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

Composition containing cell culture supernatant and method for producing same

[0001] The present 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.

[0002] With regard to the treatment of spinal cord injury and neurodegenerative diseases caused by damage to specific nerve cells, cell preparations containing human-derived nerve cells obtained by differentiating human-derived pluripotent stem cells (e.g., embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells)) have attracted attention.

[0003] As described in Patent Document 1, the inventors have created stem cells capable of differentiating into neurons, chondrocytes, osteocytes, or adipocytes, and demonstrated that the cells induced to differentiate into neurons are effective in treating a rat spinal cord injury model.

[0004] JP 2019-076008 A JP 2021-141872 A

[0005] Basso OM et al. A sensitive and reliable locomotor rating scale for open field testing in rats. J. Neurotrauma 1995; 12: 1-21.

[0006] However, by-products such as culture supernatants produced during stem cell cultivation have been discarded. Considering the impact on the environment, it is necessary to provide new uses for the culture supernatants.

[0007] The present inventors conducted extensive research to solve the above-mentioned problems. It was thought that not only do stem cells themselves have therapeutic effects, but also that various physiologically active substances secreted by stem cells, such as cytokines and exosomes, contribute to the therapeutic effects. The present inventors discovered that, because artificially cultured stem cells release these physiologically active substances into the culture medium, the culture supernatant from which the cells have been removed can also be expected to have therapeutic or cosmetic effects, 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 or cosmetic composition comprising a stem cell culture supernatant, 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 immortalized by introducing telomerase (TERT). [Item 3] The composition according to Item 1, wherein the stem cells are bound to an antibody having 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 Accession No. P-02505. [Item 5] The composition according to Item 1, wherein the stem cells include undifferentiated stem cells and / or neuronally differentiated cells.[Section 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 Item 6. The composition according to any one of Items 1 to 5, 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 factor, 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 -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. The composition according to any one of items 1 to 6, comprising at least one microRNA (miRNA) selected from the group consisting of:[Section 8] The 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, 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 Item 8. The composition according to any one of Items 1 to 7, comprising extracellular vesicles comprising 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 factor, and combinations thereof.[Section 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 The composition according to any one of Items 1 to 7, comprising extracellular vesicles containing at least one miRNA selected from the group consisting of miR-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. [Item 10] The composition according to Items 8 or 9, wherein the extracellular vesicles are exosomes of about 40 to 120 nm. [Item 11] The pharmaceutical composition according to any one of Items 1 to 10 for treating injury or disease in a subject, wherein the injury is trauma or wounding at any site or a chronic or transient functional disorder resulting therefrom, or the disease is neurological disease, depression, myasthenia gravis, dementia, Parkinson's disease, Huntington's disease, schizophrenia, ischemia, blood flow disorder, infarction, osteoporosis, immune disease, or periodontitis. [Item 12] The composition according to Item 11, wherein the subject is a mammalian subject, an avian subject, or a reptilian subject. [Item 13] The composition according to Item 12, wherein the mammalian subject is a human. [Item 14] The composition according to any one of Items 11 to 13, which is formulated for topical, oral, rectal, nasal, intravenous, intra-articular, conjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, transdermal, or subcutaneous administration. [Item 15] The pharmaceutical composition according to any one of Items 11 to 13, which is in the form of granules, fine granules, powder, hard capsules, soft capsules, syrup, emulsion, suspension or liquid, injection, infusion, transdermal absorption, transmucosal absorption, nasal drops, inhalant, or suppository. [Item 16] The cosmetic composition according to any one of Items 1 to 10, which is used for skin care, skin whitening, wrinkle reduction, texture improvement, anti-aging, and hair growth.[Item 17] The cosmetic composition according to 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. [Item 18] A method for producing the composition according to Items 1 to 17, comprising: i) culturing the seeded stem cell population; and ii) recovering the culture supernatant.

[0009] The present disclosure provides a composition comprising a stem cell culture supernatant for use in pharmaceutical or cosmetic applications, and a method for producing the same.

[0010] Figure 1 is a line graph showing changes in Basso-Beattie-Bresnahan (BBB) ​​score for each test group in a rat spinal cord injury model, namely, the group in which the culture supernatant was administered via the tail vein (◯; solid line), the group in which the culture supernatant was administered intrathecally (□; dashed line), and the control group in which the vehicle was administered via the tail vein (■; solid line).

[0011] Figure 2 shows hematoxylin-eosin (HE)-stained images of tissues collected 39 days after spinal cord injury from rats in the control group, the group administered the culture supernatant via the tail vein (intravenous injection), and the group administered the culture supernatant intrathecally (intrathecal injection) (top row: low magnification, bottom row: high magnification). Figure 2A shows HE-stained images of spinal cord tissue sections around the injury site from rats in each group. Figure 2B shows HE-stained images of brain tissue sections from rats in each group. Figure 2C shows HE-stained images of liver tissue sections from rats in each group. Figure 2D shows HE-stained images of kidney tissue sections from rats in each group. Figure 3 shows the BDNF concentration in the culture supernatants measured by ELISA. BDNF concentrations were measured in test culture supernatants of X4N cells, iPS cells, human mesenchymal stem cells (hMSCs), and culture media (RHB-A or ReproCell). FIG. 4A shows a schematic diagram of the exosome fraction of the culture supernatant. FIG. 4B shows the BDNF concentration in each fraction of the culture supernatant measured by ELISA. FIG. 5A shows the results of a test of the proliferation ability of mesenchymal stem cells when a culture supernatant according to the present disclosure was added to a mesenchymal stem cell culture system. FIG. 5B shows the results of a test of the proliferation ability of fibroblasts when a culture supernatant according to the present disclosure was added to a fibroblast culture system. FIG. 5C shows the results of a test of the proliferation ability of fibroblasts when a culture supernatant according to the present disclosure stored at -30°C, 4°C, or room temperature for one month was added to a fibroblast culture system. FIG. 6 shows the results of immunostaining of mouse ES cells that had been induced to differentiate into neurons for four days and then cultured for three days under each condition (medium only, with 10% hMSC culture supernatant added, and with 10% X4N cell culture supernatant added). TuJ: tubulin βIII antibody (neuronal differentiation marker), DAPI: cell nucleus staining, Merge: Tuj and DAPI superimposed image Figure 7 shows the results of a hair growth promotion test in mice 1 day and 9 days after shaving.

[0012] In one aspect, the present disclosure relates to a composition comprising a stem cell culture supernatant for use in pharmaceutical applications. In some embodiments, the present disclosure relates to a composition comprising a stem cell culture supernatant for use in treating injury or disease in a subject.

[0013] In one aspect, the present disclosure relates to compositions comprising stem cell culture supernatants for use in cosmetic applications. In some embodiments, the present disclosure relates to compositions comprising stem cell culture supernatants for use in skin care, anti-aging, or hair growth and restoration.

[0014] In one aspect, the present disclosure relates to a method for producing a composition of the present disclosure, comprising the steps of: i) culturing seeded stem cells; and ii) recovering the culture supernatant.

[0015] (Stem Cells) In the present disclosure, "stem cells" refer to cells that have the ability to replicate themselves without differentiation (also referred to as "self-renewal ability") and have the differentiation potential to terminally differentiate into various tissue cells. In the present disclosure, stem cells may be pluripotent stem cells that have the ability to self-renew and differentiate into multiple cell types, such as induced pluripotent stem cells or embryonic stem cells, or stem cells may be progenitor cells that have the ability to self-renew and 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 the present disclosure, "stem cells" may include cells in the process of differentiation derived from the stem cells or cells that have completed differentiation.

[0016] In some embodiments, the stem cells of the present disclosure are stem cells that express either one or both of CCN1 and CCN2.

[0017] "CCN1" is a protein belonging to the CCN family, also known as cysteine ​​rich 61 (Cyr61). "CCN2" is a protein also known as connective tissue growth factor (CTGF). The amino acid and nucleic acid sequences of these proteins are known.

[0018] As used herein, "CCN1-expressing stem cells" refers to a specific stem cell population that expresses at least two-fold more CCN1 than an unselected or untreated stem cell population.

[0019] As used herein, "CCN2-expressing stem cells" refers to a specific stem cell population that expresses CCN2 at least twice as much as an unselected or untreated stem cell population. As used herein, "CCN1 and CCN2-expressing stem cells" refers to a specific stem cell population that expresses CCN1 at least twice as much and CCN2 at least twice as much as an unselected or untreated stem cell population.

[0020] As used herein, "unselected stem cell population" refers to a stem cell population that has not been selected for the expression level of either or both of CCN1 and CCN2. 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 of CCN1 and CCN2 can be prepared by cloning stem cells from an unselected stem cell population that express at least two-fold more CCN1 and / or CCN2 than the expression levels in the unselected stem cell population.

[0022] Stem cell cloning can be carried out appropriately according to conventional methods. Examples of stem cell cloning include, but are not limited to, limiting dilution. Measurement of the expression levels of genes or proteins expressed in stem cells according to the present disclosure can be carried out according to conventional methods. Measurement of expression levels can be carried out by, but is not limited to, PCR, Northern blotting, Western blotting, immunostaining, and microarray. In some embodiments, measurement of expression levels is carried out by quantitative PCR.

[0023] As used herein, "untreated stem cell population" refers to a stem cell population that has not been subjected to a treatment that alters the expression level of either or both of CCN1 and CCN2. Treatments that alter expression levels include gene transfer, for example, introduction of a CCN1 and / or CCN2 expression cassette, or introduction of an immortalization gene. In some embodiments, the untreated stem cell population is a population of cells prepared from biological tissue according to standard methods, or commercially available stem cells.

[0024] In some embodiments, the stem cells that express either or both of CCN1 and CCN2 are stem cells that have been immortalized to an untreated stem cell population, and whose expression level of either or both of CCN1 and CCN2 is at least two-fold higher than that of an untreated stem cell population.

[0025] In some embodiments, preparing stem cells that express CCN1 comprises selecting or treating (optionally followed by selection) stem cells that express at least 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold more CCN1 than an unselected or untreated stem cell population. In other embodiments, preparing stem cells that express CCN2 comprises selecting or treating (optionally followed by selection) stem cells that express CCN2 at least 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold more CCN2 than an unselected or untreated stem cell population.

[0026] In some embodiments, preparing stem cells that express CCN1 and CCN2 comprises selecting or treating (and optionally selecting after treatment) stem cells that express at least 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold more CCN1 than an unselected or untreated stem cell population, and at least 3-fold, 5-fold, 7-fold, 9-fold, 10-fold, 15-fold, or 20-fold more CCN2.

[0027] In some embodiments, the stem cells according to the present disclosure are immortalized stem cells.

[0028] Stem cell "immortalization" can be carried out appropriately according to conventional methods. Examples of stem cell immortalization include, but are not limited to, a method involving the expression of telomerase (TERT) (preferably human-derived TERT (hTERT)), a method using SV40 T antigen, and a method involving co-expression of the hTERT catalytic subunit with p53 or RB siRNA. In some embodiments, stem cells expressing either CCN1 or CCN2, or both, are prepared by introducing hTERT into stem cells. Stem cells introduced with hTERT may, but are not limited to, transiently or constitutively express hTERT. In some embodiments, the stem cells disclosed herein are stem cells immortalized by introducing telomerase.

[0029] In some embodiments, the stem cells of the present disclosure are stem cells into which one or more reprogramming factors have been introduced.

[0030] As used herein, the term "reprogramming factor" refers to a gene that is specifically expressed in embryonic stem cells (ES cells), or a gene that plays an important role in maintaining ES cells in an undifferentiated state, or its gene product. Reprogramming factors may be in the form of, but are not limited to, genes (e.g., DNA, RNA) or proteins. 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, and Glis1. These reprogramming factors may be used alone, or in combination of two or more, three or more, four or more, five or more, for example, 1 to 10 types.

[0031] In some embodiments, the one or more reprogramming factors include a combination of Sox family and Oct family. The Sox family includes, but is not limited to, Sox1, Sox2, Sox3, Sox15, and Sox17, which may be used alone or in combination of two or more. The Oct family includes, but is not limited to, Oct3 / 4.

[0032] In some embodiments, the one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, L-Myc, NANOG, and LIN28.

[0033] As used herein, "introduction" of a reprogramming factor means placing the reprogramming factor in a state where it is present in a cell. When the reprogramming factor is a protein, introduction of one or more reprogramming factors can be carried out according to a conventional method. In one embodiment, a reprogramming factor that is a protein is introduced into a cell by lipofection, microinjection, or fusion with a cell membrane-permeable peptide.

[0034] When 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 incorporated into a vector such as a viral vector, a plasmid, or an artificial chromosome, which contains a sequence (e.g., a promoter sequence or an enhancer sequence) for driving its transcription and / or translation, and then introduced into a cell. 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, plasmids for mammalian cells; 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 the present disclosure are stem cells capable of differentiating into neurons, which are ectodermal cells, and mesodermal cells, such as chondrocytes, osteocytes, and adipocytes. In some embodiments, the stem cells according to the present disclosure are stem cells capable of differentiating into neurons.

[0036] In some embodiments, the stem cells of the present disclosure are stem cells capable of differentiating into neural cells and express one or more neural stem cell markers, including nestin, SOX2, PAX6, ABCG2, FGFR1, and Frizzled-9. In some embodiments, the stem cells of the present disclosure further express HOX4B.

[0037] In some embodiments, the stem cells according to the present disclosure are bound by the "185 antibody" described in Japanese Patent Application Laid-Open No. 2021-141872. In some embodiments, the stem cells according to the present disclosure are bound by the 185 antibody before and / or after differentiation induction. In a preferred embodiment, the stem cells according to the present disclosure are bound by the 185 antibody before differentiation induction.

[0038] In some embodiments, the stem cells are derived from a mammal, preferably a primate, more preferably a human. In some embodiments, the stem cells are derived from a mammalian biological 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 biological tissue may be prepared according to conventional methods or may be commercially available. In some embodiments, the stem cells disclosed herein are derived from the same species or the same individual as the subject to whom the composition of the present disclosure is administered.

[0039] Stem cells according to the present disclosure have the ability to differentiate into cells such as, but not limited to, neurons, chondrocytes, osteocytes, and adipocytes. In some embodiments, stem cells according to the present disclosure have the ability to differentiate into neurons. In some embodiments, stem cells according to the present disclosure have the ability to differentiate into cells such as neurons, chondrocytes, osteocytes, and adipocytes with high efficiency (e.g., after differentiation induction, the number of cells in the cell population after induction is 20% or more, 40% or more, 60% or more, 80% or more, 20% to 80%, 40% to 80%, 40% to 60%).

[0040] The stem cells according to the present disclosure are adherent cells or suspension cells. Since the amount of culture medium required for adherent cells is greater than that required for suspension cells, the stem cells according to the present disclosure are preferably adherent cells.

[0041] Stem cells according to the present disclosure have, but are not limited to, a high proliferation rate. In some embodiments, stem cells according to the present disclosure double in proliferation rate within 12 to 24 hours when cultured in a medium for primate ES cell culture.

[0042] Stem cells according to the present disclosure express and secrete effective bioactive substances into the culture medium. The stem cells according to the present disclosure secrete effective bioactive substances at higher concentrations than, for example, iPS cells. "Effective bioactive substances" or "bioactive substances" are bioactive substances effective for pharmaceutical or cosmetic applications according to the present disclosure, and include proteins including cytokines, growth factors, and extracellular matrix (ECM) secreted proteins, nucleic acid molecules including miRNA, mRNA, ncRNA, and DNA, as well as extracellular vesicles (EVs), including exosomes, that encapsulate these proteins or nucleic acid molecules. In some embodiments, the stem cells according to the present disclosure secrete extracellular vesicles containing bioactive substances including the above proteins and nucleic acid molecules.

[0043] Proteins secreted by stem cells according to the present disclosure include, 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, and 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 , 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 factor.

[0044] MiRNAs secreted by stem cells according to the present 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, These include 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, stem cells according to the present disclosure express one or more potent bioactive substances at a level at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more times higher than iPS cells. In some embodiments, stem cells according to the present disclosure express one or more proteins, including CCN1, CCN2, BDNF, VDGF, EGF, FGF, HGF, IGF, PDFG, or TGF-β, at a level at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more times higher than iPS cells. In preferred embodiments, stem cells according to the present disclosure express BDNF at a level at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more times higher than iPS cells. In some embodiments, stem cells of the present 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 about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more times higher than iPS cells.

[0046] In some embodiments, the stem cells of the present disclosure are stem cells that express either one or both of CCN1 and CCN2; are immortalized by introducing hTERT; and have one or more reprogramming factors introduced therein. In some embodiments, the stem cells of the present disclosure are X4N cells (NEcST cells) deposited on July 4, 2017, under accession number P-02505, at the National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganisms Depositary (NPMD), having an address at 2-5-8122 Kazusa Kamatari, Kisarazu City, Chiba Prefecture (292-0818). （登録商標） The deposit certificate was notified on September 15, 2017, with notification number 2017-0253.

[0047] (Stem Cell Culture Supernatant) The stem cell culture supernatant according to the present disclosure is a culture medium obtained by culturing the stem cells according to the present disclosure. Therefore, a culture supernatant usable in the present disclosure can be obtained by separating and removing cellular components after culturing the stem cells. In some embodiments, the culture supernatant according to the present disclosure may be a culture supernatant that has been appropriately subjected to various processes (e.g., centrifugation, mixing, concentration, solvent substitution, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.).

[0048] In some embodiments, the stem cell culture supernatant of the present disclosure may be included in a composition as one or more active ingredients (bioactive substances, e.g., cytokines, exosomes, miRNA) isolated or purified from the culture medium. Various components and substances in the culture supernatant, such as proteins, extracellular vesicles, nucleic acid molecules, lipid molecules, and other trace components, can be isolated and purified using conventional methods, and their concentrations can be measured using conventional methods. Isolation and purification methods include, but are not limited to, chromatography, ammonium sulfate fractionation, ultrafiltration, immunoadsorption, centrifugation, ultracentrifugation polymer precipitation, field-flow fractionation, and immunoprecipitation. Detection and concentration measurements include, but are not limited to, ELISA, Western blotting, nanoparticle analysis, vanillin analysis, qPCR, microarray analysis, or next-generation sequencing. In some embodiments, quantification of components or substances in the culture supernatant is performed using ELISA.

[0049] When culturing stem cells according to the present disclosure in a state in which they maintain their self-renewal ability and differentiation ability (e.g., the ability to differentiate into neurons, chondrocytes, osteocytes, or adipocytes), the culture conditions (e.g., medium composition, culture method, passaging method) that enable this can be appropriately determined from well-known and commonly used techniques. In some embodiments, such a medium is a xeno-free medium. In some embodiments, such a medium is a commercially available medium for culturing stem cells, such as a medium for culturing mouse ES cells (e.g., TX-WES medium, Thrombo-X), a medium for culturing primate ES cells (e.g., Primate ES Cell Medium (ReproCell)), or a medium for culturing human pluripotent stem cells (hPSCs) (e.g., NutriStem （登録商標） hPSC XF GF-free (Sartorius)), human mesenchymal stem cell (hMSC) culture medium (e.g., MSC NutriStem （登録商標） 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 human PSCs (Sartorius), or a culture medium for human MSCs (Sartorius).

[0050] In some embodiments, the culture method for culturing stem cells according to the present disclosure while maintaining their self-renewal and differentiation potential may be a method used to culture pluripotent cells, such as ES cells and iPS cells, while maintaining their differentiation potential. Culture methods include, but are not limited to, three-dimensional culture under non-adherent conditions, such as suspension culture (e.g., dispersed culture, aggregated suspension culture, etc.), 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 cells under adherent conditions in the absence of feeder cells. In cell-adherent culture vessels, the surface is coated with a cell support material (e.g., collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, vitronectin, or other materials) to improve cell adhesion. In some embodiments, the cell-adherent culture vessel is coated with, for example, 0.1% gelatin.

[0051] In some embodiments, the stem cell culture supernatant of the present disclosure is a culture medium of stem cells cultured under conditions that allow the self-renewal ability and differentiation ability of the stem cells to be maintained (undifferentiated cell culture supernatant).

[0052] Culture conditions (e.g., medium composition, differentiation induction factors, culture method, passaging method, etc.) for inducing differentiation of stem cells according to the present disclosure into target cells (e.g., neurons) can be appropriately determined from well-known and commonly used techniques. In some embodiments, such a medium is a xeno-free medium. In some embodiments, the medium for inducing differentiation of stem cells into neurons is a commercially available neuronal culture medium or neuronal differentiation medium (e.g., RHB-A Takara Bio). In other embodiments, the neuronal culture medium or neuronal differentiation induction medium contains a neuronal induction factor (e.g., brain-derived nerve growth factor (BDNF), fibroblast growth factor (FGF), a small molecule BMP inhibitor and / or a small molecule TGFβ family inhibitor).

[0053] In some embodiments, the culture method for inducing differentiation of stem cells into neural cells according to the present disclosure may be a method used for inducing differentiation of pluripotent cells, such as ES cells or iPS cells, into neural cells. The culture method includes, but is not limited to, three-dimensional culture under non-adherent conditions, two-dimensional culture under adhesive coating conditions, or a combination of three-dimensional culture and two-dimensional culture. In some embodiments, the culture method involves culturing under non-adherent conditions in the absence of feeder cells.

[0054] In some embodiments, neural cells induced from stem cells of the present disclosure have CCN1 expression levels that are at least 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 70-fold, or 100-fold lower than the expression levels of the stem cells.

[0055] In some embodiments, the medium for inducing the differentiation of stem cells according to the present disclosure into adipocytes includes, but is not limited to, a commercially available adipocyte induction medium or a commercially available animal cell medium supplemented with insulin, dexamethasone, and indomethacin. In some embodiments, the medium for inducing the differentiation of the cells according to the present disclosure into chondrocytes includes, but is not limited to, a commercially available chondrocyte induction medium or a commercially available animal cell medium supplemented with dexamethasone, ascorbic acid, and TGF-β3. In some embodiments, the medium for inducing the differentiation of the cells according to the present disclosure into bone cells includes, but is not limited to, a commercially available bone cell induction medium or a commercially available animal cell medium supplemented with dexamethasone, ascorbic acid, and β-glycerophosphate.

[0056] Whether or not the cells have differentiated into the target cells can be confirmed by conventional methods. When cells have been differentiated into neurons, this can be confirmed by, but is not limited to, staining with neuronal markers (e.g., NeuN, MAP2). When cells have been differentiated into adipocytes, this can be confirmed by, but is not limited to, oil red staining. When cells have been differentiated into chondrocytes, this can be confirmed by, but is not limited to, alcian blue staining. When cells have been differentiated into osteocytes, this can be confirmed by, but is not limited to, alkaline phosphatase staining.

[0057] In some embodiments, the stem cell culture supernatant according to the present disclosure is a culture supernatant of stem cells cultured under conditions for inducing differentiation (differentiation-induced cell culture supernatant). In some embodiments, the stem cell culture supernatant according to the present disclosure is a 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 the present disclosure is a 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 neural cells, for example, 20% to 80%, 40% to 80%, or 40% to 60% of the cell population after differentiation induction are neural cells. In some embodiments, the stem cell culture supernatant according to the present disclosure is the culture supernatant of differentiation-induced cells on day n (where n is any integer), with the day on which they were seeded in a differentiation-inducing medium being day 0. In some embodiments, the stem cell culture supernatant according to the present disclosure is the culture supernatant of differentiation-induced cells 1 day to 2 months after differentiation induction, particularly 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, or 30 days after differentiation induction. In a preferred embodiment, the stem cell culture supernatant according to the present disclosure is the culture supernatant of differentiation-induced cells 5, 6, 7, 8, 9, or 10 days after differentiation induction.

[0058] Regarding the cell culture conditions, the culture temperature is, but is not limited to, about 30°C to about 40°C, preferably about 37°C. ２ In an atmosphere containing air, e.g., CO ２ It is carried out at a concentration of about 2% to about 5%.

[0059] The stem cells of the present disclosure may be cryopreserved according to conventional methods before culturing, and then thawed before seeding, preferably just before seeding, before use.The stem cells of the present disclosure may also be cryopreserved at any time during culturing.The stem cells of the present disclosure may be frozen and stored, for example, in liquid nitrogen or a deep freezer.

[0060] In some embodiments, the stem cell culture supernatant according to the present disclosure may be a mixture of the above-described undifferentiated cell culture supernatant and differentiation-inducing cell culture supernatant. The mixture ratio can be adjusted appropriately by those skilled in the art depending on the disease and injury of the subject, and may be, for example, a mixture of undifferentiated cell culture supernatant and differentiation-inducing cell culture supernatant at 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] Without wishing to be bound by any particular theory, the culture supernatant of the stem cells disclosed herein contains a mixture of physiologically active substances including cytokines, and when administered to a subject, the physiologically active substances stimulate the differentiation and proliferation of endogenous stem cells near the damaged site, or act as an induction signal that activates the mobilization of other cells effective in tissue repair to the damaged site, thereby promoting cell proliferation and extracellular matrix formation at the damaged site.

[0062] In some embodiments, the culture supernatant according to the present disclosure contains a physiologically active substance. The physiologically active substance may be dissolved, hydrated, or dispersed in the culture supernatant, or may be contained in extracellular vesicles (EVs) in the culture supernatant. In some embodiments, the culture supernatant according to the present disclosure contains proteins, nucleic acid molecules, or extracellular vesicles containing these proteins or nucleic acid molecules that are effective for pharmaceutical or cosmetic applications according to the present disclosure. In some embodiments, the extracellular vesicles include vesicles of various sizes ranging from about 40 to about 2,000 nm, including, but not limited to, exosomes of about 40 to 120 nm, microvesicles 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 contain 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, CSF2, CSF3, CSF4, CSF5, CSF6, CSF7, CSF8, CSF9, CSF10, CSF11, CSF12, CSF13, CSF14, CSF15, CSF16, CSF17, CSF18, CSF19, CSF20, CSF21, CSF22, CSF23, CSF24, CSF25, CSF26, CSF27, CSF28, CSF39, CSF40, CSF41, CSF42, CSF50, CSF51, CSF52, CSF61, CSF62, CSF63, CSF64, CSF65, CSF76, CSF81, CSF92, CSF10, CSF111, CSF12, CSF13, CSF14, CSF15, CSF16, CSF17, CSF18, CSF19, CSF19, CSF19, CSF19, CSF21, CSF22, CSF23, CSF24, CSF25, CSF26, CSF27, CSF39, CSF41, CSF19, CSF19, CSF21, CSF22, CSF23, CSF24, CSF25, CSF31 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 factor, and combinations thereof.

[0064] In some embodiments, the culture supernatant or extracellular vesicles in the culture supernatant according to the present disclosure contain 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-542-3p, 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 of the present 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 or more times higher than the iPS cell culture supernatant. In some embodiments, the stem cell culture supernatant of the present 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 or more times higher than the iPS cell culture supernatant. In some embodiments, the stem cell culture supernatant of the present 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 or more times higher than the iPS cell culture supernatant. Preferably, the stem cell culture supernatant of the present disclosure contains one or more of 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 a concentration that is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more times higher than the iPS cell culture supernatant. In some embodiments, the stem cell culture supernatant of the present disclosure contains one or more proteins at a concentration that is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more times higher than the iPS cell culture supernatant. Preferably, the stem cell culture supernatant of the present disclosure contains CCN1, CCN2, BDNF, VDGF, EGF, FGF, HGF, IGF, PDFG, or TGF-β at a concentration that is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more times higher than that of the iPS cell culture supernatant.

[0066] In some embodiments, the culture supernatant of the present disclosure contains a physiologically active substance derived from a stem cell of the present disclosure. In some embodiments, the culture supernatant of a stem cell of the present disclosure contains one or more physiologically active substances at a concentration of at least about 0.5 pg / ml, for example, from about 0.5 pg / ml to about 10 μg / ml. In some embodiments, the culture supernatant of a stem cell of the present disclosure contains at least about 0.5 pg / ml, for example, from about 0.5 pg / ml to about 10 μg / ml of protein. In some embodiments, the culture supernatant of a stem cell of the present disclosure contains at least 1 x 10 ７ / mL, e.g., about 1 x 10 ７ ~1 x 10 １2 / mL of extracellular vesicles (particularly exosomes). Stem cell culture supernatants according to the present disclosure contain EVs at a concentration of at least 0.5 pg / ml, for example, from 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 the present disclosure can be measured as the amount of cell membrane-derived components, for example, membrane proteins such as MHC and adhesion molecules, or endosomal membrane-derived lipids (cholesterol and sphingomyelin). In some embodiments, stem cell culture supernatants according to the present disclosure contain cell membrane-derived components at a concentration of at least 0.5 pg / ml, for example, from about 0.5 pg / ml to about 10 μg / ml. In some embodiments, stem cell culture supernatants according to the present disclosure contain at least about 0.5 pg / ml of nucleic acid molecules. In some embodiments, stem cell culture supernatants according to the present disclosure contain at least about 0.5 pg / ml of miRNA.

[0067] In one embodiment, the culture supernatant of the present disclosure contains 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, e.g., at least about 1 pg / ml, at least about 10 pg / ml, at least about 100 pg / ml, at least about 1000 pg / ml, or at least about 2000 pg / ml. In one embodiment, the culture supernatant of the present disclosure contains 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 about 0.5-2000 pg / ml, about 5-1000 pg / ml, or about 50-500 pg / ml. In a preferred embodiment, 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 a preferred embodiment, 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 Compositions In one aspect, the compositions of the present disclosure are for use in pharmaceutical applications, particularly for use in treating injury or a disease or condition in a subject.

[0069] As used herein, "subject" refers to an animal having an injury or disease or condition to which a composition of the present disclosure is administered. In some embodiments, the subject includes a mammalian, avian, or reptilian subject. In some embodiments, the subject includes a primate, which may or may not be a human. In some embodiments, the subject includes a pet, such as a dog or cat, or a livestock animal, such as a cow or horse. In some embodiments, the subject is a mammalian, avian, or reptilian subject, which may or may not be a human. In a specific embodiment, the subject is a human.

[0070] As used herein, "treatment" means slowing the progression or worsening of a disease or symptom, or maintaining, alleviating, or improving the disease state. Alternatively, "treatment" means promoting recovery from injury or disease or symptom. As used herein, recovery from injury means promoting the regeneration of tissue at or near the injury site, such as bone, skin, muscle, joint, tendon, ligament, nerve, or blood vessel, or alleviating or improving symptoms and functional disorders caused by injury, such as motor dysfunction, sensory dysfunction, and autonomic nervous dysfunction.

[0071] As used herein, "injury" refers to trauma or wounding of any part of a living body, including tissues and organs, and resulting acute or chronic dysfunction (e.g., motor dysfunction, sensory dysfunction, or autonomic dysfunction). In some embodiments, the injury may be exogenous injury caused by physical or chemical stimuli or forces. In some embodiments, the injury may be endogenous injury caused by disease or the like. For example, examples of endogenous injury caused by disease include inflammation due to inflammatory disease or tissue necrosis or dysfunction associated with impaired blood flow due to vascular disease. In some embodiments, the injury may be injury to tissues such as bone, skin, muscle, joints, tendons, ligaments, nerves, or blood vessels, and / or organs such as the brain, liver, heart, lungs, kidneys, spleen, pancreas, or digestive tract. In some embodiments, the injury may involve nervous tissue, such as spinal cord injury or brain injury. In some embodiments, the injury is exogenous 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 is a nervous system disease, including depression, myasthenia gravis, ischemic brain disease (such as stroke), motor neuron disease, neurodegenerative disease, multiple sclerosis, amyotrophic lateral sclerosis, spinocerebellar degeneration, Huntington's disease, dementia (e.g., Alzheimer's disease, dementia with Lewy bodies, vascular dementia, etc.), Parkinson's disease, epilepsy, and schizophrenia, blood flow disorders, infarction, osteoporosis, immune disease, or periodontitis. In some embodiments, the disease is a nervous system disease, including depression, myasthenia gravis, dementia, Parkinson's disease, Huntington's disease, or schizophrenia. In some embodiments, the compositions of the present invention can be used to treat periodontitis or regenerate periodontal tissue.

[0073] As used herein, "use in treatment" includes administering a composition of the present disclosure to a subject for the purpose of treating disease and injury. In some embodiments, "use in treatment" includes administering a composition of the present disclosure to a subject in place of, or in conjunction with, other therapies effective in treating disease and injury. In some embodiments, "use in treatment" includes using a composition of the present disclosure in combination with, or as an alternative to, platelet-rich plasma (PRP) therapy, where, when used as an alternative, the stem cells of the present disclosure are derived from stem cells, e.g., mesenchymal stem cells, of the same species or individual as the subject to whom the composition is administered.

[0074] In one aspect, the composition of the present disclosure may be a composition used in cosmetic applications. In some embodiments, the composition of the present disclosure may be a composition used for skin care, anti-aging, skin whitening, or hair growth and restoration. In some embodiments, the composition of the present disclosure may be a composition used for improving the appearance or texture of skin, for example, skin care, skin whitening, wrinkle reduction, skin texture improvement, cell turnover promotion, or anti-aging. In some embodiments, the composition of the present disclosure may be a cosmetic for skin care, anti-aging, or skin whitening. In some embodiments, a composition comprising a culture supernatant of the present disclosure may be a hair growth or restoration agent. In some embodiments, a composition comprising a culture supernatant of the present disclosure may be a composition for cosmetic or therapeutic purposes to improve and treat acute or chronic skin wounds, lesions, scars, keloids, and the like.

[0075] In some embodiments, the culture supernatant contained in the composition of the present disclosure may be a culture supernatant obtained by separating and removing cellular components from a culture medium, or a culture supernatant preparation obtained by subjecting the culture supernatant to various treatments (e.g., centrifugation, mixing, concentration, solvent substitution, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.). Thus, it is also contemplated that the culture supernatant contained in the composition of the present disclosure may contain one or more active ingredients (e.g., cytokines, exosomes, miRNA) 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 contain 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, CSF2, CSF3, CSF4, CSF5, CSF6, CSF7, CSF8, CSF9, CSF10, CSF11, CSF12, CSF13, CSF14, CSF15, CSF16, CSF17, CSF18, CSF19, CSF20, CSF21, CSF22, CSF23, CSF24, CSF25, CSF26, CSF27, CSF28, CSF39, CSF40, CSF41, CSF42, CSF50, CSF51, CSF52, CSF61, CSF62, CSF63, CSF64, CSF65, CSF76, CSF81, CSF92, CSF10, CSF111, CSF12, CSF13, CSF14, CSF15, CSF16, CSF17, CSF18, CSF19, CSF19, CSF19, CSF19, CSF21, CSF22, CSF23, CSF24, CSF25, CSF26, CSF27, CSF39, CSF41, CSF19, CSF19, CSF21, CSF22, CSF23, CSF24, CSF25, CSF31 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 In some embodiments, the composition of the present disclosure 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 factor, and combinations thereof. In some embodiments, the composition of the present disclosure comprises at least one neuronal differentiation factor or neuronal survival factor (e.g., BDNF, NGF, GDNF, NT-3, etc.).

[0077] In some embodiments for cosmetic applications, the composition of the present disclosure comprises 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 for cosmetic applications, the composition of the present disclosure comprises 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 the present disclosure contain 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-542-3p, 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 composition of the present disclosure is liquid and comprises at least about 0.1 pg / ml of a bioactive substance. In some embodiments, the composition of the present disclosure is liquid and comprises at least about 0.1 pg / ml of a bioactive substance (extracellular vesicles, protein) at a concentration of at least about 0.1 pg / ml, e.g., about 0.5 pg / ml to 10 μg / ml, about 5 to 1000 pg / ml, or about 50 to 500 pg / ml. In some embodiments, the composition of the present disclosure is liquid and comprises at least about 0.5 pg / ml of 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 1 x 10 ７ / mL, e.g., about 1 x 10 ７ ~1 x 10 １３ , about 1×10 ８ ~1 x 10 １２ , about 1×10 ９ ~1 x 10 １１ In a preferred embodiment, the composition of the present disclosure is liquid and 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, respectively.

[0080] In some embodiments, a composition of the present disclosure comprises at least about 0.01 pg, e.g., 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 agent. In some embodiments, a composition of the present disclosure comprises at least about 0.01 pg, e.g., 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, a composition of the present disclosure comprises at least about 1 x 10 ４ , e.g., at least about 1×10 ５ pieces, at least about 1 x 10 ６ pieces, at least about 1 x 10 ７ pieces, at least about 1 x 10 ８ pieces, at least about 1 x 10 ９ pieces, at least about 1 x 10 １０ pieces, at least about 1 x 10 １１ pieces, at least about 1 x 10 １２ pieces, at least about 1 x 10 １３ pieces, at least about 1 x 10 １４ or at least about 1 x 10 １５ Contains extracellular vesicles.

[0081] The present inventors have discovered that the culture supernatant of stem cells according to the present disclosure can contain high concentrations of physiologically active substances, particularly extracellular vesicles (e.g., exosomes) containing brain-derived neurotrophic factor (BDNF) and nucleic acid molecules (e.g., miRNA) involved in cell growth and regeneration. Regarding the relationship between neurotrophic factors and disease, it has been discovered that BDNF promotes the proliferation and differentiation of tooth-related cells and blood vessels, and BDNF levels in brain tissue are reduced in individuals with Alzheimer's disease. Furthermore, BDNF knockout mice lose sensory neurons and are unable to perform coordination, balance, hearing, taste, and breathing. BDNF knockout mice also exhibit abnormalities in the cerebellum and an increased number of sympathetic neurons. Furthermore, certain types of physical exercise increase BDNF synthesis in the human brain by approximately three-fold. This phenomenon is one mechanism underlying exercise-induced neurogenesis and cognitive function improvement.

[0082] In some embodiments, compositions of the present disclosure may optionally include one or more additional ingredients, including at least some pharmaceutically acceptable carriers or excipients and other agents. Examples of pharmaceutically acceptable carriers or excipients and other agents include water, physiological saline, buffer solutions, etc. as carriers, lactose, starch, sorbitol, D-mannitol, sucrose, etc. as excipients; other agents include starch, carboxymethylcellulose, calcium carbonate, etc. as disintegrants; phosphates, citrates, acetates, etc. as buffers; gum arabic, sodium alginate, tragacanth, etc. as emulsifiers; glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, etc. as suspending agents; benzyl alcohol, chlorobutanol, sorbitol, etc. as soothing agents; propylene glycol, ascorbic acid, etc. as stabilizers; phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. as preservatives; benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. as antiseptics; and hyaluronic acid, collagen, etc. as gelling agents. The additional component may also be an active ingredient or therapeutic agent for treating injury or disease, such as an immunosuppressant, such as an anti-inflammatory agent, a steroid, or a soothing agent.

[0083] The compositions of the present disclosure may be prepared in various dosage forms and administered to a subject via an appropriate administration route, dosage frequency, and dosage interval.

[0084] In some embodiments, the compositions of the present disclosure are formulated for topical, oral, rectal, nasal, intravenous, intraarticular, intraconjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, transdermal, or subcutaneous administration. Preferably, the compositions of the present disclosure are formulated for intrathecal or intravenous administration.

[0085] The form of the composition of the present disclosure can be appropriately determined by one skilled in the art depending on the mode of use and route of administration. In some embodiments, the composition of the present disclosure may be a solid such as a powder, fine granules, or granules; a liquid such as a suspension or emulsion; or a semi-solid such as a gel. In some embodiments of compositions for pharmaceutical use, the composition of the present disclosure may be an oral pharmaceutical composition in the form of, for example, granules, fine granules, powder, hard capsules, soft capsules, syrups, emulsions, suspensions, or solutions; or a parenteral pharmaceutical composition in the form of an injection such as an intravenous, intramuscular, or subcutaneous injection, infusion, transdermal absorption, transmucosal absorption, nasal drops (e.g., nasal sprays), inhalants, or suppositories. In a preferred embodiment of a composition for pharmaceutical use, the composition of the present disclosure is in liquid form. In some embodiments of compositions for cosmetic use, the composition of the present disclosure may be any cosmetic product, including lotions, emulsions, gels, serums, general creams, packs, masks, facial cleansers, cosmetic soaps, foundations, powders, and body lotions.

[0086] In some embodiments, the compositions of the present disclosure are stored in a preserved (e.g., concentrated, frozen, dried, lyophilized) solid, liquid, or semi-solid form and can be used after preparation (e.g., melting, diluting, gelling, mixing with carriers and solvents) prior to administration to a subject.

[0087] In some embodiments, the compositions of the present disclosure are formulated as a liquid for intravenous administration and administered to a subject at a dose of ml / kg. In some embodiments, the compositions of the present disclosure are administered to a subject once every 1, 2, 3, 4, 5, 6, or 7 days, once every 1, 2, 3, or 4 weeks, or once every 1 or 2 months.

[0088] In some embodiments, the compositions of the present disclosure are formulated as a liquid for topical administration and administered to a subject at a dose of ml / kg. In some embodiments, the compositions of the present disclosure are administered to a subject once every 1, 2, 3, 4, 5, 6, or 7 days, once every 1, 2, 3, or 4 weeks, or once every 1 or 2 months.

[0089] (Method for Producing a Composition) The present disclosure relates to a method for producing a composition, comprising the steps of: i) culturing a seeded stem cell population; and ii) recovering the culture supernatant.

[0090] In some embodiments, step i) in the method for producing a composition of the present disclosure comprises: a) culturing the seeded stem cells under conditions that allow the stem cells to maintain their ability to self-renew and to differentiate into cells of interest, particularly neurons; and / or b) culturing the seeded stem cells under conditions that induce differentiation into cells of interest.

[0091] In some embodiments, step i)-a) or i)-b) is initiated about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, or 2 weeks after seeding of the stem cells. In some embodiments, after seeding of the stem cells, the stem cells can be cultured under conditions that allow them to maintain their self-renewal ability and their ability to differentiate into desired cells until step i)-a) or step i)-b) is initiated. In some embodiments, the culture of step i)-a) or i)-b) is terminated by the initiation of step ii) at any time between about 1 day and 2 months after the initiation. In some embodiments, the culture of step i)-a) or i)-b) is continued for about 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 at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more confluent. In some embodiments, at the start of step ii), the stem cells may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more confluent.

[0093] A person skilled in the art can determine the end of the culture according to step i)-a) or i)-b) (the start of step ii)) according to one or more criteria (number of days, color of the medium, presence or concentration of a particular component, confluency, cell morphology, growth rate, etc.).

[0094] Steps i)-a) and i)-b), and the subsequent step ii) may be applied independently to each of multiple culture vessels. Therefore, even if the start date and time differs for each culture vessel, culture supernatants obtained from culture vessels to which the same termination criteria for the culture of steps i)-a) and b) above are applied may be considered to be the same. Therefore, in some embodiments, the collection of culture supernatants in step ii) may include collecting the culture supernatants as bulk culture supernatants for cultures to which the same termination criteria are applied.

[0095] In some embodiments, recovering the culture supernatant in step ii) may include removing cells from the culture medium. Examples of cell removal include filtration, centrifugation, aspiration using a pipette, dropper, or aspirator, tilting the culture vessel, etc. In some embodiments, recovering the culture supernatant is carried out by centrifuging the culture medium to remove the cells.

[0096] The method for producing the composition of the present disclosure may optionally further include, after step ii), a step of performing a process for preserving the composition, and a step of preparing and performing various processes for administration.

[0097] Preferred embodiments of the present disclosure are described in detail below, but they are intended to be illustrative and not restrictive, and are not intended to limit the invention as defined in the appended claims in any way.

[0098] Example 1: X4N cells (NEcST （登録商標） A culture supernatant preparation obtained by culturing X4N cells as disclosed in JP 2019-076008 A 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 so that the bottom of the dish was covered, and the dish or culture flask was incubated at 37°C for 30 minutes, after which the dish was 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 100 / ml, and the suspension was seeded into the above 10 cm Petri dish or culture flask and incubated at 37°C, 5% CO ２Culture was initiated in the presence of PBS. The culture medium was replaced with Sartorius medium (Sartorius) the day after seeding, and culture was continued for another two days. After two days, the culture medium was collected and centrifuged at 1000 rpm for 7 minutes. The resulting supernatant was further filtered to obtain undifferentiated cell culture supernatant. Furthermore, a 10 cm Petri dish or culture flask was coated with gelatin solution (Wako Pure Chemical Industries) or vitronectin solution, which was placed so that the bottom of the dish or flask was covered, and the dish or flask was incubated at 37°C for 30 minutes, followed by washing twice with PBS. Next, 2 x 10 ５ X4N cells were suspended in 1 ml of a 50% mixture of ReproCell medium (ReproCell) and RHB-A medium (Takara Bio) to a cell concentration of 1 ml, and the suspension was seeded into the above 10 cm petri dish or culture flask and incubated at 37°C, 5% CO ２ Culture was initiated in the presence of ATP. The day after seeding, the culture medium was replaced with RHB-A medium (Takara Bio) and cultured for an additional 5 days. After 5 days, the culture medium was replaced with Sartorius medium (Sartorius) and cultured for an additional 2 days. After 2 days, the culture medium was collected and centrifuged at 1,000 rpm for 7 minutes. The resulting supernatant was further filtered to obtain the neuronal differentiation-induced cell culture supernatant. The undifferentiated cell culture supernatant and the neuronal differentiation-induced cell culture supernatant were mixed 1:1 to prepare the test culture supernatant. Furthermore, the undifferentiated cell culture supernatant, neuronal differentiation-induced cell culture supernatant, or test culture supernatant was purified and concentrated 5-fold by ultrafiltration to prepare culture supernatants.

[0099] Example 2: Rat Spinal Cord Injury Model - Evaluation of Improvement in Hindlimb Motor Function Using a Rat Spinal Cord Injury Model Twenty-four rats (Jcl: SD, SPF, female, 10 weeks old) were acclimatized for one week at a room temperature of 22±2°C, a relative humidity of 30-70%, and a 12-hour light / dark cycle. During the breeding period, the rats had free access to water and solid feed CE-2 (Feed One Co., Ltd.). After acclimatization, the rats were randomly assigned to three treatment groups (8 rats / group) stratified by weight so that the average body weights were as uniform as possible. The rats in each treatment group are shown in Table 2. After the acclimation period, rat models of spinal cord injury were prepared using the weight drop method. Specifically, the 9th and 10th thoracic vertebral arches were resected under triple anesthesia, and then a 10 g weight was dropped from a height of 50 mm to injure the spine. A rat intrathecal catheter (#0007741, short type, Alzet) was placed in the spinal cavity of the rats in the intrathecal administration group (Group 3).

[0100] The day the model was created was designated as day 0, and test substances were administered on days 1, 6, 11, 16, 21, and 31. Hindlimb motor function (BBB test) was evaluated on days 1, 7, 14, 21, 28, 35, and 39. General observations were performed at least once a day during the breeding period. Body weight measurements were also performed daily from day 0 to day 7, and once a week thereafter. After the observation period, the rats were euthanized, and tissue samples were collected for hematoxylin-eosin staining.

[0101] Statistical processing: For body weight, the mean value and standard error were calculated, and a Brtlett test was performed. For equal variance, one-way analysis of variance and Turkey-Kramer multiple comparison test were performed, and for unequal variance, a Kruskal-Wallis test and Steel-Dwass multiple comparison test were performed. For BBB scores, the mean value and standard error were calculated, and a Kruskal-Wallis test and Steel-Dwass multiple comparison test were performed. In all cases, a risk level of less than 5% (two-sided) was considered to indicate a significant difference.

[0102] Results: No significant difference in body weight change was observed between the vehicle-administered control group (Group 1) and Groups 2 and 3 administered with culture supernatant during the study period. As shown in Figure 1, significant differences in hindlimb motor function were observed between Group 2, which received intravenous administration of culture supernatant, and Group 3, which received intraspinal administration of culture supernatant, on day 21 after injury. Furthermore, on day 35 after injury, no animals in the control group had recovered to the extent that they were able to perform weight-bearing plantar landing (BBB≧8), whereas approximately half of the animals in Groups 2 and 3 had recovered to the extent that they were able to perform weight-bearing plantar landing. In particular, some animals in Group 2 had recovered to the extent that they were able to perform plantar walking (BBB=10). Therefore, both local and intravenous administration of the culture supernatant of the present disclosure promoted and improved recovery of motor function compared to the control group.

[0103] Staining of spinal cord tissue and other tissues around the injury site After the observation period in Example 1, rats were euthanized by excessive inhalation of isoflurane (concentration 5% or higher). The spine, brain, liver, and kidneys around the injury site were collected and fixed in 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 according to the following procedure: immersion in hematoxylin (Carrazi's hematoxylin, Muto Pure Chemicals Co., Ltd., Tokyo, Japan) for 5 minutes, rinsing with water for 10 minutes, immersion in eosin (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) for 30 seconds, rinsing with water, dehydration (immersion in 70% ethanol, then 90% ethanol, and then 100% ethanol), clearing with xylene, and mounting.

[0104] Results: Consistent with the data on recovery of hindlimb motor function in Example 1, the spinal cord injury site (void) was repaired by neurons in the culture supernatant-administered group (Fig. 2A; center and right panels), compared with the spinal cord tissue around the injury site in the control group (Fig. 2A, left panel). Furthermore, when the effects of administration on each tissue were observed, no effects on the spinal cord, brain, liver, or kidney tissues, such as dysplasia or inflammatory reactions, were observed in any of the control group, the group administered the culture supernatant intravenously, or the group administered the culture supernatant intrathecally (Figs. 2A-D).

[0105] Therefore, the culture supernatant of the present invention exerted its effects without causing significant side effects when administered locally (intrathecally) or systemically (intravenously), and promoted tissue recovery at the injury site. This suggests that the culture supernatant of the present invention may be a useful composition for treating injury and disease by contributing to the promotion of differentiation, proliferation, and growth of cells, particularly nerve cells.

[0106] Example 3: Measurement of physiologically active substance concentration in culture supernatants The BDNF concentrations in the test culture supernatants of X4N cells, human iPS cells, and human mesenchymal stem cells (hMSCs), prepared in the same manner as in Example 1, and in the culture medium (RHB-A medium, ReproCell) were measured using ELISA (Figure 3). The BDNF content in the culture supernatant of X4N cells was significantly higher than that in the culture supernatants of iPS cells or hMSCs.

[0107] Furthermore, the relative content of various cytokines in the test culture supernatant of X4N cells was measured using an antibody array method compared to the medium control. The results are shown in Table 3 below. It was demonstrated that the culture supernatant of X4N cells contains physiologically active substances, including various cytokines.

[0108] Example 4: Measurement of physiologically active substances in extracellular vesicles. The test culture supernatant of X4N cells and the culture supernatant of hMSCs were ultrafiltered using a 30 kDa filter to obtain the residue (exosome fraction) and filtrate. RNA was extracted from the exosome fraction, and the expression levels of miRNA in exosomes were analyzed by microarray analysis. The results are shown in Table 4 below. The miRNAs in the table are annotated as miRNAs involved in cell growth and regeneration. In X4N cells, these miRNAs were present in exosomes in the culture supernatant at least twice as much as in human mesenchymal stem cells.

[0109] We also investigated whether the physiologically active substances secreted by X4N cells were concentrated in the culture supernatant or extracellular vesicles (exosomes). A portion of the exosome fraction from the X4N cells was treated with NaOH + Triton X to disrupt the exosomes. The BDNF concentrations in the resulting X4N cell culture supernatant filtrate fraction, exosome fraction (disrupted), exosome fraction (untreated), and culture supernatant were quantified by ELISA (Figures 4A and 4B). Compared to the other fractions, the amount of BDNF in the exosome fraction (disrupted) was significantly higher.

[0110] These results suggest that X4N cells secrete extracellular vesicles containing physiologically active substances and that the physiologically active substances are concentrated in the extracellular vesicles. Without wishing to be bound by any particular theory, the recovery effect observed in the spinal cord injury rat model by both local and intravenous administration of the culture supernatant may be due to the culture supernatant of the present disclosure containing extracellular vesicles containing physiologically active substances, which may enable more stable delivery of the physiologically active substances to cells near the target site (e.g., the injury site).

[0111] Example 5: Measurement of exosome count in culture supernatant The test culture supernatant of X4N cells was filtered through a 0.22 μm pore size PVDF filter (Millex GV, Millipore), and the filtrate was used as a sample stock solution. The sample stock solution was diluted 50-fold with PBS to obtain Sample 1, and the same was diluted 40-fold to obtain Sample 2. Each sample was subjected to particle analysis using a Nanosight 300 machine, a Nanosight syringe pump, Nanosight NTA3.3 software, and an 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 particle analysis, peaks were observed at particle sizes of 68 nm and 114 nm in Sample 1, and the particle concentration was 2.04 × 10 １０ In sample 2, peaks were observed at particle sizes of 64 nm and 119 nm, and the particle concentration was 1.98 × 10 １０ The exosomes produced by X4N cells were relatively homogeneous with a particle diameter of around 100 nm.

[0112] Number of exosomes in the culture supernatant of X4N cells (up to × 10 １０ The exosome concentration (cells / ml) is much higher than that of other cell culture supernatants per cell count. While MSC and iPS culture supernatants did not show any cell proliferation effect unless they were concentrated, the effect of adding unconcentrated X4N cell supernatant was observed, which is thought to be due to the high concentration of exosomes and physiologically active substances such as BDNF.

[0113] Example 6: Verification of the proliferation-promoting effect of X4N cell culture supernatant on mesenchymal stem cells and fibroblasts. The test culture supernatant of X4N cells, the culture supernatant of undifferentiated cells, and hMSC culture medium (Sartorius) were each diluted to a predetermined concentration (10%, 5%, 1%) and diluted to 2 × 10 ５The hMSCs were cultured at a concentration of 1000 cells / ml and cultured for 3 days. The difference in proliferation rate of the hMSCs in each culture was examined by counting the number of cells using a cell counter (Thermo Fisher) (Fig. 5A). Addition of 10% of the test culture supernatant of X4N cells promoted proliferation approximately 2-fold compared to the culture system without the addition of the supernatant.

[0114] Similarly, 10% X4N cell test culture supernatant or iPS cell culture supernatant was added to a human dermal fibroblast culture system, and the viable cell count after 3 days of culture was measured using a cell counter (Thermo Fisher) (Figure 5B). The results showed that the culture system supplemented with X4N cell culture supernatant exhibited approximately twice the proliferation-promoting effect compared to the system supplemented with iPS cell culture supernatant. 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 iPS medium (Company S), ReproCell iPS medium (Company R), and fibroblast medium (Takara Bio)) that had been stored for 1 month at -30°C, 4°C, and room temperature. The viable cell count 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 storage at room temperature for 1 month.

[0115] Example 7: Verification of the effect of X4N cell culture supernatant on promoting neuronal differentiation of mouse ES cells Mouse ES cells were cultured in a Neurobasal medium containing N2 / B27. （商標） After differentiation in culture medium for 4 days, allowing spheroid formation, the cells were seeded onto laminin-coated glass-bottom plates and cultured for 3 days under each condition (medium alone, 10% hMSC culture supernatant, or 10% X4N cell culture supernatant). The X4N cell culture supernatant used was a purified, concentrated test culture supernatant obtained by ultrafiltration, diluted 5-fold with PBS. After culture, the cells were stained with tubulin βIII antibody and DAPI and observed under a fluorescent microscope. As a result, culturing with X4N cell culture supernatant significantly promoted neural differentiation compared to culture medium alone or hMSC culture supernatant (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 the following test substances were applied once a day to five mice per group: Group 1: purified and concentrated test culture supernatant of X4N cells; Group 2: 1% minoxidil (Riaup （登録商標） ) (positive control); and Group 3: 50% ethanol (negative control). The degree of hair growth was visually scored. The total score for each group on the 9th day was calculated based on the results of the X4N purified and concentrated test culture supernatant, Riap （登録商標） and 50% ethanol were 4, 10, and 2, respectively, and the culture supernatant of X4N cells showed twice the hair growth activity compared to the negative control.

[0117] The present disclosure provides a composition comprising a stem cell culture supernatant for pharmaceutical or cosmetic use, for example, for use in treating injury or disease in a subject, and a method for producing the same. The present disclosure can be used in a variety of fields, including the pharmaceutical and cosmetic fields.

Claims

1. A pharmaceutical or cosmetic composition comprising a culture supernatant of stem cells, wherein the stem cells express either CCN1 or CCN2 or both, and the stem cells are immortalized.

2. The composition of claim 1, wherein the stem cells are bound to an antibody having the CDR sequences of SEQ ID NOs: 1-6.

3. The composition of claim 1, wherein the stem cells are X4N cells deposited under accession number P-02505.

4. The composition of claim 1, wherein the stem cells include undifferentiated stem cells and / or neuronally differentiated cells.

5. 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 2. The composition of 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 factor, and combinations thereof.

6. 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 of claim 1, comprising at least one microRNA (miRNA) selected from the group consisting of: 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.

7. 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, 2. The composition of claim 1, comprising extracellular vesicles comprising 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 factor, and combinations thereof.

8. 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 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.

9. The pharmaceutical composition according to claim 1 for treating an injury or disease in a subject, wherein the injury is trauma or wounding at any site or a chronic or transient functional disorder resulting therefrom, or the disease is a neurological disease, depression, myasthenia gravis, dementia, Parkinson's disease, Huntington's disease or schizophrenia, ischemia, blood flow disorder, infarction, osteoporosis, an immune disease or periodontitis.

10. The composition of claim 9, wherein the subject is a mammalian subject, an avian subject, or a reptilian subject.

11. The composition of claim 9, formulated for topical, oral, rectal, nasal, intravenous, intraarticular, conjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, transdermal or subcutaneous routes of administration.

12. The pharmaceutical composition according to claim 9, which is in the form of granules, fine granules, powder, hard capsules, soft capsules, syrup, emulsion, suspension or liquid, injection, infusion, transdermal absorption agent, transmucosal absorption agent, nasal drops, inhalant or suppository.

13. The cosmetic composition according to claim 1, which is used for skin care, skin whitening, wrinkle improvement, skin texture improvement, anti-aging, and hair growth.

14. The cosmetic composition according to claim 13, which is in the form of a lotion, milky lotion, gel, serum, cream, pack, mask, facial cleanser, cosmetic soap, foundation, face powder, or body lotion.

15. A method for producing the composition according to any one of claims 1 to 14, comprising: i) culturing the seeded stem cell population; and ii) recovering the culture supernatant.

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