Composition containing cell culture supernatant and method for producing the same

Abstract

Provided is a novel use for a stem cell culture supernatant. Provided is a composition that contains a stem cell culture supernatant and that is for use in medical or cosmetic applications for a subject.

Description

[Technical Field] 【0001】 This disclosure relates to a composition comprising a stem cell culture supernatant for use in pharmaceutical or cosmetic applications, and a method for producing the same. [Background technology] 【0002】 Cell therapies containing human-derived nerve cells obtained by differentiating human-derived pluripotent stem cells (e.g., embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells)) are attracting attention for the treatment of spinal cord injury and neurodegenerative diseases caused by damage to specific nerve cells. 【0003】 As described in Patent Document 1, the present inventors have created stem cells capable of differentiating into nerve cells, chondrocytes, osteocytes, or adipocytes, and have demonstrated that these cells, which have been induced to differentiate into nerve cells, are effective in treating a rat spinal cord injury model. [Prior art documents] [Patent Documents] 【0004】 [Patent Document 1] Japanese Patent Publication No. 2019-076008 [Patent Document 2] Japanese Patent Publication No. 2021-141872 [Non-patent literature] 【0005】 [Non-Patent Document 1] Basso OM et al. A sensitive and reliable locomotor rating scale for open field testing in rats. J.Neurotrauma 1995; 12: 1-21. [Overview of the project] [Problems that the invention aims to solve] 【0006】 However, by-products such as the culture supernatant generated during the culture of stem cells were discarded. Considering the impact on the environment and the like, it is necessary to provide new uses for the culture supernatant. 【Means for Solving the Problems】 【0007】 The inventors of the present invention conducted intensive studies to solve the above problems. It was considered that stem cells not only bring a therapeutic effect by themselves, but also various bioactive substances such as cytokines and exosomes secreted by stem cells contribute to the therapeutic effect. Since the artificially cultured stem cells release these bioactive substances into the culture medium, the inventors found that the culture supernatant from which the cells were removed could also be expected to have a therapeutically or cosmetically effective effect, and thus completed the present invention. 【0008】 The present disclosure relates to the following compositions and methods for producing the compositions: [Item 1] A pharmaceutical composition or a cosmetic composition containing the culture supernatant of stem cells, wherein the stem cells express either or both of CCN1 and CCN2, and the stem cells are immortalized. [Item 2] The composition according to Item 1, wherein the stem cells are stem cells immortalized by introducing telomerase (TERT). [Item 3] The composition according to Item 1, wherein the stem cells are bound to an antibody having the CDR sequences of SEQ ID NOs: 1 to 6. [Item 4] The composition according to Item 1, wherein the stem cells are X4N cells deposited under the accession number P-02505. [Item 5] The composition according to Item 1, wherein the stem cells include undifferentiated stem cells and / or nerve-differentiated cells. [Item 6] The culture supernatant contains BDNF, BMP, BMP1, BMP10, BMP2, BMP4, BMP5, BMP7, CLEC, COL, COL11A1, COL11A2, COL14A1, COL18A1, COL1A1, COL1A. 2, COL22A1, COL2A1, COL3A1, COL4A2, COL5A1, COL5A2, COL6A1, COL6A2, CSF, CSF1, CXCL1, CXCL12, DKK1, EDN1, EFEMP, EF EMP1, ELN, ENO1, FGF, FGF1, FGF18, FGF7, FGF9, FN, FN1, FRZB, FSTL1, GDF, GDF10, GDF11, GDF15, GDF2, GMFB, GRN, HDGF, H GF, IGF, IGF2, IGFBP, IGFBP5, IGFBP7, IL, INHBA, JAG1, KGF, KIF20B, KRT, KRT1, KRT10, KRT2, KRT9, LGALS3BP, LGBDNF, LO XL2, MANF, MIA, MIF, MMP, MMP1, MMP10, MMP11, MMP14, MMP15, MMP2, MMP23, MMP9, MMP, NGF, NID2, NRG1, NT3, PCOLCE, PDGF , PDGF-C, PDGF-D, PTN, PTX3, SCG, SCG2, SDF-1, SFRP, SFRP1, SFRP2, SFRP3, SFRP5, SHANK3, SPARC, TBX, TBX20, TBX5, TGF A composition according to any one of items 1 to 5, comprising at least one protein selected from the group consisting of -α, TGF-β, TGF-β1, TGF-β2, THBS, THBS1, THBS2, TIMP, TIMP1, TIMP2, TIMP3, TIMP4, VCAN, VEGF, VEGFA, VEGFB, VEGFC, VEGFD, VIM, WNT5A, elastin, glycoprotein, keratin, laminin, basement membrane factors, and combinations thereof. [Section 7] The culture supernatant contains miR-363, miR499, miR-1, miR-21, miR-126, miR-34, miR-16, hsa-miR-1-3p, hsa-miR-675-3p, h sa-miR-10395-3p, hsa-miR-133b, hsa-miR-208b-3p, hsa-miR-4488, hsa-miR-450b-5p, hsa-miR-499b A composition according to any one of items 1 to 6, comprising at least one microRNA (miRNA) selected from the group consisting of -3, hsa-miR-542-3p, hsa-miR-642a-3p and hsa-miR-9901, gga-mir-302a, gga-mir-302b, gga-mir-302c, gga-mir-302d, eca-mir-369, mmu-mir-19a, mmu-mir-20a and combinations thereof. [Section 8] The composition according to any one of items 1 to 7, comprising extracellular vesicles containing at least one protein selected from the group consisting of BDNF, BMP, BMP1, BMP10, BMP2, BMP4, BMP5, BMP7, CLEC, COL, COL11A1, COL11A2, COL14A1, COL18A1, COL1A1, COL1A2, COL22A1, COL2A1, COL3A1, COL4A2, COL5A1, COL5A2, COL6A1, COL6A2, CSF, CSF1, CXCL1, CXCL12, DKK1, EDN1, EFEMP, EFEMP1, ELN, ENO1, FGF, FGF1, FGF18, FGF7, FGF9, FN, FN1, FRZB, FSTL1, GDF, GDF10, GDF11, GDF15, GDF2, GMFB, GRN, HDGF, HGF, IGF, IGF2, IGFBP, IGFBP5, IGFBP7, IL, INHBA, JAG1, KGF, KIF20B, KRT, KRT1, KRT10, KRT2, KRT9, LGALS3BP, LGBDNF, LOXL2, MANF, MIA, MIF, MMP, MMP1, MMP10, MMP11, MMP14, MMP15, MMP2, MMP23, MMP9, MMP, NGF, NID2, NRG1, NT3, PCOLCE, PDGF, PDGF-C, PDGF-D, PTN, PTX3, SCG, SCG2, SDF-1, SFRP, SFRP1, SFRP2, SFRP3, SFRP5, SHANK3, SPARC, TBX, TBX20, TBX5, TGF-α, TGF-β, TGF-β1, TGF-β2, THBS, THBS1, THBS2, TIMP, TIMP1, TIMP2, TIMP3, TIMP4, VCAN, VEGF, VEGFA, VEGFB, VEGFC, VEGFD, VIM, WNT5A, elastin, glycoprotein, keratin, laminin, basement membrane factors and combinations thereof. [Item 9] The culture supernatant contains miR-363, miR499, miR-1, miR-21, miR-126, miR-34, miR-16, hsa-miR-1-3p, hsa-miR-675-3p, hsa-miR- 10395-3p, hsa-miR-133b, hsa-miR-208b-3p, hsa-miR-4488, hsa-miR-450b-5p, hsa-miR-499b-3, hsa-miR-542 A composition according to any one of items 1 to 7, comprising an extracellular vesicle containing at least one miRNA selected from the group consisting of -3p, hsa-miR-642a-3p and hsa-miR-9901, gga-mir-302a, gga-mir-302b, gga-mir-302c, gga-mir-302d, eca-mir-369, mmu-mir-19a, mmu-mir-20a and combinations thereof. [Section 10] The composition according to item 8 or 9, wherein the extracellular vesicles are exosomes approximately 40-120 nm in size. [Section 11] A pharmaceutical composition according to paragraphs 1 to 10 for treating injury or disease in a subject, wherein the injury is trauma or wound in any site or chronic or transient functional impairment resulting therefrom, or the disease is a neurological disorder, depression, myasthenia gravis, dementia, Parkinson's disease, Huntington's disease or schizophrenia, ischemia, circulatory disorder, infarction, osteoporosis, immune disease or periodontitis. [Section 12] The composition according to item 11, wherein the target is a mammal, a bird, or a reptile. [Section 13] The composition according to item 12, wherein the target mammal is human. [Section 14] A composition according to any one of claims 11 to 13, formulated for use via topical, oral, rectal, nasal, intravenous, intra-articular, conjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, transdermal, or subcutaneous administration routes. [Section 15] The pharmaceutical compositions described in items 11 to 13, which are in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions or liquids, injections, drips, transdermal agents, transmucosal agents, nasal drops, inhalants or suppositories. [Section 16] A cosmetic composition as described in any of items 1 to 10, used for skincare, skin whitening, wrinkle improvement, texture improvement, anti-aging, and hair growth. [Section 17] The cosmetic composition described in item 16, which is in the form of a lotion, emulsion, gel, serum, cream, pack, mask, facial cleanser, cosmetic soap, foundation, powder, or body lotion. [Section 18] i) the process of culturing the seeded stem cell population; and ii) Step of collecting the culture supernatant A method for producing the compositions described in items 1 to 17, including the composition described in items 1 to 17. [Effects of the Invention] 【0009】 This disclosure provides a composition comprising a stem cell culture supernatant for use in pharmaceutical or cosmetic applications, and a method for producing the same. [Brief explanation of the drawing] 【0010】 [Figure 1] Figure 1 is a line graph showing the changes in Basso-Beattie-Bresnahan (BBB) ​​scores for each test group in a rat spinal cord injury model: the group administered culture supernatant via tail vein (○; solid line), the group administered culture supernatant intrathecally (□; dashed line), and the control group administered a solvent via tail vein (■; solid line). 【0011】 [Figure 2A] Figure 2 shows hematoxylin-eosin (HE) stained images of rat tissue collected 39 days after spinal cord injury from the control group, the group administered culture supernatant via tail vein (intravenous injection), and the group administered culture supernatant intrathecally (intrathecal injection) (upper panel: low magnification, lower panel: high magnification). Figure 2A shows HE stained images of spinal cord tissue sections around the injury site from rats in each group. [Figure 2B] Figure 2B shows HE-stained images of brain tissue sections from rats in each group. [Figure 2C] Figure 2C shows HE-stained images of liver tissue sections from rats in each group. [Figure 2D] Figure 2D shows HE-stained images of kidney tissue sections from rats in each group. [Figure 3] Figure 3 shows the BDNF concentration in the culture supernatant measured by ELISA. BDNF concentrations were measured in the test culture supernatant of X4N cells, the culture supernatant of iPS cells, the culture supernatant of human mesenchymal stem cells (hMSCs), and culture medium (RHB-A or ReproCELL). [Figure 4A] Figure 4A shows a schematic diagram of the exosome fraction of the culture supernatant. [Figure 4B] Figure 4B shows the BDNF concentrations in each fraction of the culture supernatant, as measured by the ELISA method. [Figure 5A] Figure 5A shows the results of a mesenchymal stem cell proliferation test when the culture supernatant according to this disclosure was added to the mesenchymal stem cell culture system. [Figure 5B] Figure 5B shows the results of a fibroblast proliferation test when the culture supernatant according to this disclosure was added to a fibroblast culture system. [Figure 5C] Figure 5C shows the results of a fibroblast proliferation test when the culture supernatant according to this disclosure, stored at -30°C, 4°C, or room temperature for one month, was added to a fibroblast culture system. [Figure 6] Figure 6 shows the immunostaining results of mouse ES cells that had been induced to differentiate into neurons for 4 days, after being cultured for 3 days under various conditions (culture medium only, with 10% hMSC culture supernatant, and with 10% X4N cell culture supernatant). TuJ: Tubulin βIII antibody (neuronal differentiation marker), DAPI: Cell nucleus staining, Merge: Overlay image of TuJ and DAPI. [Figure 7] Figure 7 shows the results of hair growth promotion tests in mice one and nine days after shaving. [Modes for carrying out the invention] 【0012】 In one embodiment, the disclosure relates to a composition comprising a stem cell culture supernatant for use in a pharmaceutical application. In some embodiments, the disclosure relates to a composition comprising a stem cell culture supernatant for use in the treatment of injury or disease in a subject. 【0013】 In one embodiment, the disclosure relates to a composition comprising stem cell culture supernatant for use in cosmetic applications. In several embodiments, the disclosure relates to a composition comprising stem cell culture supernatant for use in skincare, anti-aging, or hair growth and hair restoration. 【0014】 In one embodiment, the present disclosure relates to a method for producing the composition of the present disclosure, comprising the steps of i) culturing seeded stem cells; and ii) collecting the culture supernatant. 【0015】 (stem cells) In this disclosure, “stem cells” means cells that have the ability to replicate themselves without differentiation (also called “self-renewal ability”) and that have the ability to differentiate into various tissue cells. In this disclosure, stem cells may be pluripotent stem cells that have self-renewal ability and can differentiate into multiple cell types, such as induced pluripotent stem cells or embryonic stem cells, or stem cells may be progenitor cells that have self-renewal ability and the ability to differentiate into limited tissue cells. It is known that stem cells can be induced to differentiate into desired cell types by stimulation with a specific combination of factors. Therefore, in the context of this disclosure, “stem cells” may include cells in the differentiation process or cells that have completed differentiation derived from such stem cells. 【0016】 In some embodiments, the stem cells relating to this disclosure are stem cells expressing either CCN1 or CCN2, or both. 【0017】 "CCN1," also known as cysteine-rich 61 (Cyr61), is a protein belonging to the CCN family. "CCN2" is a protein also known as connective tissue growth factor (CTGF). The amino acid and nucleic acid sequences of these proteins are publicly known. 【0018】 In this specification, "stem cells expressing CCN1" means a specific population of stem cells that expresses at least twice as much CCN1 compared to an unselected or untreated population of stem cells. 【0019】 In this specification, "stem cells expressing CCN2" means a specific population of stem cells that expresses at least twice as much CCN2 as an unselected or untreated population of stem cells. In this specification, "stem cells expressing CCN1 and CCN2" means a specific population of stem cells that expresses at least twice as much CCN1 and at least twice as much CCN2 as an unselected or untreated population of stem cells. 【0020】 In this specification, “unselected stem cell population” means a stem cell population that has not been selected with respect to the expression levels of either CCN1 or CCN2, or both. In some embodiments, the unselected stem cell population is a population of cells prepared from biological tissue according to a conventional method, or commercially available stem cells. 【0021】 In some embodiments, stem cells expressing either or both CCN1 and CCN2 can be prepared by cloning stem cells from an unselected stem cell population in which the expression level of either or both CCN1 and CCN2 is at least twice as high as that of the unselected stem cell population. 【0022】 Stem cell cloning can be carried out as appropriate according to conventional methods. Examples of stem cell cloning methods include, but are not limited to, limiting dilution. The expression levels of genes or proteins expressed in the stem cells relating to this disclosure can be measured according to conventional methods. Expression levels can be measured by, but are not limited to, PCR, Northern blotting, Western blotting, immunohistochemistry, or microarrays. In some embodiments, expression levels are measured by quantitative PCR. 【0023】 In this specification, “untreated stem cell population” means a stem cell population that has not been treated to alter the expression levels of either or both CCN1 and CCN2. Treatment to alter expression levels includes gene transfer, such as the introduction of a CCN1 and / or CCN2 expression cassette, or the introduction of an immortalization gene. In some embodiments, the untreated stem cell population is a population of cells prepared from living tissue according to a conventional method, or commercially available stem cells. 【0024】 In some embodiments, stem cells expressing either or both CCN1 and CCN2 are stem cells in which the expression level of either or both CCN1 and CCN2 is at least twice as high as that of the untreated stem cell population, achieved by immortalizing an untreated stem cell population. 【0025】 In some embodiments, the step of preparing stem cells expressing CCN1 includes selecting or processing (and optionally selecting) stem cells that express at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more CCN1 than an unselected or untreated stem cell population. In other embodiments, the step of preparing stem cells expressing CCN2 includes selecting or processing (and optionally selecting after processing) stem cells that express at least 5, 10, 20, 30, 40, or 50 times more CCN2 than an unselected or untreated stem cell population. 【0026】 In some embodiments, the step of preparing stem cells expressing CCN1 and CCN2 includes selecting or processing (or, in some cases, selecting after processing) stem cells that express at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more CCN1 than that of an unselected or untreated stem cell population, and at least 3, 5, 7, 9, 10, 15, or 20 times more CCN2 than that of an unselected or untreated stem cell population. 【0027】 In some embodiments, the stem cells relating to this disclosure are immortalized stem cells. 【0028】 The "immortalization treatment" of stem cells can be carried out as appropriate according to conventional methods. Examples of stem cell immortalization treatment include, but are not limited to, methods involving the expression of telomerase (TERT) (preferably human-derived TERT (hTERT)), methods utilizing the SV40T antigen, and methods involving the co-expression of an hTERT catalytic subunit with p53 or RBsiRNA. In some embodiments, stem cells expressing either or both CCN1 and CCN2 are prepared by introducing hTERT into the stem cells. Stem cells into which hTERT has been introduced may, but are not limited to, transiently or constitutively express hTERT. In some embodiments, the stem cells according to this disclosure are stem cells immortalized by the introduction of telomerase. 【0029】 In some embodiments, the stem cells of this disclosure are stem cells into which one or more reprogramming factors have been introduced. 【0030】 In this specification, "reprogramming factor" means a gene specifically expressed in embryonic stem cells (ES cells) or a gene or gene product that plays an important role in maintaining ES cells in an undifferentiated state. Reprogramming factors may be in the form of genes (e.g., DNA, RNA) or proteins, but are not limited to these. Examples of reprogramming factors include, but are not limited to, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1. These reprogramming factors may be used individually, or in combination of two or more, three or more, four or more, five or more, or, for example, one to ten types. 【0031】 In some embodiments, one or more reprogramming factors include a combination of the Sox family and the Oct family. The Sox family includes, but is not limited to, Sox1, Sox2, Sox3, Sox15, and Sox17, which may be used individually or in combination of two or more. The Oct family includes, but is not limited to, Oct3 / 4. 【0032】 In some embodiments, one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, L-Myc, NANOG, and LIN28. 【0033】 In this specification, "introduction" of a reprogramming factor means placing the reprogramming factor into the cell. If the reprogramming factor is a protein, the introduction of one or more reprogramming factors can be carried out according to conventional methods. In one embodiment, a protein reprogramming factor is introduced into the cell by lipofection, microinjection, or fusion with a cell membrane-permeable peptide. 【0034】 If the reprogramming factor is a gene (e.g., DNA or RNA), the introduction of one or more reprogramming factors can be carried out according to conventional methods. For example, a reprogramming factor that is a gene may be introduced into cells by being incorporated into a vector such as a viral vector, plasmid, or artificial chromosome that contains sequences (e.g., promoter sequences or enhancer sequences) to activate its transcription and / or translation. Examples of viral vectors include, but are not limited to, Sendai virus vectors. Examples of artificial chromosome vectors include, but are not limited to, human artificial chromosomes (HACs). Examples of plasmids include, but are not limited to, mammalian cell plasmids; gene modification and transcription / translation of gene products can be carried out according to conventional methods. 【0035】 In some embodiments, the stem cells according to this disclosure are stem cells that have the ability to differentiate into ectoderm-derived cells such as nerve cells, and into mesoderm-derived cells such as chondrocytes, osteocytes, and adipocytes. 【0036】 In some embodiments, the stem cells according to this disclosure are stem cells capable of differentiating into nerve cells and express one or more neural stem cell markers. The one or more neural stem cell markers include nestin, SOX2, PAX6, ABCG2, FGFR1, and Frizzled-9. In some embodiments, the stem cells according to this disclosure further express HOX4B. 【0037】 In some embodiments, the stem cells according to this disclosure are bound with the “185 antibody” described in Japanese Patent Publication No. 2021-141872. In some embodiments, the stem cells according to this disclosure are bound with the 185 antibody before and / or after differentiation induction. In a preferred embodiment, the stem cells according to this disclosure are bound with the 185 antibody before differentiation induction. [Table 1] 【0038】 In some embodiments, the stem cells are derived from mammals, preferably primates, and more preferably humans. In some embodiments, the stem cells are derived from mammalian tissue, such as bone marrow, dental pulp, umbilical cord tissue, adipose tissue, or placental tissue. In some embodiments, the stem cells are mesenchymal stem cells. Stem cells derived from living tissue may be prepared by conventional methods or commercially available products may be used. In some embodiments, the stem cells relating to this disclosure are derived from the same species or the same individual as the subject to whom the composition relating to this disclosure is administered. 【0039】 The stem cells relating to this disclosure have the ability to differentiate into nerve cells, chondrocytes, osteocytes, and adipocytes, among others, but are not limited to these. In some embodiments, the stem cells relating to this disclosure have the ability to differentiate into nerve cells. In some embodiments, the stem cells relating to this disclosure have the ability to differentiate into nerve cells, chondrocytes, osteocytes, and adipocytes with high efficiency (for example, 20% or more, 40% or more, 60% or more, 80% or more, 20% to 80%, 40% to 80%, and 40% to 60% of the cell population after differentiation induction). 【0040】 The stem cells relating to this disclosure are adherent cells or suspension cells. Since adherent cells require a larger volume of culture medium relative to the number of cells compared to suspension cells, the stem cells relating to this disclosure are preferably adherent cells. 【0041】 The stem cells relating to this disclosure, though not limited to these, have a rapid proliferation rate. In some embodiments, the stem cells relating to this disclosure double in size in 12 to 24 hours when cultured using primate ES cell culture medium. 【0042】 The stem cells relating to this disclosure express and secrete effective bioactive substances into the culture medium. The stem cells relating to this disclosure secrete effective bioactive substances at a higher concentration than, for example, iPS cells. "Effective bioactive substance" or "bioactive substance" refers to a bioactive substance effective in the pharmaceutical or cosmetic applications relating to this disclosure, and includes proteins including cytokines, growth factors, and extracellular matrix (ECM) secretory proteins, nucleic acid molecules including miRNA, mRNA, ncRNA, and DNA, and extracellular vesicles (EVs) including exosomes that encapsulate these proteins or nucleic acid molecules. In some embodiments, the stem cells relating to this disclosure secrete extracellular vesicles containing the above-mentioned proteins and nucleic acid molecules, which constitute the bioactive substance. 【0043】 The proteins secreted by stem cells relating to this disclosure are, but are not limited to, BDNF, BMP, BMP1, BMP10, BMP2, BMP4, BMP5, BMP7, CLEC, COL, COL11A1, COL11A2, COL14A1, COL18A1, COL1A1, COL1A2, COL22A1, COL2A1, COL3A1, COL4A2, COL5A1, COL5A2, COL6A1, COL6A2, CSF, CSF 1, CXCL1, CXCL12, DKK1, EDN1, EFEMP, EFEMP1, ELN, ENO1, FGF, FGF1, FGF18, FGF7, FGF9, FN, FN1, FRZB, FSTL1, GDF, GD F10, GDF11, GDF15, GDF2, GMFB, GRN, HDGF, HGF, IGF, IGF2, IGFBP, IGFBP5, IGFBP7, IL, INHBA, JAG1, KGF, KIF20B, KRT, KRT1, KRT10, KRT2, KRT9, LGALS3BP, LGBDNF, LOXL2, MANF, MIA, MIF, MMP, MMP1, MMP10, MMP11, MMP14, MMP15, MMP2, MM P23, MMP9, MMP, NGF, NID2, NRG1, NT3, PCOLCE, PDGF, PDGF-C, PDGF-D, PTN, PTX3, SCG, SCG2, SDF-1, SFRP, SFRP1, SFRP2 It contains SFRP3, SFRP5, SHANK3, SPARC, TBX, TBX20, TBX5, TGF-α, TGF-β, TGF-β1, TGF-β2, THBS, THBS1, THBS2, TIMP, TIMP1, TIMP2, TIMP3, TIMP4, VCAN, VEGF, VEGFA, VEGFB, VEGFC, VEGFD, VIM, WNT5A, elastin, glycoprotein, keratin, laminin, or basement membrane factors. 【0044】 The miRNAs secreted by stem cells relating to this disclosure include, but are not limited to, miR-363, miR499, miR-1, miR-21, miR-126, miR-34, miR-16, hsa-miR-1-3p, hsa-miR-675-3p, hsa-miR-10395-3p, hsa-miR-133b, hsa-miR-208b-3p, hsa-miR-4488, This includes hsa-miR-450b-5p, hsa-miR-499b-3, hsa-miR-542-3p, hsa-miR-642a-3p and hsa-miR-9901, gga-mir-302a, gga-mir-302b, gga-mir-302c, gga-mir-302d, eca-mir-369, mmu-mir-19a or mmu-mir-20a. 【0045】 In some embodiments, the stem cells of the Disclosure express one or more effective bioactive substances at levels at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 times, or higher, compared to iPS cells. In some embodiments, the stem cells of the Disclosure express one or more proteins, including CCN1, CCN2, BDNF, VDGF, EGF, FGF, HGF, IGF, PDFG, or TGF-β, at levels at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 times, or higher, compared to iPS cells. In preferred embodiments, the stem cells of the Disclosure express BDNF at levels at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 times, or higher, compared to iPS cells. In some embodiments, the stem cells relating to this disclosure express miRNAs including gga-mir-302a, gga-mir-302b, gga-mir-302c, gga-mir-302d, eca-mir-369, mmu-mir-19a, mmu-mir-20a, or dre-mir-93 at levels at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 times, or more, compared to iPS cells. 【0046】 In some embodiments, the stem cells relating to this disclosure are stem cells that express either or both of CCN1 and CCN2; are immortalized by introducing hTERT; and are introduced with one or more reprogramming factors. In some embodiments, the stem cells relating to this disclosure are X4N cells (NEcST) deposited on July 4, 2017, with accession number P-02505, at the Patent Microorganism Depository Center (NPMD), Biotechnology Center, National Institute of Technology and Evaluation, located at 2-5-8122 Kazusa Kamatari, Kisarazu, Chiba, 292-0818, Japan. (登録商標) It is a cell, and its certificate of acceptance was notified on September 15, 2017, under notification number 2017-0253. 【0047】 (Stem cell culture supernatant) The stem cell culture supernatant according to this disclosure is a culture medium obtained by culturing the stem cells according to this disclosure as described above. Therefore, a culture supernatant usable in this disclosure can be obtained by separating and removing cellular components after culturing the stem cells. In some embodiments, the culture supernatant according to this disclosure may be a culture supernatant that has been subjected to various treatments as appropriate (e.g., centrifugation, mixing, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.). 【0048】 In some embodiments, the stem cell culture supernatant according to this disclosure may be included in the composition as one or more active ingredients (bioactive substances, e.g., cytokines, exosomes, miRNAs) isolated or purified from the culture medium. Various components and substances in the culture supernatant, e.g., proteins, extracellular vesicles, nucleic acid molecules, lipid molecules, and other trace components, can be isolated and purified according to conventional methods, and their concentrations can be measured according to conventional methods. Methods for isolation and purification include, but are not limited to, chromatography, ammonium sulfate fractionation, ultrafiltration, immunoadsorption, centrifugation, ultracentrifugation polymer precipitation, field flow fractionation, and immunoprecipitation. Methods for detection and concentration measurement include, but are not limited to, ELISA, Western blotting, nanoparticle analysis, vanillin, qPCR, microarray, or next-generation sequencing analysis. In some embodiments, the quantification of components or substances in the culture supernatant is performed using ELISA. 【0049】 When culturing the stem cells relating to this disclosure while maintaining their self-renewal ability and differentiation ability (e.g., ability to differentiate into nerve cells, chondrocytes, osteocytes, or adipocytes), the culture conditions (composition of the culture medium, culture method, subculturing method, etc.) that enable this can be appropriately set from known and conventional techniques. In some embodiments, such a medium is a xenofree medium. In some embodiments, such a medium is a commercially available culture medium for stem cells, for example, mouse ES cell culture medium (e.g., TX-WES medium, Thrombo-X), primate ES cell culture medium (e.g., Primate ES Cell Medium (ReproCELL)), human pluripotent stem cell (hPSC) culture medium (e.g., NutriStem (登録商標) hPSC XF GF-free (Sartorius Corporation), culture medium for human mesenchymal stem cells (hMSCs) (e.g., MSC NutriStem) (登録商標) The medium is XF Medium (Sartorius), or a culture medium for neural stem cells. In some embodiments, the medium is a culture medium for primate ES cells (ReproCELL), a culture medium for hPSCs (Sartorius), or a culture medium for hMSCs (Sartorius). 【0050】 In some embodiments, the culture method for culturing stem cells according to this disclosure while maintaining their self-renewal and differentiation capabilities may be a method used for culturing differentiated pluripotent cells such as ES cells and iPS cells while maintaining their differentiation capabilities. The culture method is not limited to, but may include, three-dimensional culture under non-adherent conditions, such as suspension culture (e.g., dispersed culture, aggregated suspension culture), or two-dimensional culture under adherent conditions, such as plate culture, or a combination of three-dimensional and two-dimensional culture. In some embodiments, the culture method involves culturing under adherent conditions in the absence of feeder cells. In cell-adherent incubators, the surface is coated with a cell-supporting substance (e.g., collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, or vitronectin) to improve adhesion to cells. In some embodiments, the cell-adherent incubator is coated with, for example, 0.1% gelatin. 【0051】 In some embodiments, the stem cell culture supernatant according to this disclosure is a culture medium of stem cells cultured under conditions that allow for the maintenance of the stem cells' self-renewal and differentiation capabilities (undifferentiated cell culture supernatant). 【0052】 The culture conditions (composition of culture medium, differentiation-inducing factors, culture method, subculturing method, etc.) for inducing differentiation of stem cells according to this disclosure into target cells (e.g., nerve cells) can be appropriately set from known and conventional techniques. In some embodiments, such a culture medium is a xenofree medium. In some embodiments, the culture medium for inducing differentiation of stem cells into nerve cells is a commercially available nerve cell culture medium or nerve differentiation medium (e.g., RHB-A Takara Bio). In other embodiments, the nerve cell culture medium or nerve differentiation induction medium contains nerve cell-inducing factors (e.g., brain-derived nerve growth factor (BDNF), fibroblast growth factor (FGF), small molecule BMP inhibitors and / or the small molecule TGFβ family inhibitors). 【0053】 In some embodiments, the culture method for inducing differentiation of stem cells into nerve cells according to this disclosure may be a method used for inducing differentiation of differentiated pluripotent cells such as ES cells and iPS cells into nerve cells. The culture method is not limited to, but may include three-dimensional culture under non-adherent conditions, two-dimensional culture under adherent covering conditions, or culture combining three-dimensional and two-dimensional culture. In some embodiments, the culture method is performed in the absence of feeder cells and under non-adherent conditions. 【0054】 In some embodiments, nerve cells derived from the stem cells relating to this disclosure have an expression level of CCN1 that is at least 2, 3, 5, 10, 20, 30, 50, 70, or 100 times lower than the expression level of the stem cells. 【0055】 In some embodiments, the culture medium for inducing differentiation of the stem cells according to this disclosure into adipocytes is, but is not limited to, a commercially available adipocyte induction medium or a commercially available animal cell medium containing insulin, dexamethasone, and indomethacin. In some embodiments, the culture medium for inducing differentiation of the cells according to this disclosure into chondrocytes is, but is not limited to, a commercially available chondrocyte induction medium or a commercially available animal cell medium containing dexamethasone, ascorbic acid, and TGF-β3. In some embodiments, the culture medium for inducing differentiation of the cells according to this disclosure into osteocytes is, but is not limited to, a commercially available osteocyte induction medium or a commercially available animal cell medium containing dexamethasone, ascorbic acid, and β-glycerophosphate. 【0056】 Confirmation of differentiation into the target cell type can be performed according to standard methods. If differentiated into nerve cells, confirmation can be performed by staining with nerve cell markers (e.g., NeuN, MAP2), although this is not limited to these methods. If differentiated into adipocytes, confirmation can be performed by oil red staining, although this is not limited to these methods. If differentiated into chondrocytes, confirmation can be performed by Alcian blue staining, although this is not limited to these methods. If differentiated into osteocytes, confirmation can be performed by alkaline phosphatase staining, although this is not limited to these methods. 【0057】 In some embodiments, the stem cell culture supernatant according to this disclosure is the culture supernatant of stem cells cultured under conditions for differentiation induction (differentiation-inducing cell culture supernatant). In some embodiments, the stem cell culture supernatant according to this disclosure is the culture supernatant of a stem cell population in which 20% or more, 40% or more, 60% or more, or 80% or more of the cell population after differentiation induction are differentiated cells, for example, 20% to 80%, 40% to 80%, or 40% to 60% of the cell population after differentiation induction are differentiated cells. In some embodiments, the stem cell culture supernatant according to this disclosure is the culture supernatant of a stem cell population in which 20% or more, 40% or more, 60% or more, or 80% or more of the cell population after differentiation induction are nerve cells, for example, 20% to 80%, 40% to 80%, or 40% to 60% of the cell population after differentiation induction are nerve cells. In some embodiments, the stem cell culture supernatant according to the disclosure is the culture supernatant of differentiated cells on day n, with the day of seeding in the differentiation induction medium being day 0 (where n is any integer). In some embodiments, the stem cell culture supernatant according to the disclosure is the culture supernatant of differentiated cells on day 1 to 2 months after differentiation induction, particularly on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 after differentiation induction. In preferred embodiments, the stem cell culture supernatant according to the disclosure is the culture supernatant of differentiated cells on day 5, 6, 7, 8, 9, or 10 after differentiation induction. 【0058】 Regarding the conditions for cell culture, the culture temperature is not limited, but is approximately 30°C to 40°C, preferably 37°C. Culture is carried out in an atmosphere of CO2-containing air, for example, at a CO2 concentration of approximately 2% to 5%. 【0059】 The stem cells of this disclosure may be cryopreserved according to conventional methods before culture and thawed before seeding, preferably immediately before seeding, for use. The stem cells of this disclosure may also be cryopreserved at any point during culture. The stem cells of this disclosure may be frozen and stored, for example, in liquid nitrogen or in a deep freezer. 【0060】 In some embodiments, the stem cell culture supernatant according to this disclosure may be a culture supernatant obtained by mixing the above-mentioned undifferentiated cell culture supernatant and differentiation-inducing cell culture supernatant. The mixing ratio can be appropriately adjusted by those skilled in the art depending on the disease and injury of the subject, for example, the undifferentiated cell culture supernatant to differentiation-inducing cell culture supernatant may be mixed in a ratio of 1:10 to 10:1 (e.g., 1:10, 1:5, 3:10, 2:5, 1:2, 3:5, 7:10, 4:5, 1:1, 5:4, 10:7, 5:3, 2:1, 5:2, 10:3, 5:1, or 10:1). 【0061】 While we do not wish to be bound by any particular theory, the culture supernatant of stem cells relating to this disclosure contains a mixture of bioactive substances, including cytokines. When administered to a subject, these bioactive substances may stimulate the differentiation and proliferation of endogenous stem cells near the site of injury, or act as inductive signals to activate the recruitment of other tissue-repairing cells to the site of injury, thereby promoting cell proliferation and the formation of the extracellular matrix at the site of injury. 【0062】 In some embodiments, the culture supernatant according to this disclosure contains bioactive substances. The bioactive substances may be dissolved, hydrated, or dispersed in the culture supernatant, or contained in extracellular vesicles (EVs) in the culture supernatant. In some embodiments, the culture supernatant according to this disclosure contains proteins, nucleic acid molecules, or extracellular vesicles containing these proteins or nucleic acid molecules that are effective in the pharmaceutical or cosmetic applications according to this disclosure. In some embodiments, the extracellular vesicles include, but are not limited to, vesicles of various sizes ranging from about 40 to about 2,000 nm, such as exosomes of about 40 to 120 nm, microendoplasmic reticulum of about 50 to 1,000 nm, and apoptotic bodies of 500 to 2,000 nm. 【0063】 In some embodiments, the culture supernatant or extracellular vesicles in the culture supernatant according to the present disclosure are BDNF, BMP, BMP1, BMP10, BMP2, BMP4, BMP5, BMP7, CLEC, COL, COL11A1, COL11A2, COL14A1, COL18A1, COL1A1, COL1A2, COL22A1, COL2A1, COL3A1, COL4A2, COL5A1, COL5A2, COL6A1, COL6A2, CSF, CSF1, C XCL1, CXCL12, DKK1, EDN1, EFEMP, EFEMP1, ELN, ENO1, FGF, FGF1, FGF18, FGF7, FGF9, FN, FN1, FRZB, FSTL1, GDF, GDF10, GDF1 1, GDF15, GDF2, GMFB, GRN, HDGF, HGF, IGF, IGF2, IGFBP, IGFBP5, IGFBP7, IL, INHBA, JAG1, KGF, KIF20B, KRT, KRT1, KRT10, KR T2, KRT9, LGALS3BP, LGBDNF, LOXL2, MANF, MIA, MIF, MMP, MMP1, MMP10, MMP11, MMP14, MMP15, MMP2, MMP23, MMP9, MMP, NGF, N ID2, NRG1, NT3, PCOLCE, PDGF, PDGF-C, PDGF-D, PTN, PTX3, SCG, SCG2, SDF-1, SFRP, SFRP1, SFRP2, SFRP3, SFRP5, SHANK3, SPA It contains at least one protein selected from the group consisting of RC, TBX, TBX20, TBX5, TGF-α, TGF-β, TGF-β1, TGF-β2, THBS, THBS1, THBS2, TIMP, TIMP1, TIMP2, TIMP3, TIMP4, VCAN, VEGF, VEGFA, VEGFB, VEGFC, VEGFD, VIM, WNT5A, elastin, glycoprotein, keratin, laminin, basement membrane factors, and combinations thereof. 【0064】 In some embodiments, the culture supernatant or extracellular vesicles in the culture supernatant according to this disclosure are miR-363, miR499, miR-1, miR-21, miR-126, miR-34, miR-16, hsa-miR-1-3p, hsa-miR-675-3p, hsa-miR-10395-3p, hsa-miR-133b, hsa-miR-208b-3p, hsa-miR-4488, hsa-miR-450b It contains at least one nucleic acid molecule selected from the group consisting of -5p, hsa-miR-499b-3, hsa-miR-542-3p, hsa-miR-642a-3p and hsa-miR-9901, gga-mir-302a, gga-mir-302b, gga-mir-302c, gga-mir-302d, eca-mir-369, mmu-mir-19a, mmu-mir-20a and combinations thereof. 【0065】 In some embodiments, the stem cell culture supernatant according to this disclosure contains one or more effective bioactive substances at a concentration of at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 times or more compared to the iPS cell culture supernatant. In some embodiments, the stem cell culture supernatant according to this disclosure contains extracellular vesicles at a concentration of at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 times or more compared to the iPS cell culture supernatant. In some embodiments, the stem cell culture supernatant according to this disclosure contains one or more nucleic acid molecules (e.g., DNA, mRNA, miRNA, and ncRNA) at a concentration of at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 times or more compared to the iPS cell culture supernatant. Preferably, the stem cell culture supernatant according to this disclosure contains one or more gga-mir-302a, gga-mir-302b, gga-mir-302c, gga-mir-302d, eca-mir-369, mmu-mir-19a, mmu-mir-20a, or dre-mir-93 at concentrations at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 times, or more, compared to the iPS cell culture supernatant. In some embodiments, the stem cell culture supernatant according to this disclosure contains one or more proteins at concentrations at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 times, or more, compared to the iPS cell culture supernatant. Preferably, the culture supernatant of stem cells according to this disclosure contains CCN1, CCN2, BDNF, VDGF, EGF, FGF, HGF, IGF, PDFG, or TGF-β at concentrations at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 times or more compared to the culture supernatant of iPS cells. 【0066】 In some embodiments, the culture supernatant according to this disclosure contains a bioactive substance derived from the stem cells according to this disclosure. In some embodiments, the culture supernatant of the stem cells according to this disclosure contains one or more bioactive substances at a concentration of at least about 0.5 pg / ml, for example, about 0.5 pg / ml to about 10 μg / ml. In some embodiments, the culture supernatant of the stem cells according to this disclosure contains at least about 0.5 pg / ml, for example, about 0.5 pg / ml to about 10 μg / ml of protein. In some embodiments, the culture supernatant of the stem cells according to this disclosure contains at least 1 × 10⁻⁶ 7 / mL, for example, about 1 × 10⁻⁶ 7 ~1 × 10 12 It contains extracellular vesicles (particularly exosomes) at a concentration of at least 0.5 pg / ml, for example, about 0.5 pg / ml to about 10 μg / ml. In some embodiments, the amount of extracellular vesicles in the stem cell culture supernatant according to this disclosure can be measured as the amount of cell membrane-derived components, such as membrane proteins such as MHC and adhesion molecules, or lipids derived from the endosomal membrane (cholesterol and sphingomyelin). In some embodiments, the stem cell culture supernatant according to this disclosure contains cell membrane-derived components at a concentration of at least 0.5 pg / ml, for example, about 0.5 pg / ml to about 10 μg / ml. In some embodiments, the stem cell culture supernatant according to this disclosure contains at least about 0.5 pg / ml of nucleic acid molecules. In some embodiments, the stem cell culture supernatant according to this disclosure contains at least about 0.5 pg / ml of miRNA. 【0067】 In one embodiment, the culture supernatant according to the Disclosure contains secreted proteins selected from the group consisting of CCN1, CCN2, BDNF, VDGF, EGF, FGF, HGF, IGF, PDFG, and TGF-β at concentrations of at least about 0.5 pg / ml, for example, at least about 1 pg / ml, at least about 10 pg / ml, at least about 1000 pg / ml, or at least about 2000 pg / ml. In one embodiment, the culture supernatant according to the Disclosure contains secreted proteins selected from the group consisting of CCN1, CCN2, BDNF, VDGF, EGF, FGF, HGF, IGF, PDFG, and TGF-β at concentrations of about 0.5 to 2000 pg / ml, about 5 to 1000 pg / ml, or about 50 to 500 pg / ml. In preferred embodiments, the culture supernatant according to the present disclosure contains BDNF at a concentration of about 0.5 to 2000 pg / ml, more preferably about 5 to 1000 pg / ml, and even more preferably about 50 to 500 pg / ml. In preferred embodiments, the culture supernatant according to the present disclosure contains BDNF and / or CCN1 at a concentration of about 0.5 to 2000 pg / ml, more preferably about 5 to 1000 pg / ml, and even more preferably about 50 to 500 pg / ml. 【0068】 (Pharmaceutical or cosmetic composition) In one embodiment, the compositions of the present disclosure are compositions for use in pharmaceutical applications, particularly for use in the treatment of injury, disease, or symptoms in a subject. 【0069】 In this specification, “Subject” means an animal having injury, disease, or symptoms to which the composition of this disclosure is administered. In some embodiments, the subject includes mammals, birds, and reptiles. In some embodiments, the subject includes or excludes primates, including humans. In some embodiments, the subject includes pets such as dogs and cats, and livestock such as cattle and horses. In some embodiments, the subject is a mammal, bird, or reptile, including or excluding humans. In a specific embodiment, the subject is a human. 【0070】 In this specification, “treatment” means slowing the progression or exacerbation of a disease or symptom, maintaining, alleviating, or improving the disease. Alternatively, “treatment” means promoting the recovery of an injury or disease or symptom. In this specification, recovery of an injury means promoting the regeneration of tissues at or near the site of the injury, such as bone, skin, muscle, joint, tendon, ligament, nerve or blood vessel, or alleviating or improving symptoms and functional impairments resulting from the injury, such as motor dysfunction, sensory dysfunction or autonomic dysfunction. 【0071】 As used in this disclosure, “injury” means trauma or wound in any part of the living body, including tissues and organs, and the resulting acute or chronic functional impairment (e.g., motor dysfunction, sensory dysfunction, or autonomic dysfunction). In some embodiments, injury may be extrinsic injury caused by physical or chemical stimuli or forces. In some embodiments, injury may be intrinsic injury caused by disease, etc. Examples of intrinsic injury caused by disease include inflammation due to inflammatory diseases, or tissue necrosis or dysfunction due to impaired blood flow caused by vascular diseases. In some embodiments, injury may be injury to tissues such as bones, skin, muscles, joints, tendons, ligaments, nerves, or blood vessels, and / or injury to organs such as the brain, liver, heart, lungs, kidneys, spleen, pancreas, or digestive tract. In some embodiments, injury may be injury involving nerve tissue, such as spinal cord injury or brain injury. In some embodiments, injury may be extrinsic spinal cord injury, spinal cord injury caused by cancer, or spinal cord injury caused by vascular disease. 【0072】 In some embodiments, the disease or symptom includes neurological disorders such as depression, myasthenia gravis, ischemic brain disease (such as stroke), motor neurological disorders, neurodegenerative diseases, multiple sclerosis, amyotrophic lateral sclerosis, spinocerebellar degeneration, Huntington's disease, dementia (e.g., Alzheimer's disease, Lewy body dementia, vascular dementia), Parkinson's disease, epilepsy, and schizophrenia, blood flow disorders, infarction, osteoporosis, immune disorders, or periodontitis. In some embodiments, the disease is a neurological disorder including depression, myasthenia gravis, dementia, Parkinson's disease, Huntington's disease, or schizophrenia. In some embodiments, the compositions of the present invention may be used in the treatment of periodontitis or in the regeneration of periodontal tissue. 【0073】 In this specification, therapeutic use includes administering the compositions of the Disclosure to a subject for the treatment of diseases and injuries. In some embodiments, therapeutic use includes administering the compositions of the Disclosure to a subject in lieu of or in parallel with other effective treatments for diseases and injuries. In some embodiments, therapeutic use includes using the compositions of the Disclosure in combination with or as a substitute for platelet-rich plasma (PRP) therapy, wherein, when used as a substitute, the stem cells relating to the Disclosure are derived from the same species or the same individual as the subject to which the composition is administered, for example, mesenchymal stem cells. 【0074】 In one embodiment, the compositions of the Disclosure may be compositions used in cosmetic applications. In some embodiments, the compositions of the Disclosure may be compositions used for skin care, anti-aging, skin whitening, or hair growth and hair restoration purposes. In some embodiments, the compositions of the Disclosure may be compositions used for improving the appearance and texture of the skin, for example, skin care, skin whitening, wrinkle reduction, texture improvement, or promoting cell turnover, or for inhibiting aging. In some embodiments, the compositions of the Disclosure may be cosmetics for skin care, anti-aging, or whitening purposes. In some embodiments, compositions comprising the culture supernatant of the Disclosure may be hair growth agents or hair restoration agents. In some embodiments, compositions comprising the culture supernatant of the Disclosure may be compositions for the improvement and treatment of acute or chronic wounds, lesions, scars, keloids, etc., of the skin for cosmetic or therapeutic purposes. 【0075】 In some embodiments, the culture supernatant contained in the compositions of the present disclosure may be the culture supernatant obtained by separating and removing cellular components from the culture medium, or a culture supernatant preparation obtained by various treatments of the culture supernatant (e.g., centrifugation, mixing, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.). Therefore, it is also conceivable that the culture supernatant contained in the compositions of the present disclosure may be one or more active ingredients (e.g., cytokines, exosomes, miRNAs) isolated or purified from the culture supernatant. 【0076】 In some embodiments, the culture supernatant or extracellular vesicles in the culture supernatant according to the present disclosure are BDNF, BMP, BMP1, BMP10, BMP2, BMP4, BMP5, BMP7, CLEC, COL, COL11A1, COL11A2, COL14A1, COL18A1, COL1A1, COL1A2, COL22A1, COL2A1, COL3A1, COL4A2, COL5A1, COL5A2, COL6A1, COL6A2, CSF, CSF1, C XCL1, CXCL12, DKK1, EDN1, EFEMP, EFEMP1, ELN, ENO1, FGF, FGF1, FGF18, FGF7, FGF9, FN, FN1, FRZB, FSTL1, GDF, GDF10, GDF1 1, GDF15, GDF2, GMFB, GRN, HDGF, HGF, IGF, IGF2, IGFBP, IGFBP5, IGFBP7, IL, INHBA, JAG1, KGF, KIF20B, KRT, KRT1, KRT10, KR T2, KRT9, LGALS3BP, LGBDNF, LOXL2, MANF, MIA, MIF, MMP, MMP1, MMP10, MMP11, MMP14, MMP15, MMP2, MMP23, MMP9, MMP, NGF, N ID2, NRG1, NT3, PCOLCE, PDGF, PDGF-C, PDGF-D, PTN, PTX3, SCG, SCG2, SDF-1, SFRP, SFRP1, SFRP2, SFRP3, SFRP5, SHANK3, SPA The composition comprises at least one protein selected from the group consisting of RC, TBX, TBX20, TBX5, TGF-α, TGF-β, TGF-β1, TGF-β2, THBS, THBS1, THBS2, TIMP, TIMP1, TIMP2, TIMP3, TIMP4, VCAN, VEGF, VEGFA, VEGFB, VEGFC, VEGFD, VIM, WNT5A, elastin, glycoprotein, keratin, laminin, basement membrane factors, and combinations thereof. In some embodiments, the composition of the present disclosure comprises at least one neuronal differentiation-inducing factor or neuronal survival-maintaining factor (e.g., BDNF, NGF, GDNF, NT-3, etc.). 【0077】 In some embodiments relating to cosmetic applications, the compositions of the present disclosure include at least one protein selected from the group consisting of IGFBP, HGF, VEGF, PDGF, EGF, KGF (FGF-7), PDGFR, TGF-α, TGF-β, and combinations thereof. In preferred embodiments relating to cosmetic applications, the compositions of the present disclosure include IGFBP, HGF, VEGF, PDGF, EGF, KGF (FGF-7), PDGFR, TGF-α, and TGF-β. 【0078】 In some embodiments, the culture supernatant or extracellular vesicles in the culture supernatant according to this disclosure are miR-363, miR499, miR-1, miR-21, miR-126, miR-34, miR-16, hsa-miR-1-3p, hsa-miR-675-3p, hsa-miR-10395-3p, hsa-miR-133b, hsa-miR-208b-3p, hsa-miR-4488, hsa-miR-450b It contains at least one nucleic acid molecule selected from the group consisting of -5p, hsa-miR-499b-3, hsa-miR-542-3p, hsa-miR-642a-3p and hsa-miR-9901, gga-mir-302a, gga-mir-302b, gga-mir-302c, gga-mir-302d, eca-mir-369, mmu-mir-19a, mmu-mir-20a and combinations thereof. 【0079】 In some embodiments, the compositions of the present disclosure are liquid and contain a bioactive substance at least about 0.1 pg / ml. In some embodiments, the compositions of the present disclosure are liquid and contain a bioactive substance (extracellular vesicles, proteins) at a concentration of at least about 0.1 pg / ml, such as from about 0.5 pg / ml to 10 μg / ml, from about 5 to 1000 pg / ml, or from about 50 to 500 pg / ml. In some embodiments, the compositions of the present disclosure are liquid and contain a secreted protein selected from the group consisting of CCN1, CCN2, BDNF, VDGF, EGF, FGF, HGF, IGF, PDFG, and TGF-β at a concentration of at least about 0.5 pg / ml, such as from about 0.5 pg / ml to 10 μg / ml, from about 5 to 1000 pg / ml, or from about 50 to 500 pg / ml. In some embodiments, the compositions of the present disclosure are liquid and contain extracellular vesicles at a concentration of about 1×10 7 / mL, such as about 1×10 7 to 1×10 13 , about 1×10 8 to 1×10 12 , about 1×10 9 to 1×10 11 / mL. In a preferred embodiment, the compositions of the present disclosure are liquid and contain BDNF and / or CCN1 at concentrations of about 0.5 to 2000 pg / ml, more preferably about 5 to 1000 pg / ml, and even more preferably about 50 to 500 pg / ml, respectively. 【0080】 In some embodiments, the compositions of the Disclosure contain at least about 0.01 pg, for example, at least about 0.1 pg, at least about 1 pg, at least about 10 pg, at least about 100 pg, at least about 1 μg, at least about 10 μg, at least about 100 μg, or at least about 1 g of a bioactive substance. In some embodiments, the compositions of the Disclosure contain at least about 0.01 pg, for example, at least about 0.1 pg, at least about 1 pg, at least about 10 pg, at least about 100 pg, at least about 1 μg, at least about 10 μg, at least about 100 μg, or at least about 1 g of a protein selected from the group consisting of CCN1, CCN2, BDNF, VDGF, EGF, FGF, HGF, IGF, PDFG, and TGF-β. In some embodiments, the compositions of the Disclosure contain at least about 1 × 10⁻¹⁶ 4 pieces, for example, at least about 1 × 10 5 each, at least about 1 × 10 6 each, at least about 1 × 10 7 each, at least about 1 × 10 8 each, at least about 1 × 10 9 each, at least about 1 × 10 10 each, at least about 1 × 10 11 each, at least about 1 × 10 12 each, at least about 1 × 10 13 each, at least about 1 × 10 14 pieces, or at least about 1 × 10 15 It contains individual extracellular vesicles. 【0081】 The inventors have found that the culture supernatant of stem cells according to this disclosure may contain high concentrations of bioactive substances, particularly brain-derived neurotrophic factor (BDNF) and extracellular vesicles (e.g., exosomes) containing nucleic acid molecules (e.g., miRNA) involved in cell growth and regeneration. Regarding the association between neurotrophic factors and disease, BDNF has been found to promote the proliferation and differentiation of tooth-related cells and blood vessels, and it is known that BDNF levels in brain tissue are reduced in people with Alzheimer's disease. Furthermore, BDNF knockout mice lack sensory nerves and are unable to perform coordinated movements, balance movements, auditory perception, taste, and respiratory movements. BDNF knockout mice also exhibit cerebellar abnormalities, with an increased number of sympathetic neurons. Moreover, certain types of physical exercise can increase BDNF synthesis in the human brain by approximately three times. This phenomenon is one of the mechanisms of exercise-induced neurogenesis and exercise-induced improvement of cognitive function. 【0082】 In some embodiments, the compositions of the present disclosure may optionally include one or more additional components. These additional components may include at least several pharmaceutically acceptable carriers or excipients and other agents. Examples of pharmaceutically acceptable carriers or excipients and other agents include: carriers such as water, physiological saline, and buffer solutions; excipients such as lactose, starch, sorbitol, D-mannitol, and sucrose; other agents such as disintegrants such as starch, carboxymethylcellulose, and calcium carbonate; buffers such as phosphates, citrates, and acetates; emulsifiers such as gum arabic, sodium alginate, and tragacanth; suspending agents such as glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate; analgesics such as benzyl alcohol, chlorobutanol, and sorbitol; stabilizers such as propylene glycol and ascorbic acid; preservatives such as phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben; antiseptics such as benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol; and gelling agents such as hyaluronic acid and collagen. Additional ingredients may also be ingredients or therapeutic agents effective in treating injury or disease, such as anti-inflammatory agents, immunosuppressants, steroids, and analgesics. 【0083】 The compositions of this disclosure can be prepared in various dosage forms and administered to subjects via an appropriate route of administration, number of doses, and interval between doses. 【0084】 In some embodiments, the compositions of the present disclosure are prepared for topical, oral, rectal, nasal, intravenous, intra-articular, intraconjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, transdermal, or subcutaneous administration routes. Preferably, the compositions of the present disclosure are prepared for intrathecal or intravenous administration. 【0085】 The form of the compositions of this disclosure may be appropriately determined by those skilled in the art depending on the manner of use and route of administration. In some embodiments, the compositions of this disclosure may be solids such as powders, granules, or fine particles; liquids such as suspensions or emulsions; or semi-solids such as gels. In some embodiments of compositions for pharmaceutical use, the compositions of this disclosure may be orally administered pharmaceutical compositions in the form of granules, fine particles, powders, hard capsules, soft capsules, syrups, emulsions, suspensions, or liquids; or parenterally administered pharmaceutical compositions in the form of injections for intravenous, intramuscular, or subcutaneous administration, drips, transdermal agents, transmucosal agents, nasal drops (such as nasal sprays), inhalants, suppositories, etc. In preferred embodiments of compositions for pharmaceutical use, the compositions of this disclosure are liquids. In some embodiments of compositions for cosmetic use, the compositions of this disclosure may be any cosmetic, including lotions, emulsions, gels, serums, general creams, packs, masks, facial cleansers, cosmetic soaps, foundations, face powders, and body lotions. 【0086】 In some embodiments, compositions of the present disclosure may be stored in solid, liquid, or semi-solid form after preservation treatment (e.g., concentration, freeze, dry, freeze-dried) and may be used after preparation treatment (e.g., melting, dilution, gelling, mixing with a carrier and solvent) before administration to a subject. 【0087】 In some embodiments, the compositions of the present disclosure are prepared as liquids for intravenous administration and administered to subjects at doses of ml / kg. In some embodiments, the compositions of the present disclosure are administered to subjects at a frequency of once every 1, 2, 3, 4, 5, 6, and 7 days, once every 1, 2, 3, and 4 weeks, and once every 1 and 2 months. 【0088】 In some embodiments, the compositions of the present disclosure are prepared as liquids for topical administration and administered to subjects at doses of ml / kg. In some embodiments, the compositions of the present disclosure are administered to subjects at a frequency of once every 1, 2, 3, 4, 5, 6, and 7 days, once every 1, 2, 3, and 4 weeks, and once every 1 and 2 months. 【0089】 (Method of manufacturing the composition) This disclosure relates to a method for producing a composition, comprising the steps of i) culturing a seeded population of stem cells; and ii) recovering the culture supernatant. 【0090】 In some embodiments, step i) in a method for producing the composition of the present disclosure includes a) culturing seeded stem cells under conditions that allow the self-renewal ability of the stem cells and the ability to differentiate into target cells, particularly nerve cells; and / or b) culturing them under conditions that induce differentiation into target cells. 【0091】 In some embodiments, step i)-a) or i)-b) is initiated approximately 1, 2, 3, 4, 5, 6, 7 days, 1 week, or 2 weeks after stem cell seeding. In some embodiments, after stem cell seeding and until step i)-a) or step i)-b) is initiated, the stem cells may be cultured under conditions that allow them to maintain their self-renewal ability and differentiation ability into the cells of interest. In some embodiments, the culture in step i)-a) or i)-b) is terminated by the initiation of step ii) at any point between approximately 1 day and 2 months after the initiation. In some embodiments, the culture in step i)-a) or i)-b) is continued for approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or 2 months. 【0092】 In some embodiments, at the start of step i)-a) or i)-b), the stem cells may be confluent at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, at the start of step ii), the stem cells may be confluent at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. 【0093】 A person skilled in the art can determine the end of the culture in step i)-a) or i)-b) (the start of step ii) according to one or more criteria (number of days, color of the culture medium, presence or concentration of a specific component, confluence, cell morphology, growth rate, etc.). 【0094】 Steps i)-a) and i)-b), and the subsequent step ii), may be applied independently to multiple culture vessels. Therefore, even if the start date and time differ for each culture vessel, the culture supernatant obtained from culture vessels to which the same termination criteria for steps i)-a) and b) above were applied may be considered the same. Accordingly, in some embodiments, the collection of the culture supernatant in step ii) may include collecting it as bulk culture supernatant for cultures to which the same termination criteria were applied. 【0095】 In some embodiments, the recovery of the culture supernatant in step ii) may include a step of removing cells from the culture medium. Examples of cell removal steps include filtration, centrifugation, aspiration using a pipette, dropper or aspirator, and tilting the culture vessel. In some embodiments, the recovery of the culture supernatant is performed by centrifugation of the culture medium to remove cells. 【0096】 The method for producing the composition of the present disclosure may optionally further include, after step ii), a step of processing the composition for preservation, a step of preparing it for administration and processing it in various ways. 【0097】 Preferred embodiments relating to this disclosure will be described in detail below, but these are all illustrative and not restrictive, and do not in any way limit the invention described in the attached claims. [Examples] 【0098】 Example 1: X4N cells (NEcST (登録商標) Preparation of cell culture supernatant A culture supernatant preparation obtained by culturing X4N cells as disclosed in Japanese Patent Publication No. 2019-076008 was prepared according to the following procedure: A 0.1% gelatin solution (Wako Pure Chemical Industries) was placed in a 10 cm petri dish or culture flask until the bottom was submerged, and the mixture was incubated at 37°C for 30 minutes, then washed twice with PBS. Next, 1 x 10 5 X4N cells were suspended in 1 ml of ReproCELL medium (ReproCELL) to a cell concentration of 1 / ml, seeded in the above 10 cm petri dish or culture flask, and culture was started at 37°C in the presence of 5% CO2. The culture medium was replaced with Sartorius medium (Sartorius) the day after seeding, and cultured for another 2 days. After 2 days, the culture medium was collected, centrifuged at 1000 rpm for 7 minutes, and the supernatant obtained was further filtered to obtain the undifferentiated cell culture supernatant. Furthermore, the 10 cm petri dish or culture flask was coated by adding enough gelatin solution (Wako Pure Chemical Industries) or vitronectin solution to cover the bottom, incubating at 37°C for 30 minutes, and then washing twice with PBS. Next, 2 x 10 5 X4N cells were suspended in 1 ml of a medium mixture of 50% Reprocell medium (Reprocell Inc.) and 50% RHB-A medium (Takara Bio) to achieve a cell concentration of 1 / ml. This medium was then seeded into the 10 cm petri dish or culture flask and cultured at 37°C in the presence of 5% CO2. The culture medium was replaced with RHB-A medium (Takara Bio) the day after seeding and cultured for a further 5 days. After 5 days, the culture medium was replaced with Sartorius medium (Sartorius Inc.) and cultured for a further 2 days. After 2 days, the culture medium was collected, centrifuged at 1000 rpm for 7 minutes, and the resulting supernatant was further filtered to obtain the neuronal differentiation-inducing cell culture supernatant. The undifferentiated cell culture supernatant and the neuronal differentiation-inducing cell culture supernatant were mixed in a 1:1 ratio to prepare the test culture supernatant. Furthermore, the undifferentiated cell culture supernatant, neuronal differentiation-inducing cell culture supernatant, or test culture supernatant was purified and concentrated 5-fold by ultrafiltration to prepare the culture supernatant. 【0099】 Example 2: Rat spinal cord injury model • Evaluation of hindlimb motor function improvement using a rat spinal cord injury model. Twenty-four rats (Jcl:SD, SPF, female, 10 weeks old) were acclimatized for one week at room temperature of 22±2°C, relative humidity of 30-70%, and 12 hours of light and 12 hours of darkness. During this time, the rats had free access to water and solid feed CE-2 (Feed One Co., Ltd.). After acclimatization, these rats were randomly assigned to three treatment groups (8 rats / group) based on body weight stratification to ensure the average body weight was as uniform as possible. Table 2 shows the results for each treatment group of rats. [Table 2] After the acclimatization period, a rat model of spinal cord injury was created using the weight drop method. Specifically, the vertebral arches of the 9th and 10th thoracic vertebrae were resected under triple anesthesia, and then the spine was injured by immediately dropping a 10g weight from a height of 50mm. In the intrathecal administration group (Group 3), rats were implanted in the spinal cavity with a rat intrathecal administration catheter (#0007741, Short type, Alzet). 【0100】 The test substance was administered on days 1, 6, 11, 16, 21, and 31, with the model creation day being day 0. Hindlimb motor function (BBB test) was evaluated on days 1, 7, 14, 21, 28, 35, and 39. The general condition was observed at least once a day during rearing. Body weight was measured daily from day 0 to day 7, and then once a week thereafter. After the observation period, the rats were euthanized, and tissue samples were collected for staining with hematoxylin and eosin. 【0101】 • Statistical processing For weight, the mean and standard error were calculated, and Brtlett's test was performed. For equal variances, one-way ANOVA and Turkey-Kramer multiple comparison tests were performed, or for unequal variances, Kruskal-Wallis tests and Steel-Dwass multiple comparison tests were performed. For BBB scores, the mean and standard error were calculated, and Kruskal-Wallis tests and Steel-Dwass multiple comparison tests were performed. A significance level of less than 5% (two-sided) was considered statistically significant for all results. 【0102】 ·result There was no significant difference in body weight change during the study period between the control group (Group 1) administered with the solvent and Groups 2 or 3 administered with the culture supernatant. As shown in Figure 1, regarding hindlimb motor function, a significant difference was observed between Group 2, which received intravenous administration of the culture supernatant, and the control group at 35 days post-injury, and between Group 3, which received intraspinal administration of the culture supernatant, and the control group at 21 days post-injury. Furthermore, at 35 days post-injury, no individuals in the control group recovered beyond the level of being able to bear weight on the soles of their feet (BBB≧8), but approximately half of the individuals in Groups 2 and 3 recovered to the level of being able to bear weight on the soles of their feet, and in particular, some individuals in Group 2 recovered to the level of being able to walk on their soles (BBB=10). Therefore, both local and intravenous administration of the culture supernatant as disclosed herein resulted in accelerated and improved recovery of motor function compared to the control group. 【0103】 • Staining of spinal cord tissue and other tissues surrounding the injury site. After the observation period in Example 1, the rats were euthanized by excessive inhalation of isoflurane (concentration of 5% or more). The spine, brain, liver, and kidneys around the injured area were collected and fixed with 4% paraformaldehyde at room temperature for 12 hours. The tissue samples were embedded in paraffin, cut into 5 μm sections, and stained with hematoxylin and eosin (Figures 2A-D). Staining was performed using the following procedure: immersion in hematoxylin (Carrazi' hematoxylin, Muto Pure Chemicals Co., Ltd, Tokyo, Japan) for 5 minutes, rinsing with water for 10 minutes, immersion in eosin (eosin, Muto Pure Chemicals Co., Ltd, Tokyo, Japan) for 30 seconds, rinsing with water, dehydration (immersion sequentially in 70% ethanol, then 90% ethanol, then 100% ethanol), xylene clearing, and mounting. 【0104】 ·result Consistent with the data on the recovery of hind limb motor function in Example 1, the spinal cord injury site (void) was repaired by nerve cells in the culture supernatant administration group (Figure 2A; center, right) compared to the spinal cord tissue around the injury site in the control group (Figure 2A left). Furthermore, when observing the effects on each tissue administered, no effects on the brain, liver, or kidney tissue were observed in any of the groups—the control group, the group that received the culture supernatant intravenously, or the group that received the culture supernatant intrathecally—in addition to the spinal cord (Figure 2A-D). 【0105】 Therefore, the culture supernatant according to the present invention exerted its effects without causing significant side effects whether administered locally (intrathecally) or systemically (intravenously), promoting tissue recovery at the site of injury. Thus, it is suggested that the culture supernatant according to the present invention may be a useful composition for treating injury and disease by contributing to the promotion of cell differentiation, proliferation, and growth, particularly of nerve cells. 【0106】 Example 3: Measurement of the concentration of physiologically active substances in the culture supernatant Using the ELISA method, the concentrations of BDNF in the culture supernatant of X4N cells, human iPS cells, and human mesenchymal stem cells (hMSCs), as well as in culture media (RHB-A medium, ReproCELL medium), were measured (Figure 3). The culture supernatant of X4N cells had a significantly higher BDNF content compared to the culture supernatant of iPS cells or hMSCs. 【0107】 Furthermore, the relative content of various cytokines in the test culture supernatant of X4N cells was measured using antibody array analysis compared to the culture medium control. The results are shown in Table 3 below. It was shown that the culture supernatant of X4N cells contains various physiologically active substances, including cytokines. [Table 3] 【0108】 Example 4: Measurement of bioactive substances in extracellular vesicles The culture supernatants of X4N cells and hMSCs were ultrafiltered using a 30kDa filter to obtain the residue (exosome fraction) and filtrate. RNA extraction was performed on the exosome fraction, and the expression levels of miRNAs in the exosomes were analyzed by microarray analysis. The results are shown in Table 4 below. [Table 4] The miRNAs listed in the table are annotated as being involved in cell growth and regeneration. In X4N cells, these miRNAs were present at least twice as much in exosomes in the culture supernatant compared to human mesenchymal stem cells. 【0109】 Furthermore, we investigated whether the bioactive substances secreted by X4N cells were concentrated in the culture supernatant or in extracellular vesicles (exosomes). A portion of the exosome fraction of the above-mentioned X4N cells was treated with NaOH + TritonX to disrupt the exosomes. The concentration of BDNF was quantified by ELISA in the filtrate fraction, exosome fraction (disrupted), exosome fraction (untreated), and culture supernatant of the obtained X4N cell culture supernatant (Figures 4A, 4B). Compared to the other fractions, the amount of BDNF was significantly higher in the exosome fraction (disrupted). 【0110】 These results suggest that X4N cells secrete extracellular vesicles containing bioactive substances, and that these bioactive substances are concentrated within these extracellular vesicles. While we do not wish to be bound by any particular theory, the reason why the regenerative effect was observed in the spinal cord injury rat model with intravenous administration in addition to local administration of the culture supernatant may be that the culture supernatant of this disclosure contains extracellular vesicles containing bioactive substances, allowing the bioactive substances to be delivered more stably to cells proximal to the target site (e.g., the injury site). 【0111】 Example 5: Measurement of exosome count in culture supernatant The supernatant of X4N cell cultures was filtered through a PVDF filter (Millex GV, Millipore) with a pore size of 0.22 μm, and the filtrate was used as the stock sample. Sample 1 was prepared by diluting the stock sample 50-fold with PBS, and Sample 2 was prepared by diluting it 40-fold. Particle analysis was performed on each sample using a Nanosight 300 machine, Nanosight syringe pump, Nanosight NTA3.3 software, and O-Ring top-plate (Malvern Panalytical Inc.) under the following measurement conditions: Camera Level: 16, Screen Gain: 1.0, Syringe Pump Speed ​​(flow rate): 100, Capture time: 60 sec, Repeat: 4 times (total: 5 times). As a result of the particle analysis, peaks were observed in Sample 1 at particle sizes of 68 nm and 114 nm, and the particle concentration was 2.04 × 10⁶ 10 The particle density was 1.98 × 10¹⁶ particles / ml. In sample 2, peaks were observed at particle sizes of 64 nm and 119 nm, and the particle concentration was 1.98 × 10¹⁶. 10 The concentration was 1 cell / ml. The exosomes produced by X4N cells were relatively homogeneous, with a particle size of approximately 100 nm. 【0112】 Number of exosomes in the culture supernatant of X4N cells (~×10) 10 The concentration of cells / ml is significantly higher compared to the culture supernatant of other cells per the same number of cells. While cell proliferation was not observed in MSC and iPS cell culture supernatants without concentration, the X4N cell supernatant showed an effect with the addition of unconcentrated culture supernatant, which is thought to be due to the high exosome concentration and the concentration of bioactive substances such as BDNF. 【0113】 Example 6: Verification of the proliferation-promoting effect of X4N cell culture supernatant on mesenchymal stem cells and fibroblasts. X4N cell culture supernatant, undifferentiated cell culture supernatant, and hMSC culture medium (Sartorius) were prepared at the specified concentrations (10%, 5%, and 1%), respectively, in a 2 × 10⁶ solution. 5The difference in the growth rate of hMSCs in each culture after adding the test culture supernatant of X4N cells to an hMSC culture system seeded at cells / ml and culturing for 3 days was investigated by measuring the number of cells using a cell counter (Thermo Fisher) (Figure 5A). Adding 10% of the test culture supernatant of X4N cells resulted in approximately twice the growth promotion effect compared to the culture system without the additive. 【0114】 Similarly, 10% of the test culture supernatant of X4N cells or iPS cell culture supernatant was added to a culture system of human dermal fibroblasts, and the number of viable cells after 3 days of culture was measured using a cell counter (Thermo Fisher) (Figure 5B). As a result, the culture system with X4N cell culture supernatant showed approximately twice the proliferation-promoting effect compared to the system with iPS cell culture supernatant. Furthermore, to confirm the stability of the culture supernatant disclosed herein, human dermal fibroblasts were cultured for 3 days using X4N cell culture supernatant and other media (Sartorius medium for iPS cells (S), ReproCELL medium for iPS cells (R), and fibroblast medium (Takara Bio)) that had been stored for 1 month at -30°C, 4°C, and room temperature. The number of viable cells after culture was measured using a cell counter (Thermo Fisher) (Figure 5C). It was found that the X4N cell culture supernatant maintained its fibroblast proliferation-promoting effect even after being stored at room temperature for 1 month. 【0115】 Example 7: Verification of the effect of X4N cell culture supernatant on promoting neural differentiation of mouse ES cells. Mouse ES cells were treated with Neurobasal cells containing N2 / B27. (商標)After differentiating cells in culture medium for 4 days while inducing spheroid formation, the cells were seeded on laminin-coated glass-bottom plates and cultured for 3 days under each condition (culture medium only, with 10% hMSC culture supernatant added, and with 10% X4N cell culture supernatant added). For the X4N cell culture supernatant, purified concentrated test culture supernatant obtained by ultrafiltration was used, diluted 5-fold with PBS. After culturing, cells were stained with tubulin βIII antibody and DAPI and observed using a fluorescence microscope. As a result, significant promotion of neural differentiation was observed when X4N cell culture supernatant was added compared to culture in culture medium only and when hMSC culture supernatant was added (Figure 6). 【0116】 Example 8: Verification of the hair growth promoting effect of X4N cell culture supernatant in rats. On day 0, the backs of C3H mice were shaved, and five mice in each group were treated with the following test substances once a day: Group 1: Test culture supernatant of purified and concentrated X4N cells; Group 2: 1% minoxidil (Rogaine) (登録商標) )(positive control); and Group 3: 50% ethanol (negative control). The degree of hair growth was visually evaluated and scored. The total score for each group on day 9 was calculated using X4N purified concentrated culture supernatant and RiUP. (登録商標) The concentrations were 4, 10, and 2 in 50% ethanol, respectively, and the culture supernatant of X4N cells showed twice the hair growth activity compared to the negative control. [Industrial applicability] 【0117】 This disclosure provides compositions comprising stem cell culture supernatants for use in pharmaceutical or cosmetic applications, such as in the treatment of injury or disease in a subject, and methods for producing the same. This disclosure is applicable to various fields, including the pharmaceutical and cosmetic fields.

Claims

[Claim 1] A composition comprising a culture supernatant of stem cells, wherein the composition is a pharmaceutical composition for treating injury or disease in a subject, or a cosmetic composition for skin care, texture improvement, anti-aging, or hair growth and hair restoration, wherein the stem cells are stem cells that are derived from mesenchymal stem cells, immortalized by introduction of telomerase (TERT), express both CCN1 and CCN2, and have been introduced with a combination of OCT3 / 4, SOX2, KLF4, L-Myc and LIN28, and are capable of differentiating into nerve cells. [Claim 2] The composition according to claim 1, wherein the stem cells are X4N cells deposited under accession number P-02505. [Claim 3] The composition according to claim 1, wherein the stem cells include undifferentiated stem cells and / or differentiated nerve cells. [Claim 4] The culture supernatant contains BDNF, BMP, BMP1, BMP10, BMP2, BMP4, BMP5, BMP7, CLEC, COL, COL11A1, COL11A2, COL14A1, COL18A1, COL1A1, COL1. A2, COL22A1, COL2A1, COL3A1, COL4A2, COL5A1, COL5A2, COL6A1, COL6A2, CSF, CSF1, CXCL1, CXCL12, DKK1, EDN1, EFEMP, E FEMP1, ELN, ENO1, FGF, FGF1, FGF18, FGF7, FGF9, FN, FN1, FRZB, FSTL1, GDF, GDF10, GDF11, GDF15, GDF2, GMFB, GRN, HDGF , HGF, IGF, IGF2, IGFBP, IGFBP5, IGFBP7, IL, INHBA, JAG1, KGF, KIF20B, KRT, KRT1, KRT10, KRT2, KRT9, LGALS3BP, LGBDNF , LOXL2, MANF, MIA, MIF, MMP, MMP1, MMP10, MMP11, MMP14, MMP15, MMP2, MMP23, MMP9, MMP, NGF, NID2, NRG1, NT3, PCOLCE, PDGF, PDGF-C, PDGF-D, PTN, PTX3, SCG, SCG2, SDF-1, SFRP, SFRP1, SFRP2, SFRP3, SFRP5, SHANK3, SPARC, TBX, TBX20, TBX5 The composition according to claim 1, comprising at least one protein selected from the group consisting of TGF-α, TGF-β, TGF-β1, TGF-β2, THBS, THBS1, THBS2, TIMP, TIMP1, TIMP2, TIMP3, TIMP4, VCAN, VEGF, VEGFA, VEGFB, VEGFC, VEGFD, VIM, WNT5A, elastin, glycoprotein, keratin, laminin, basement membrane factors, and combinations thereof. [Claim 5] The culture supernatant contains miR-363, miR499, miR-1, miR-21, miR-126, miR-34, miR-16, hsa-miR-1-3p, hsa-miR-675-3p, h sa-miR-10395-3p, hsa-miR-133b, hsa-miR-208b-3p, hsa-miR-4488, hsa-miR-450b-5p, hsa-miR-499b The composition according to claim 1, comprising at least one microRNA (miRNA) selected from the group consisting of -3, hsa-miR-542-3p, hsa-miR-642a-3p and hsa-miR-9901, gga-mir-302a, gga-mir-302b, gga-mir-302c, gga-mir-302d, eca-mir-369, mmu-mir-19a, mmu-mir-20a and combinations thereof. [Claim 6] Culture supernatant contains BDNF, BMP, BMP1, BMP10, BMP2, BMP4, BMP5, BMP7, CLEC, COL, COL11A1, COL11A2, COL14A1, COL18A1, COL1A1, COL1A2 , COL22A1, COL2A1, COL3A1, COL4A2, COL5A1, COL5A2, COL6A1, COL6A2, CSF, CSF1, CXCL1, CXCL12, DKK1, EDN1, EFEMP, EFEM P1, ELN, ENO1, FGF, FGF1, FGF18, FGF7, FGF9, FN, FN1, FRZB, FSTL1, GDF, GDF10, GDF11, GDF15, GDF2, GMFB, GRN, HDGF, HGF , IGF, IGF2, IGFBP, IGFBP5, IGFBP7, IL, INHBA, JAG1, KGF, KIF20B, KRT, KRT1, KRT10, KRT2, KRT9, LGALS3BP, LGBDNF, LOXL 2, MANF, MIA, MIF, MMP, MMP1, MMP10, MMP11, MMP14, MMP15, MMP2, MMP23, MMP9, MMP, NGF, NID2, NRG1, NT3, PCOLCE, PDGF, P DGF-C, PDGF-D, PTN, PTX3, SCG, SCG2, SDF-1, SFRP, SFRP1, SFRP2, SFRP3, SFRP5, SHANK3, SPARC, TBX, TBX20, TBX5, TGF-α, The composition according to claim 1, comprising an extracellular vesicle containing at least one protein selected from the group consisting of TGF-β, TGF-β1, TGF-β2, THBS, THBS1, THBS2, TIMP, TIMP1, TIMP2, TIMP3, TIMP4, VCAN, VEGF, VEGFA, VEGFB, VEGFC, VEGFD, VIM, WNT5A, elastin, glycoprotein, keratin, laminin, basement membrane factors, and combinations thereof. [Claim 7] The culture supernatant contains miR-363, miR499, miR-1, miR-21, miR-126, miR-34, miR-16, hsa-miR-1-3p, hsa-miR-675-3p, hsa-miR -10395-3p, hsa-miR-133b, hsa-miR-208b-3p, hsa-miR-4488, hsa-miR-450b-5p, hsa-miR-499b-3, hsa-miR-5 The composition according to claim 1, comprising an extracellular vesicle containing at least one miRNA selected from the group consisting of 42-3p, hsa-miR-642a-3p and hsa-miR-9901, gga-mir-302a, gga-mir-302b, gga-mir-302c, gga-mir-302d, eca-mir-369, mmu-mir-19a, mmu-mir-20a and combinations thereof. [Claim 8] A pharmaceutical composition according to claim 1 for treating injury or disease in a subject, wherein the injury is trauma or wound in any site or chronic or transient functional impairment resulting therefrom, or the disease is a neurological disorder, depression, myasthenia gravis, dementia, Parkinson's disease, Huntington's disease or schizophrenia, ischemia, circulatory disorder, infarction, osteoporosis, immune disease or periodontitis. [Claim 9] The composition according to claim 8, wherein the target is a mammal, a bird, or a reptile. [Claim 10] The composition according to claim 8, formulated for use via local, oral, rectal, nasal, intravenous, intra-articular, conjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, transdermal, or subcutaneous administration routes. [Claim 11] The pharmaceutical composition according to claim 8, which is in the form of granules, fine granules, powder, hard capsule, soft capsule, syrup, emulsion, suspension or liquid, injection, drip, transdermal, transmucosal, nasal, inhalation or suppository. [Claim 12] A cosmetic composition according to claim 1 for skincare, skin texture improvement, anti-aging, or hair growth and hair restoration, which is in the form of a lotion, emulsion, gel, serum, cream, pack, mask, facial cleanser, cosmetic soap, foundation, powder, or body lotion. [Claim 13] i) The process of culturing the seeded stem cell population; and ii) Steps to collect the culture supernatant A method for producing the composition according to claims 1 to 12, including the method described above.

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