocytes.

10. The method for producing highly proliferative cells according to any one of claims 6 to 9, wherein, The inhibitor includes at least one compound selected from the group consisting of TGFβ receptor inhibitors and ROCK inhibitors.

11. The method for producing highly proliferative cells according to any one of claims 6 to 10, wherein, In (i) above, the culture in the presence of the inhibitor is carried out using a culture medium containing the inhibitor, and the concentration of the inhibitor in the culture medium is in the range of 0.001 μM to 100 μM.

12. A method for producing a cell secretion from a highly proliferative cell, which includes: (I) A culture of highly proliferative cells derived from ectodermal cells according to any one of claims 1 to 5, or a culture of highly proliferative cells obtained by carrying out the production method according to any one of claims 6 to 11; and (II) isolating a cell secretion secreted by the highly proliferative cells from the culture.

13. The method for manufacturing a cell secretion according to claim 12, wherein, The cell secretion contains exosomes.

14. A cell secretion, which is a cell secretion containing at least one of a protein and an miRNA, wherein the protein contains a combination of glycoprotein M6B (Q13491), HLA class II histocompatibility antigen DRα chain (P01903), tweety homolog 1 (Q9H313), fibrillin 2 (P35556), HLA class II histocompatibility antigen DRB1β chain (P01911), and S100 calcium-binding protein A8 (P05109), the miRNA contains a combination of hsa-miR-206 (MIMAT0000462), hsa-miR-204-5p (MIMAT0000265), hsa-miR-128-3p (MIMAT0000424), hsa-miR-363-3p (MIMAT0000707), and hsa-miR-323a-3p (MIMAT0000755).

Images