Pharmaceutical or cosmetic composition containing culture supernatant of ips cells or differentiated cells derived from ips cells

Abstract

The present invention provides: a composition containing a culture supernatant of iPS cells or cells derived from iPS cells; a method for producing the same; a pharmaceutical or cosmetic composition containing the culture supernatant; and a method for producing the same.

Description

A pharmaceutical or cosmetic composition containing the culture supernatant of iPS cells or differentiated cells derived from iPS cells.

[0001] The present invention relates to a composition comprising the culture supernatant of iPS cells or cells derived from iPS cells.

[0002] Active research and development is underway on cell populations containing differentiated cells obtained by inducing differentiation of pluripotent stem cells. These cell populations (pluripotent stem cell processed products) are attracting attention for their usefulness as pharmaceuticals (cell-based therapies) or as research tools in drug discovery and development. While human cells are primarily used when used as pharmaceuticals, when used as research tools, they are not limited to human cells and a wide range of cells from various organisms are used. Depending on the purpose, pluripotent stem cells such as iPS cells and ES cells are widely used.

[0003] In recent years, it has become known that hundreds of different humoral factors (secretome: proteins, growth factors, exosomes, extracellular matrix, etc.) are secreted from cells in the supernatant (culture supernatant) obtained when culturing stem cells or inducing differentiation of various stem cells into specific cells. These proteins include cytokines and chemokines, and because they have the property of acting on other cells and activating their functions, they are thought to be useful in the prevention and treatment of diseases. Research is being conducted not only on processed products of pluripotent stem cells, but also on the culture supernatant obtained during cell culture. In the medical field, there are attempts to use culture supernatant obtained by culturing various cells to treat and prevent many diseases, including intractable diseases. This is useful for restoring the function of cells damaged by injury or aging and for the regeneration of tissues and organs, and can be used as regenerative medicine. Furthermore, the culture supernatant is beginning to attract attention not only in the medical field but also in the field of beauty (the field of regenerative beauty). There is a great social demand for improving skin aging, blemishes, and wrinkles associated with aging, as well as promoting hair growth. To address these issues, cosmetics and other products using cell culture supernatants have been proposed.

[0004] For example, Patent Document 1 provides a cosmetic product containing a culture supernatant obtained by culturing horse-derived mesenchymal stem cells, and states that the culture supernatant of these mesenchymal stem cells is useful for improving blemishes and wrinkles. However, the present invention includes a collection step of collecting cells from racehorses or competition horses and a selection step of selecting mesenchymal stem cells from the collected cells, which presents a problem in that access to racehorses or competition horses is limited when utilizing these cells.

[0005] In addition, Patent Document 2 discloses a cosmetic composition for skin whitening, wrinkle improvement, or regeneration containing exosomes contained in the culture supernatant obtained when stem cells are differentiated into adipocytes. Patent Document 3 discloses a hair growth promoting sheet using the culture supernatant of dental pulp stem cells. However, all of these use somatic stem cells collected from donors or cells obtained by differentiating them, and there is a problem that, for commercial use, only physicians / dentists can collect the human cells / tissues that are the starting materials for the invention. Furthermore, there is a problem that the properties differ from donor to donor, and there are limitations in proliferative capacity and differentiation capacity.

[0006] The present invention has been made in view of the above, and provides cosmetic compositions and pharmaceutical compositions that can be safely used in humans, as well as methods for producing the same.

[0007] Japanese Patent Publication No. 2018-024595, Japanese Patent Publication No. 2018-184446, WO-A1-2016 / 175164

[0008] The object of this invention is to provide novel pharmaceutical and cosmetic compositions using culture supernatants of iPS cells and differentiated cells derived from iPS cells, as well as methods for producing them.

[0009] As a result of diligent research, the inventors have discovered that the culture supernatant of iPS cells and cells differentiated from iPS cells contains substances effective for medical and cosmetic effects, and have completed the present invention. That is, the present invention includes the following embodiments.

[0010] Item 1. A pharmaceutical or cosmetic composition comprising a cell culture supernatant containing iPS cells or differentiated cells derived from iPS cells. Item 2. The composition according to Item 1, wherein the differentiated cells are at least one type of cell selected from the group consisting of cardiomyocytes, nerve cells, mesenchymal stem cells, and hepatocytes. Item 3. The composition according to Item 1 or 2, wherein the iPS cells are of human origin. Item 4. The composition according to any one of Items 1 to 3, used for the treatment of wounds, ischemia, blood flow disorders, infarction, osteoporosis, immune diseases, neurological diseases, metabolic diseases, periodontitis, or regeneration of periodontal tissue. Item 5. The composition according to Item 4, wherein the ischemia, blood flow disorder, or infarction is arteriosclerosis, thrombosis, angina pectoris, myocardial infarction, cerebral infarction, ulcer, or necrosis. Item 6. The composition according to Item 5, wherein the differentiated cells are cardiomyocytes and / or mesenchymal stem cells. Item 7. The composition according to Item 4, wherein the metabolic disease is diabetes mellitus or non-alcoholic fatty liver disease. Item 8. The composition according to claim 7, wherein the differentiated cells are cardiomyocytes. Claim 9. The composition according to any one of claims 1 to 3, used for anti-inflammatory and / or immune tolerance. Claim 10. The composition according to claim 9, wherein the differentiated cells are mesenchymal stem cells. Claim 11. The composition according to any one of claims 1 to 3, used for skin whitening, improvement of blemishes, wrinkles, sagging or depressions, or for hair growth or hair regeneration. Claim 12. The composition according to claim 11, wherein the differentiated cells are cardiomyocytes and / or mesenchymal stem cells, and is for hair growth or hair regeneration. Claim 13. The composition according to claim 11, wherein the differentiated cells are mesenchymal stem cells, and is for improvement of wrinkles, sagging or depressions. Claim 14. The composition according to any one of claims 1 to 3, wherein the differentiated cells are nervous system cells, and is for anti-aging of the brain and nervous system. Claim 15. The composition according to any one of claims 1 to 5, 9 and 11, wherein the culture supernatant of the iPS cells comprises at least one protein selected from the group consisting of VGF, FGF, PDGF, BMP and TGF-α. Claim 16. The composition according to any one of claims 1 to 9, 11 and 12, wherein the differentiated cells are cardiomyocytes and the culture supernatant comprises at least one protein selected from the group consisting of FGF, KGF, IGF, VEGF and DKK.17. The composition according to any one of claims 1 to 7, 9 to 13, wherein the differentiated cells are mesenchymal stem cells and the culture supernatant contains at least one protein selected from the group consisting of HGF, FGF, SDF-1, PDGF, and DKK. 18. The composition according to any one of claims 1 to 5, 9, 11, and 14, wherein the differentiated cells are neural cells and the culture supernatant contains at least one protein selected from the group consisting of FGF, PDGF, TIMP, MMP, and GDF. 19. The composition according to claims 15 to 18, wherein the culture supernatant is concentrated with the protein. 20. The composition according to any one of claims 1 to 9, 11, 12, 15, and 19, wherein the culture supernatant of iPS cells contains at least one microRNA selected from the group consisting of miR-21-5p, miR-182-5p, miR-148b-3p, miR-7-5p, and miR-378a-3p. Item 21. The composition according to any one of items 1 to 9, 11, 12, 16, and 19, wherein the differentiated cells are cardiomyocytes, and the culture supernatant contains at least one microRNA selected from the group consisting of miR-1-3p, miR-143-3p, miR-21-5p, miR-218-5p, and miR-30a-5p. Item 22. The composition according to any one of items 1 to 7, 9 to 13, 17, and 19, wherein the differentiated cells are mesenchymal stem cells, and the culture supernatant contains at least one microRNA selected from the group consisting of miR-100-5p, miR-21-5p, miR-143-3p, miR-30a-5p, and miR-27a-3p. Item 23. The composition according to any one of claims 1 to 9, 11, 12, 15, 19, and 20, wherein the culture supernatant of the iPS cells contains VGF, FGF, PDGF, BMP, TGF-α, miR-21-5p, miR-182-5p, miR-148b-3p, miR-7-5p, and miR-378a-3p. Claim 24. The composition according to any one of claims 1 to 9, 11, 12, 16, 19, and 21, wherein the differentiated cells are cardiomyocytes, and the culture supernatant contains FGF, KGF, IGF, VEGF, DKK, miR-1-3p, miR-143-3p, miR-21-5p, miR-218-5p, and miR-30a-5p.Item 25. The composition according to any one of items 1 to 7, 9 to 13, 17, 19 and 22, wherein the differentiated cells are mesenchymal stem cells and the culture supernatant contains HGF, FGF, SDF-1, PDGF, DKK, miR-100-5p, miR-21-5p, miR-143-3p, miR-30a-5p and miR-27a-3p. Item 26. The composition according to any one of items 1 to 5, 7, 9, 11, 14, 18 and 19, wherein the differentiated cells are nervous system cells and the culture supernatant contains FGF, PDGF, TIMP, MMP and GDF. Item 27. The composition according to any one of items 1 to 26, wherein the culture supernatant is obtained by culturing cells in serum-free medium and / or does not contain animal-derived components. Item 28. A method for treating wounds, ischemia, blood flow disorders, infarctions, or cosmetic procedures, comprising the step of applying a composition containing the culture supernatant of iPS cells or differentiated cells derived from iPS cells to a target. Item 29. Cosmetic use of a composition containing the culture supernatant of cells including iPS cells or differentiated cells derived from iPS cells. Item 30. Use of the culture supernatant of cells including iPS cells or differentiated cells derived from iPS cells in the manufacture of a pharmaceutical product for the treatment of wounds, ischemia, blood flow disorders, infarctions, neurological diseases, or osteoporosis.

[0011] According to the present invention, novel pharmaceutical compositions and cosmetic compositions using culture supernatants of iPS cells and differentiated cells derived from iPS cells, as well as methods for producing them, are provided.

[0012] This figure shows the differentiation induction stages from iPS cells to cardiomyocytes. This figure shows the evaluation results of the culture supernatant of iPS-CM on the proliferation of human dermal papilla cells. This figure shows the flowchart of the hair growth experiment. This figure shows the results of the hair growth experiment on day 10 and day 14 after administration of the culture supernatant, and the proteins contained in the culture supernatant. This figure shows the results of a mouse hair growth test using the culture supernatant of iPS-MSCs. (A) Shows the administration sites for topical administration and intradermal injection. (B) Results on day 13 after administration. (C) Results on day 16 after administration. This figure shows the results of a human hair growth test using the culture supernatant of iPS-CMs. (A) Photographs comparing the degree of hair growth of the subject before treatment and at 9 weeks after administration. (B) Figures showing hair width per 1 cm and (C) number of hairs per 1 cm. (D) Shows the change in hair width and (E) number of hairs over time at four measured sites. (F) Shows the results for each measurement site before treatment and at 9 weeks after administration. This figure evaluates the effect of iPS-CM culture supernatant on intracellular lipid accumulation. (A) shows the staining results of intracellular lipid droplets and (B) shows the stained image of intracellular lipid droplets. This figure evaluates the effect of iPS-CM culture supernatant on a mouse diabetes model. (A) shows the flow chart of the test. (B) shows the changes in fasting blood glucose levels. (C) shows the results of an oral glucose tolerance test measuring the change in blood glucose levels over time. This figure shows the results of measuring the amount of exosomes in iPS-MSC supernatant. This figure shows the results of a cell proliferation test of human normal dermal fibroblasts using iPS-CM culture supernatant. This figure shows the degree of FGF expression in iPS-CM culture supernatant. This figure shows the results of a proliferation test of human fibroblasts using iPS-MSC culture supernatant. This figure shows the results of a wound-healing assay using human fibroblasts. This figure shows the results of a wound-healing assay using a human keratinocyte cell line. This figure shows the evaluation results of angiogenesis using iPS-CM and iPS-MSC culture supernatant. (A) Microscopic image and (B) Cell area percentage calculated from the obtained image. This figure shows the results of GO analysis performed on the top 20 humoral factor genes that showed above-average expression. This figure shows the results of GO analysis performed on the top 20 extracellular matrix-related genes that showed above-average expression.This figure shows a heat map of proteins detected during the differentiation induction of iPS cells into cardiomyocytes. This figure shows the ELISA detection results of HGF contained in the culture supernatant of cells differentiated from iPS cells into mesenchymal stem cells.

[0013] The cosmetic composition according to the present invention is a cosmetic composition used for skin whitening, improvement of blemishes and / or wrinkles, or hair growth or regeneration. When administered to humans, this composition acts on skin cells and keratinocytes, improving the condition of the skin, thereby improving blemishes and wrinkles, and providing moisture and firmness to the skin. It also has a skin whitening effect. Furthermore, it acts on hair, having an effect on hair growth and prevention of hair loss.

[0014] The inventors of this invention have surprisingly discovered that the culture supernatant of iPS cell-derived cardiomyocytes is effective in promoting hair growth and / or hair restoration. Furthermore, they have found that the culture supernatant of iPS cell-derived mesenchymal stem cells (MSCs) is effective in improving wrinkles, sagging, or depressions, thus completing the present invention. In addition, they discovered that the culture supernatant of iPS cell-derived mesenchymal stem cells (MSCs) releases a much larger amount of exosomes compared to somatic cell-derived MSCs (umbilical cord blood-derived MSCs, adipose-derived MSCs, and dental pulp-derived MSCs), and for the first time, they have found a significant difference in the amount of exosomes released by iPS cell-derived MSCs and other MSCs, even though they are all MSCs. This result indicates that the properties of cells obtained by differentiation induction from iPS cells are different from those of other somatic cell-derived cells, etc.

[0015] The pharmaceutical composition according to the present invention is a therapeutic or preventive composition for wounds, ischemia, blood flow disorder-related diseases, infarction, osteoporosis, immune diseases, neurological diseases, metabolic diseases, periodontitis, or periodontal tissue regeneration, comprising a cell culture supernatant obtained by culturing cells selected from the group consisting of iPS cells, cardiomyocytes, nerve cells, hepatocytes, and mesenchymal stem cells. When this pharmaceutical composition is administered to humans, a wound healing effect is obtained. Furthermore, because it promotes angiogenesis, therapeutic or preventive effects are obtained for poor blood circulation, blood flow disorders, ischemia, and infarcted areas. In addition, it acts on immune cells, resulting in anti-inflammatory effects and effects that contribute to immune tolerance.

[0016] 1. iPS cells The iPS cells used to obtain the culture supernatant in this invention are not particularly limited and can be used in a wide range of applications. While the iPS cells are not particularly limited, human-derived iPS cells (h-iPS cells) are preferred in terms of safety.

[0017] 2. Differentiated cells are not particularly limited, but examples of differentiated cells include cardiomyocytes, nervous cells, hepatocytes, mesenchymal stem cells, retinal cells such as retinal pigment epithelial cells, blood (hematopoietic) cells, hepatocytes, pancreatic beta cells, kidney cells, chondrocytes, germ cells, intestinal epithelial cells, vascular endothelial cells, skin cells (fibroblasts, keratinocytes), pigment cells, and hair follicle stem cells. Among these, cardiomyocytes, nervous cells, hepatocytes, and mesenchymal stem cells are particularly preferred.

[0018] Methods for inducing differentiation from iPS cells into differentiated cells can be appropriately used using conventionally known methods. The following describes methods for inducing differentiation into each cell type, but these are not the only methods available.

[0019] 2.1. Cardiomyocytes Methods for inducing differentiation of h-iPS cells into cardiomyocytes include, but are not limited to, the methods described in Biochemical and Biophysical Research Communications, 425 (2012) 321-327, or the methods described in Proc Natl Acad Sci USA, 2013 Aug 27;110(35):E3281-90.

[0020] 2.2. Mesenchymal Stem Cells Methods for inducing differentiation from h-iPS cells into mesenchymal stem cells include, but are not limited to, the methods described in World J Stem Cells, 2021 Aug 26; 13(8): 1094-1111.

[0021] 2.3. Neural Cells Methods for inducing differentiation of h-iPS cells into neural cells include, but are not limited to, the methods described in Hum Mol Genet, 2006;15(17):2623-2635. or the methods described in Nat Biotechnol, 2009 Mar; 27(3):275-280. Neural cells include nerve cells, neural progenitor cells, neural stem cells, and other cells that constitute the nervous system.

[0022] 2.4. Hepatocytes Methods for inducing differentiation of h-iPS cells into hepatocytes include, but are not limited to, the methods described in Hepatology 51, 297-305 (2010) or the methods described in Proc Natl Acad Sci USA., 2014 Nov 25;111(47):16772-7.

[0023] 3. Culture Supernatant In this invention, the term "culture supernatant" refers to the culture medium obtained by culturing cells. For example, after culturing iPS cells and cells differentiated from iPS cells using an appropriate culture vessel and culture medium, the cells adhering to the culture vessel are detached, and the remaining culture medium is used as is, or centrifuged as necessary, to obtain the culture supernatant. Since the culture supernatant obtained in this way is substantially free of cells, it does not cause immune rejection. For this reason, it can be used even if it is the culture supernatant of allogeneic iPS cells or cells differentiated from allogeneic iPS cells, which has the advantage of having a very wide range of applicability. Although the culture supernatant usable in this invention can be obtained by separating and removing cells after culturing, the method of separation and removal can be any known method such as centrifugation, and is not particularly limited.

[0024] In this invention, the culture supernatant may be a culture supernatant that has been subjected to various treatments as appropriate (e.g., centrifugation, concentration, dialysis, freezing, drying, freeze-drying, dilution, desalting, storage, etc.). Concentrating the culture supernatant has the advantage of saving space during storage. Furthermore, freezing and / or drying the culture supernatant allows for long-term storage. Thus, if the culture supernatant is prepared in advance and stored, it can be used immediately when needed, which is extremely convenient and advantageous.

[0025] More specifically, the culture supernatant used in the present invention can be obtained by a method comprising 1) a differentiation induction step into target cells, 2) a purification (concentration) step of the differentiated cells, and 3) a recovery step of the culture supernatant. This method may include a broad-culture step after 1) the differentiation induction step if necessary. Furthermore, the method for obtaining the culture supernatant may also include 4) a concentration step of the culture supernatant.

[0026] 1) In the differentiation induction process to target cells, human iPS cells (QHJI14s04) are placed in a 10cm dish at a rate of 1 × 10 5 ~1 x 10 6 cells / cm 2 Seeds are sown and cultured under conditions of 37°C and 5% CO2. The day of sowing is designated as Day 0, and the culture medium is changed on Day 1.

[0027] 2) In the purification (concentration) process of differentiated cells, the proportion of differentiated cells can be increased by using differentiation markers of the target cells as indicators. For example, in the induction of differentiation into cardiomyocytes, the proportion of differentiated cells can be increased by using cardiac troponin T (TnT), a cardiomyocyte differentiation marker, as an indicator. By culturing in glucose-free DMEM medium from Day 16 to Day 22, and in α-MEM medium from Day 23 to Day 25, cells that have completed differentiation into cardiomyocytes can be concentrated. This is advantageous because it allows for the efficient secretion of cardiomyocyte-specific components into the culture supernatant.

[0028] 3) In the culture supernatant recovery process, the culture supernatant is recovered when the culture medium is changed. The culture medium is changed once every two days from Day 4 to Day 15, which is the differentiation induction process, and once a day from Day 17 to Day 25, which is the purification process. For example, if the target cells are cardiomyocytes, the cardiomyocytes obtained between Day 4 and Day 7 are called "differentiated cardiomyocytes," the cardiomyocytes obtained between Day 8 and Day 16 are called "pre-purification cardiomyocytes," and the cardiomyocytes obtained between Day 17 and Day 25 are called "purified cardiomyocytes." The culture supernatants obtained during these periods are called "culture supernatant obtained from differentiated cardiomyocytes," "culture supernatant obtained from pre-purification cardiomyocytes," and "culture supernatant obtained from purified cardiomyocytes," respectively. The culture supernatants obtained in this way can be frozen and stored at -30°C.

[0029] 4) In the culture supernatant concentration step, the culture supernatant obtained above can be concentrated, for example, by ultrafiltration. Concentrating the culture supernatant by ultrafiltration is advantageous because it removes waste products from the culture supernatant and increases the content of active ingredients. The culture supernatant thus concentrated is rich in the proteins necessary for the effects of the present invention to be achieved. Examples of such proteins include at least one protein selected from the group consisting of PDGF, VGF, TGF-α, TGF-β, CSF, MMP, TIMP, VEGF, FGF, KGF, IGF, GDF, ANGPT, BMP, SFRP, ELN, COL, CLEC, INHBA, DKK, SCG, GRN, HDGF, GMFB, EFEMP, FN, SPARC, KRT, THBS, IGFBP, PCOLCE, VCAN, LGALS3BP, HGF, SDF-1 (CXCL12)NTF3, and IL.

[0030] In the above, the cell culture form is not particularly limited. It may be adherent culture, suspension culture, or multilayer sheet culture.

[0031] The culture time can be set appropriately depending on the type of cells, but 12 to 168 hours is preferred, 18 to 72 hours is more preferred, and 24 to 48 hours is even more preferred.

[0032] It is preferable that the culture supernatant is serum-free, as its safety is enhanced by its absence. For example, a serum-free culture supernatant can be prepared by culturing iPS cells or cells differentiated from iPS cells in a serum-free medium (serum-free medium). A serum-free culture supernatant can also be obtained by performing one or more subculturing cycles, with the last or last few subculturing cycles being in serum-free medium. Alternatively, a serum-free culture supernatant can also be obtained by removing serum from the collected culture supernatant using methods such as dialysis or solvent exchange with a column.

[0033] The culture supernatant preferably does not contain animal-derived components. Animal-derived components include, but are not limited to, feeder cells, extracellular matrix such as gelatin, animal serum such as that of cattle, and growth factors produced from animal cells.

[0034] As described in this invention, the culture supernatant obtained when culturing iPS cells and cells obtained by differentiating iPS cells contains hundreds of types of protein components, including signaling molecules that contribute to cell activation, such as cytokines and exosomes, and proteins that constitute the extracellular matrix (ECM). For example, it contains growth factors such as Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Hepatocyto Growth Factor (HGF), and Transforming Growth Factor (TGF), as well as exosomes. The culture supernatant containing these cytokines and exosomes is thought to have the function of repairing damaged tissue (wound healing), promoting angiogenesis, and protecting tissue. It is also useful for skin whitening, improving blemishes and wrinkles, improving skin's moisturizing ability and viscoelasticity, and promoting hair growth or regeneration.

[0035] The pharmaceutical or cosmetic composition containing the culture supernatant of the present invention preferably contains proteins such as growth factors and cytokines secreted from iPS cells or cells obtained by differentiation induction from iPS cells. Examples of such proteins include FSTL1, HGF, BMPs (BMP1, BMP2, BMP4, BMP5, BMP7, BMP10, etc.), DKK (DKK1, DKK3, etc.), FGF (FGF18, FGF1, FGF9, FGF13, etc.), KGF (FGF7), IGF (IGF2, etc.), CSF (CSF1, etc.), NTF (NTF3, etc.), VEGF (VEGFA, VEGFB, VEGFC, VEGFD, etc.), VGF, TGF-α, TGF-β (TGF-β1, TGF-β2, etc.), PDGF (PDGF-C, PDGF-D, etc.), GDF (GDF2, GDF10, GDF11, GDF15, etc.), ANGPT (ANGPT1, ANGPT2, etc.), SFRP (SFRP1, SFRP2, FRZB It contains at least one protein selected from the group consisting of (SFRP3), SFRP5, CLEC11A, TIMP1, INHBA, SCG2, GRN, HDGF, GMFB, EFEMP1, MIA, SDF-1 (CXCL12), MANF, PTN, JAG1, TGFB1, NRG1, CSF1, MIF, EDN1, CXCL1, NELL (NELL1, NELL2, etc.), PTPRZ1, SEMA3A, NCAM1, DCC, NTN1, IL6ST, WNT5A, and WNT5B. These cytokines have wound healing and anti-inflammatory effects. They are also useful in treating ischemia, blood flow disorders, or infarction because they promote angiogenesis. Furthermore, they are useful for skin whitening, improving blemishes and wrinkles, and promoting hair growth.

[0036] In a pharmaceutical or cosmetic composition containing the culture supernatant of pre-purification cardiomyocytes, it is preferable that at least one protein selected from the group consisting of VEGFD, FGF18, KGF (FGF7), VEGFC, PDGF-C, GDF2, GDF10, GDF11, DKK1, and SFRP5 is contained as a protein such as a growth factor or cytokine. Particularly, it is more preferable that VEGFD is contained. Further, at least one protein selected from the group consisting of IGF2, CSF1, VEGFA, TGF-β1, TGF-β2, PDGF-D, GDF15, DKK3, ANGPT1, BMP1, BMP2, BMP4, BMP5, BMP7, BMP10, SFRP1, SFRP2, and FRZB (SFRP3) may also be contained.

[0037] In a pharmaceutical or cosmetic composition containing the culture supernatant of post-purification cardiomyocytes, it is preferable that at least one protein selected from the group consisting of FGF1, FGF9, VEGFB, FGF18, KGF (FGF7), VEGFC, PDGF-C, GDF2, GDF10, GDF11, DKK1, and SFRP5 is contained as a protein such as a growth factor or cytokine. Particularly, it is more preferable that at least one protein selected from the group consisting of FGF1, FGF9, and VEGFB is contained. Further, at least one protein selected from the group consisting of IGF2, CSF1, VEGFA, TGF-β1, TGF-β2, PDGF-D, GDF15, DKK3, ANGPT1, BMP1, BMP2, BMP4, BMP5, BMP7, BMP10, SFRP1, SFRP2, and FRZB (SFRP3) may also be contained.

[0038] In a pharmaceutical or cosmetic composition containing the culture supernatant of differentiated cardiomyocytes, it is preferable that at least one protein selected from the group consisting of NTF3, ANGPT2, IGF2, CSF1, VEGFA, TGF-β1, TGF-β2, PDGF-D, GDF15, DKK3, ANGPT1, BMP1, BMP2, BMP4, BMP5, BMP7, BMP10, SFRP1, SFRP2, and FRZB (SFRP3) is contained as a protein such as a growth factor or cytokine. Particularly, it is more preferable that NTF3 and / or ANGPT2 is contained.

[0039] A pharmaceutical or cosmetic composition containing the culture supernatant of mesenchymal stem cells obtained by differentiation induction from iPS cells produced in the present invention preferably contains at least one protein selected from the group consisting of IGFBP5, IGFBP7, FGF, FSTL1, CLEC11A, TIMP1, INHBA, SCG2, BMP1, GRN, HDGF, PDGFC, GMFB, EFEMP1, MIA, CXCL12, MANF, PTN, JAG1, DKK1, TGFB1, NRG1, CSF1, MIF, EDN1, CXCL1, WNT5A, WNT5B, and GDF2 as a growth factor or cytokine.

[0040] In the present invention, a pharmaceutical or cosmetic composition containing the culture supernatant of nervous system cells obtained by differentiation induction from iPS cells is preferably made to include at least one protein selected from the group consisting of FGF, PDGF, TIMP, MMP, GDF, NELL (NELL1, NELL2, etc.), PTPRZ1, SEMA3A, NCAM1, DCC, NTN1, and IL6ST, as a growth factor or cytokine.

[0041] The pharmaceutical or cosmetic composition containing the culture supernatant according to the present invention preferably contains exosomes secreted from iPS cells or cells obtained by differentiation induction from iPS cells. The microRNAs (hereinafter referred to as miRNAs) contained in the exosomes include miR-302a-5p, miR-148a-3p, miR-302b-3p, miR-302b-5p, miR-302c-3p, miR-302a-3p, miR-302d-3p, miR-151a-3p, miR-21-5p, miR-302c-5p, miR-26a-5p, miR-92a-3p, miR-302d-5p, miR-363-3p, miR-320a-3p, miR-182-5p, miR-1290, miR-363-5p, miR-1298-5p, miR-148b-3p, miR-7-5p, miR-378a-3p, miR-423-3p, miR-423-5p, miR-222-3p, miR-3529-3p, miR-183-5p, miR -106b-3p, miR-25-5p, miR-1246, miR-122-5p, miR-122b-3p, miR-143-3p, miR-218-5p, miR-126-3p, miR-192-5p, miR-99a-5p, miR-30a-5 p, miR-12136, miR-30d-5p, miR-27b-3p, miR-100-5p, miR-146b-5p, let-7i-5p, miR-574-5p, miR-199b-5p, miR-99b-5p, miR-133a-3p, mi R-24-3p, miR-1-3p, miR-30e-5p, miR-619-5p, miR-499a-5p, let-7c-5p, let-7a-5p, let-7e-5p, miR-128-3p, miR-3184-3p, miR-7704, mi Preferably, it contains at least one miRNA selected from the group consisting of R-221-3p, let-7g-5p, let-7f-5p, let-7b-5p, miR-27a-3p, miR-199a-3p, miR-29a-3p, miR-199b-3p, miR-155-5p, miR-199a-5p, miR-23a-3p, miR-22-3p and miR-181a-5p, and more preferably miR-21-5p, miR-182-5p, miR-148b-3p, miR-7-5p, miR-378a-3p,More preferably, it contains at least one miRNA selected from the group consisting of miR-1-3p, miR-143-3p, miR-218-5p, miR-30a-5p, miR-100-5p, and miR-27a-3p.

[0042] In a pharmaceutical or cosmetic composition containing the culture supernatant of pre-purified cardiomyocytes, as the miRNA contained in exosomes, at least one miRNA selected from the group consisting of miR-122-5p, miR-148a-3p, miR-122b-3p, miR-21-5p, miR-143-3p, miR-218-5p, miR-151a-3p, miR-26a-5p, miR-126-3p, miR-1290, miR-302a-5p, miR-192-5p, miR-99a-5p, miR-30a-5p, miR-12136, miR-30d-5p, miR-320a-3p, miR-27b-3p, miR-100-5p, miR-146b-5p, let-7i-5p, miR-574-5p, miR-148b-3p, miR-199b-5p, miR-99b-5p, miR-378a-3p, miR-133a-3p, miR-24-3p, miR-1-3p, and miR-30e-5p is preferably contained, and more preferably at least one miRNA selected from the group consisting of miR-1-3p, miR-143-3p, miR-21-5p, miR-218-5p, and miR-30a-5p is contained.

[0043] In a pharmaceutical or cosmetic composition containing the culture supernatant of purified cardiomyocytes, the miRNAs contained in the exosomes include miR-148a-3p, miR-143-3p, miR-1-3p, miR-122-5p, miR-21-5p, miR-218-5p, miR-151a-3p, miR-619-5p, miR-378a-3p, miR-99a-5p, miR-122b-3p, miR-26a-5p, miR-30a-5p, miR-30d-5p, miR-100-5p, miR-320a-3p, miR-133a-3p, miR-499a-5p, It is preferable that at least one miRNA selected from the group consisting of miR-12136, let-7c-5p, let-7a-5p, let-7i-5p, miR-27b-3p, miR-30e-5p, let-7e-5p, miR-128-3p, miR-423-5p, miR-3184-3p, miR-7704, and miR-92a-3p is included, and it is more preferable that at least one miRNA selected from the group consisting of miR-1-3p, miR-143-3p, miR-21-5p, miR-218-5p, and miR-30a-5p is included.

[0044] In pharmaceutical or cosmetic compositions containing the culture supernatant of iPS cells, the miRNAs contained in the exosomes include miR-302a-5p, miR-148a-3p, miR-302b-3p, miR-302b-5p, miR-302c-3p, miR-302a-3p, miR-302d-3p, miR-151a-3p, miR-21-5p, miR-302c-5p, miR-26a-5p, miR-92a-3p, miR-302d-5p, miR-363-3p, miR-320a-3p, miR-182-5p, miR-1290, miR-363-5p, and m It is preferable that the group includes at least one selected from the group consisting of iR-1298-5p, miR-148b-3p, miR-7-5p, miR-378a-3p, miR-423-3p, miR-423-5p, miR-222-3p, miR-3529-3p, miR-183-5p, miR-106b-3p, miR-25-5p, and miR-1246, and it is more preferable that it includes at least one miRNA selected from the group consisting of miR-21-5p, miR-182-5p, miR-148b-3p, miR-7-5p, and miR-378a-3p.

[0045] In a pharmaceutical or cosmetic composition containing the culture supernatant of mesenchymal stem cells obtained by differentiation induction from iPS cells, the miRNAs contained in the exosomes are let-7i-5p, miR-100-5p, miR-21-5p, miR-151a-3p, miR-221-3p, miR-222-3p, miR-143-3p, let-7a-5p, miR-26a-5p, miR-148a-3p, let-7g-5p, let-7f-5p, miR-30a-5p, let-7b-5p, miR-320a-3p, miR-27a-3p, miR-30d-5p, and miR-99a-5p. Preferably, at least one miRNA selected from the group consisting of miR-199a-3p, miR-99b-5p, miR-24-3p, miR-29a-3p, miR-199b-3p, miR-155-5p, miR-574-5p, miR-199a-5p, miR-23a-3p, miR-22-3p, miR-423-3p, and miR-181a-5p is included, and more preferably, at least one miRNA selected from the group consisting of miR-100-5p, miR-21-5p, miR-143-3p, miR-30a-5p, and miR-27a-3p is included.

[0046] The pharmaceutical or cosmetic composition according to the present invention, which contains a culture supernatant, preferably contains extracellular matrix secreted from iPS cells or cells obtained by differentiation induction from iPS cells. Examples of extracellular matrix include collagen (COL1A1, COL2A1, COL3A1, COL5A1, COL6A1, COL11A1, COL14A1, COL18A1, COL22A1, COL1A2, COL4A2, COL5A2, COL6A2, COL11A2, etc.), collagen 2, laminin, fibronectin (FN1), elastin (ELN), TIMP (TIMP1, TIMP2, TIMP3, TIMP4, etc.), MMPs (MMP1, MMP2, MMP9, This includes at least one selected from the group consisting of MMP10, MMP11, MMP14, MMP15, MMP23, etc.), SPARC, keratin (KRT1, KRT9, KRT2, KRT10, etc.), THBS2, PCOLCE, LGALS3BP, PTX3, THBS1, AGRN, LOXL2, VIM, NID2, ENO1, IGFBP5, IGFBP7, KIF20B, SHANK3, YWHAE, proteoglycans (VCAN, etc.), glycoproteins, and basement membrane factors. Furthermore, at least one ECM remodeling enzyme, which also acts as a regulatory factor for these, is included, selected from the group consisting of transglutaminase, collagen crosslinker, and matrix metalloproteinase. These extracellular matrix components and their regulatory factors act on the skin and skin cells to improve blemishes and wrinkles, increase skin hydration, provide moisture and firmness to the skin, create elasticity, and improve skin extensibility. They can also improve sagging and depressions. Sagging and depressions are caused, for example, by aging. Thus, the culture supernatant of the present invention is effective for anti-aging.

[0047] In pharmaceutical or cosmetic compositions containing the culture supernatant of unpurified cardiomyocytes, it is particularly preferable that the extracellular matrix contains at least one selected from the group consisting of ELN, MMP1, MMP2, MMP9, MMP10, MMP11, MMP14, MMP15, MMP23, TIMP1, TIMP2, TIMP3, TIMP4, COL1A1, COL2A1, COL3A1, COL5A1, COL6A1, COL11A1, COL14A1, COL18A1, COL22A1, COL1A2, COL4A2, COL5A2, COL6A2, and COL11A2.

[0048] In pharmaceutical or cosmetic compositions containing the culture supernatant of purified cardiomyocytes, it is particularly preferable that the extracellular matrix contains at least one selected from the group consisting of ELN, MMP1, MMP2, MMP9, MMP10, MMP11, MMP14, MMP15, MMP23, TIMP1, TIMP2, TIMP3, TIMP4, COL1A1, COL2A1, COL3A1, COL5A1, COL6A1, COL11A1, COL14A1, COL18A1, COL22A1, COL1A2, COL4A2, COL5A2, COL6A2, and COL11A2.

[0049] In pharmaceutical or cosmetic compositions containing culture supernatant of differentiated cardiomyocytes, it is particularly preferable that the extracellular matrix contains at least one selected from the group consisting of MMP1, MMP2, MMP9, MMP10, MMP11, MMP14, MMP15, MMP23, TIMP1, TIMP2, TIMP3, TIMP4, COL1A1, COL2A1, COL3A1, COL5A1, COL6A1, COL11A1, COL14A1, COL18A1, COL22A1, COL1A2, COL4A2, COL5A2, COL6A2, and COL11A2.

[0050] The pharmaceutical or cosmetic composition containing the culture supernatant of mesenchymal stem cells obtained by differentiation induction from iPS cells produced in the present invention preferably contains, in particular, at least one protein selected from the group consisting of collagen (COL1A1, COL1A2, COL3A1, COL6A2, COL5A2, etc.), fibronectin (FN1), MMPs (MMP1, MMP2, etc.), keratin (KRT1, KRT9, KRT2, KRT10, etc.), SPARC, THBS2, PCOLCE, VCAN, LGALS3BP, PTX3, THBS1, AGRN, LOXL2, VIM, NID2, ENO1, and KIF20B as an extracellular matrix. In addition, SHANK3 and / or YWHAE may be included as other proteins.

[0051] The pharmaceutical or cosmetic composition containing the culture supernatant of the present invention preferably contains proteins secreted from iPS cells or cells obtained by differentiation induction from iPS cells.The pharmaceutical or cosmetic composition according to the present invention contains the following proteins: FSTL1, HGF, BMPs (BMP1, BMP2, BMP4, BMP5, BMP7, BMP10, etc.), DKK (DKK1, DKK3, etc.), FGF (FGF18, FGF1, FGF9, FGF13, etc.), KGF (FGF7), IGF (IGF2, etc.), CSF (CSF1, etc.), NTG (NTF3, etc.), VEGF (VEGFA, VEGFB, VEGFC, VEGFD, etc.), VGF, TGF-α, TGF-β (TGF-β1, TGF-β2, etc.), PD GF (PDGF-C, PDGF-D, etc.), GDF (GDF2, GDF10, GDF11, GDF15, etc.), ANGPT (ANGPT1, ANGPT2, etc.), SFRP (SFRP1, SFRP2, FRZB (SFRP3), SFRP5, etc.), CLEC11A, TIMP1, INHBA, SCG2, GRN, HDGF, GMFB, EFEMP1, MIA, SDF-1 (CXCL12), MANF, PTN, JAG1, TGFB1, NRG1, CSF1, MIF, EDN1, CXCL1, NELL (NELL1, NELL2, etc.) ), PTPRZ1, SEMA3A, NCAM1, DCC, NTN1, IL6ST, WNT5A, WNT5B, Collagen (COL1A1, COL2A1, COL3A1, COL5A1, COL6A1, COL11A1, COL14A1, COL18A1, COL22A1, COL1A2, COL4A2, COL5A2, COL6A2, COL11A2, etc.), Collagen II, Laminin, Fibronectin (FN1), Elastin (ELN), TIMP (TIMP1, TIMP2, TIMP3, TIMP4, etc.), MMPs (MMP It is preferable that the product contains at least one protein selected from the group consisting of 1, MMP2, MMP9, MMP10, MMP11, MMP14, MMP15, MMP23, etc.), SPARC, keratin (KRT1, KRT9, KRT2, KRT10, etc.), THBS2, PCOLCE, LGALS3BP, PTX3, THBS1, AGRN, LOXL2, VIM, NID2, ENO1, IGFBP5, IGFBP7, KIF20B, SHANK3, YWHAE, proteoglycan (VCAN), glycoprotein, and basement membrane factors.

[0052] In the present invention, the culture supernatant obtained by culturing pre-purified cardiomyocytes preferably contains at least one of the following proteins: VEGFD, FGF18, KGF (FGF7), VEGFC, PDGF-C, GDF2, GDF10, GDF11, DKK1, SFRP5, IGF2, CSF1, VEGFA, TGF-β1, TGF-β2, PDGF-D, GDF15, DKK3, ANGPT1, BMP1, BMP2, BMP4, BMP5, BMP7, BMP10, SFRP1, SFRP2, and FRZB Preferably, the product contains at least one protein selected from the group consisting of (SFRP3), Elastin, MMPs (MMP1, MMP2, MMP9, MMP10, MMP11, MMP14, MMP15, MMP23, etc.), TIMP1, TIMP2, TIMP3, TIMP4, COL1A1, COL2A1, COL3A1, COL5A1, COL6A1, COL11A1, COL14A1, COL18A1, COL22A1, COL1A2, COL4A2, COL5A2, COL6A2, and COL11A2.

[0053] In the present invention, it is preferable that the culture supernatant obtained by culturing purified cardiomyocytes contains at least one of the following proteins: FGF1, FGF9, VEGFB, FGF18, KGF (FGF7), VEGFC, PDGF-C, GDF2, GDF10, GDF11, DKK1, SFRP5, IGF2, CSF1, VEGFA, TGF-β1, TGF-β2, PDGF-D, GDF15, DKK3, ANGPT1, BMP1, BMP2, BMP4, BMP5, BMP7, BMP10, SFRP1, SFRP2, FRZB It is preferable to include at least one protein selected from the group consisting of (SFRP3), Laminin, ELN, MMP1, MMP2, MMP9, MMP10, MMP11, MMP14, MMP15, MMP23, TIMP1, TIMP2, TIMP3, TIMP4, COL1A1, COL2A1, COL3A1, COL5A1, COL6A1, COL11A1, COL14A1, COL18A1, COL22A1, COL1A2, COL4A2, COL5A2, COL6A2, and COL11A2.

[0054] In the present invention, it is preferable that the culture supernatant obtained by culturing differentiating cardiomyocytes contains at least one of the following proteins. It is preferable to include at least one protein selected from the group consisting of NTF3, ANGPT2, IGF2, CSF1, VEGFA, TGF-β1, TGF-β2, PDGF-D, GDF15, DKK3, ANGPT1, BMP1, BMP2, BMP4, BMP5, BMP7, BMP10, SFRP1, SFRP2, FRZB (SFRP3), MMP1, MMP2, MMP9, MMP10, MMP11, MMP14, MMP15, MMP23, TIMP1, TIMP2, TIMP3, TIMP4, COL1A1, COL2A1, COL3A1, COL5A1, COL6A1, COL11A1, COL14A1, COL18A1, COL22A1, COL1A2, COL4A2, COL5A2, COL6A2, and COL11A2.

[0055] In the present invention, it is preferable that the culture supernatant obtained by culturing cardiomyocytes obtained by differentiating iPS cells contains at least one protein selected from the group consisting of FGF, KGF, IGF, VEGF, and DKK, and more preferably contains FGF, KGF, IGF, VEGF, and DKK.

[0056] In the present invention, it is preferable that the culture supernatant obtained when mesenchymal stem cells obtained by differentiating iPS cells are cultured contains at least one of the following proteins: IGFBP5, IGFBP7, FGF, FSTL1, CLEC11A, TIMP1, INHBA, SCG2, BMP1, GRN, HDGF, PDGFC, GMFB, EFEMP1, MIA, CXCL12, MANF, PTN, JAG1, DKK1, TGFB1, NRG1, CSF1, MIF, EDN1, CXCL1, WNT5A, WNT5B, GDF2, collagen (COL1A1, COL1A2, COL3A1, COL6A2, C It is preferable that the protein contains at least one protein selected from the group consisting of OL5A2, fibronectin (FN1), MMPs (MMP1, MMP2, etc.), keratin (KRT1, KRT9, KRT2, KRT10, etc.), SPARC, THBS2, PCOLCE, VCAN, LGALS3BP, PTX3, THBS1, AGRN, LOXL2, VIM, NID2, ENO1, KIF20B, SHANK3, and YWHAE.

[0057] In the present invention, it is preferable that the culture supernatant obtained when mesenchymal stem cells obtained by differentiating iPS cells are cultured contains at least one protein selected from the group consisting of HGF, FGF, SDF-1, PDGF, and DKK, and it is even more preferable that it contains HGF, FGF, SDF-1, PDGF, and DKK.

[0058] In the present invention, it is preferable that the culture supernatant obtained when culturing nerve cells obtained by differentiating iPS cells contains at least one of the following substances: It is preferable that it contains at least one protein selected from the group consisting of FGF, PDGF, TIMP, MMP, GDF, NELL, PTPRZ1, SEMA3A, NCAM1, DCC, NTN1, and IL6ST. In the present invention, it is preferable that the culture supernatant obtained when culturing nerve cells obtained by differentiating iPS cells contains at least one protein selected from the group consisting of FGF, PDGF, TIMP, MMP, and GDF, and it is even more preferable that it contains FGF, PDGF, TIMP, MMP, and GDF.

[0059] The proteins present in the cell culture supernatant obtained by the manufacturing method of the present invention are useful for wound healing, improvement of blemishes, wrinkles, sagging and depressions, skin whitening, and hair growth or regeneration. Furthermore, because they promote angiogenesis, they are useful in the treatment of ischemia, blood flow disorder-related diseases, or infarction. The pharmaceutical and cosmetic compositions according to the examples of the present invention include the cell culture supernatant obtained by the manufacturing method of the present invention, and the culture supernatant preferably contains at least one protein selected from the group consisting of PDGF, VGF, TGF-α, TGF-β, CSF, MMP, TIMP, VEGF, FGF, KGF, IGF, NTF, GDF, ANGPT, BMP, SFRP, ELN, COL, CLEC, INHBA, DKK, SCG, GRN, HDGF, GMFB, EFEMP, FN, SPARC, KRT, THBS, IGFBP, PCOLCE, VCAN, LGALS3BP, HGF, SDF-1, NTF3, and IL. More preferably, the effective protein includes at least one protein selected from the group consisting of FGF, DKK, PDGF, TGF-β, CSF, MMP, TIMP, BMP, and COL, but is not limited to these and may include thousands of other proteins.

[0060] The pharmaceutical or cosmetic composition according to the present invention, which contains the culture supernatant of cardiomyocytes obtained by differentiation induction from iPS cells, preferably contains at least one protein selected from the group consisting of PDGF, TGF-β, CSF, MMP, TIMP, VEGF, FGF, KGF, IGF, GDF, ANGPT, BMP, SFRP, ELN, COL, IL, CLEC, INHBA, DKK, SCG, GRN, HDGF, GMFB, EFEMP, FN, SPARC, KRT, THBS, IGFBP, PCOLCE, VCAN, LGALS3BP, SDF-1, and NTF3. A pharmaceutical or cosmetic composition containing the culture supernatant of mesenchymal stem cells obtained by differentiation induction from iPS cells according to the present invention preferably contains at least one protein selected from the group consisting of PDGF, TGF-β, CSF, MMP, TIMP, VEGF, HGF, FGF, IGF, GDF, ANGPT, BMP, SFRP, COL, IL, CLEC, INHBA, DKK, SCG, GRN, HDGF, PDGF, GMFB, EFEMP, FN, SPARC, KRT, THBS, IGFBP, PCOLCE, VCAN, LGALS3BP, and SDF-1. A pharmaceutical or cosmetic composition containing the culture supernatant of nervous system cells obtained by differentiation induction from iPS cells according to the present invention preferably contains at least one protein selected from the group consisting of FGF, PDGF, TIMP, MMP, and GDF.

[0061] The pharmaceutical and cosmetic compositions containing the culture supernatant of the present invention contain growth factors such as cytokines, anti-inflammatory molecules, and hormones secreted from iPS cells or cells obtained by differentiation induction from iPS cells. Through the actions of the aforementioned cytokines, growth factors, and extracellular matrix, angiogenesis is promoted and vascular protective effects are obtained. These compositions are also useful for the treatment of diabetes and non-alcoholic fatty liver disease (NASH). Furthermore, they promote cell proliferation and are effective in wound healing. They also contribute to immunomodulation and are expected to have anti-inflammatory effects. In addition, they are useful for skin whitening, improvement of blemishes and wrinkles, and hair growth and regeneration.

[0062] Wound healing is a regenerative and repair response to rebuild damaged tissues and cells. The mechanism of wound healing goes through four stages: (1) bleeding and coagulation phase (hemostasis phase), (2) inflammation phase, (3) proliferation phase, and (4) reconstruction phase (remodeling phase). Specifically, (1) is the period when a coagulation clot from bleeding seals the defect and stops the bleeding; (2) is the period when inflammatory cells (neutrophils, monocytes, macrophages, etc.) migrate to the wound and attack necrotic and crushed tissue; (3) is the period when fibroblasts migrate from the periphery, rebuild the extracellular matrix, angiogenesis occurs, and granulation tissue is formed; and (4) is the period when collagen production becomes sufficient, the number of fibroblasts decreases, scarring becomes less severe, and epidermal cells migrate, causing the wound to contract and close.

[0063] Cell growth factors involved in wound healing include PDGF, TGF-β, EGF, FGF, TNF-α, IL-1, GM-CSF, IFN, and ET, while extracellular matrix components include collagen, elastin, reticulin, laminin, proteoglycans, fibronectin, and tenascin. As described above, factors that act on wound healing include those that promote the production of collagen and elastin in the skin, thus promoting the regeneration of new skin cells and tissues that are even better than the original skin, leading to cosmetic benefits. Furthermore, certain cytokines and growth factors involved in wound healing are expected to supplement the growth factors lacking in dormant hair follicles, create new capillaries in the scalp to improve blood flow, and supply oxygen and nutrients to the hair bulb. Certain cytokines and growth factors are expected to stimulate hair matrix cells within the hair bulb to activate the dermal papilla, and also create new capillaries in the scalp to improve blood flow and promote hair growth.

[0064] DKK1 (Dickkopf1) works by acting on melanocytes, which are skin cells, to suppress melanin production and achieve a skin-whitening effect. It also acts on epidermal cells to promote their stratification, making it useful for wrinkle reduction. Furthermore, DDK1 inhibits the expression of receptors for melanin uptake in epidermal cells, thus contributing to skin whitening.

[0065] FGF18 (Fibroblast Growth Factor 18) is useful for angiogenesis and wound healing. Furthermore, it is thought to be useful for promoting hair growth and suppressing hair loss by accelerating the transition to the growth phase of the hair cycle.

[0066] PDGF (Platelet-Derived Growth Factor) promotes the proliferation and migration of vascular endothelial cells and fibroblasts, and is involved in angiogenesis. Because its expression increases during inflammation and wound healing, it is useful in the treatment of blood flow-related diseases and wound healing. PDGF promotes collagen production by stimulating fibroblast proliferation, making it useful in preventing or improving wrinkles. Furthermore, by acting on the skin, subcutaneous fat, facial muscles, and ligaments together with various cell growth factors such as TGF-β and VEGF, it is expected to be effective in improving wrinkles and sagging. In addition, it also promotes the proliferation of hair matrix cells, making it useful for hair growth and regeneration.

[0067] TGF-β (Transforming Growth Factor β) is involved in the production of proteins that make up the extracellular matrix, the formation of capillary lumenes, and the expression of VEGF. Furthermore, TGF-β acts on smooth muscle and fibroblasts, promoting PDGF production, thus promoting anti-inflammatory effects and being useful in the treatment of blood flow disorders and wound healing. In addition, by promoting collagen production through the proliferation of fibroblasts, it is useful in improving skin firmness and preventing or improving wrinkles. Moreover, TGF-β, along with various cell growth factors such as PDGF, TGF-β, and VEGF, is expected to be effective in improving wrinkles and sagging when acted on the skin, subcutaneous fat, facial muscles, and ligaments.

[0068] Colony-stimulating factor (CSF) is a type of cytokine that promotes the proliferation and differentiation of immune cells. Because it acts on immune cells, it is expected to have anti-inflammatory effects. By promoting cell proliferation, it also promotes skin repair.

[0069] MMP (Matrix Metalloproteinase) is a proteolytic enzyme that breaks down the extracellular matrix and is involved in its turnover, thus contributing to skin regeneration and offering potential cosmetic benefits. It also plays a role in releasing VEGF and promotes angiogenesis by disrupting the structure of blood vessel walls.

[0070] TIMP (Tissue Inhibitor of Metalloproteinase) plays a role in inhibiting MMP and regulating MMP activity, and TIMP secretion is useful in treating arteriosclerosis and pulmonary fibrosis. Furthermore, because it is involved in collagen and elastin synthesis in the dermis, it is useful in preventing or treating wrinkles.

[0071] VEGF (Vascular Endothelial Growth Factor) is a dimeric glycoprotein. It primarily binds as a ligand to vascular endothelial growth factor receptors (VEGFRs) on the surface of vascular endothelial cells, influencing cell migration, proliferation, and lumen formation, stimulating differentiation, and increasing microvessel permeability. Because it is involved in angiogenesis and wound healing, it is considered useful in the treatment of ischemia, blood flow disorders, and infarction. Furthermore, because it is involved in the activation of immune cells such as monocytes and macrophages, it is thought to have anti-inflammatory effects and be involved in immune tolerance. It can suppress inflammation in the skin, including the epidermis and dermis, and further enhance regenerative and repair capabilities. In addition, VEGF is one of the factors released from dermal papilla cells and is known as a growth phase maintenance factor in the hair cycle, making it useful for hair growth and regeneration. VEGF delivers nutrients to the hair follicles, promoting hair growth and regeneration.

[0072] FGF (Fibroblast Growth Factor) is a peptide that shows high affinity for heparin and is involved in the proliferation and differentiation of endothelial cells, smooth muscle cells, and fibroblasts, making it useful in wound healing and angiogenesis. FGF18 is important for regulating hair growth and skin maintenance. FGF13 is involved in the regulation of neuronal excitability in inhibitory interneurons and in seizure susceptibility.

[0073] KGF (Epidermal Growth Factor, also known as FGF-7) promotes hair growth and development by stimulating the proliferation and division of hair matrix cells, and also has angiogenesis and wound healing effects. Furthermore, by increasing keratinocytes, it is thought that skin turnover is properly regulated, improving skin condition. As a result, it has the effect of improving blemishes and wrinkles.

[0074] IGF (Insulin-like Growth Factor) is a peptide hormone with a structure similar to insulin. It has growth-regulating functions, insulin-like activity, and cell division-promoting effects, and is associated with cosmetic and wound-healing benefits. It also acts on hair matrix cells to promote their division, thus playing a role in hair growth. When IGF-1 is expressed, the hair growth phase is extended and the resting phase (when hair falls out) is shortened, which is thought to prevent hair loss and thinning, and promote hair growth and regeneration.

[0075] GDF (Growth and Differentiation Factor) is a growth factor involved in development and cardioprotection. In particular, GDF11 plays a role in slowing down the aging process and promoting the production of proteins important for skin regeneration, such as collagen and elastin, and is expected to have cosmetic and anti-aging effects.

[0076] ANGPT (angiopoietin) is a growth factor that promotes vascular formation and angiogenesis. Because it promotes blood vessel formation, it is useful in wound healing and the treatment of ischemia, blood flow disorders, or infarction.

[0077] BMP (Bone Morphogenetic Protein) is involved in all aspects of development, including bone and cartilage formation, mesoderm induction, and organ formation such as the heart. Furthermore, BMP is a key protein in maintaining the dormant state of hair follicle stem cells and is involved in the hair growth cycle.

[0078] SFRP (Secreted Frizzled-Related Protein) is an extracellular WNT-binding protein that plays a crucial role in stem cell proliferation and regeneration by regulating (promoting or inhibiting) WNT signaling, and is therefore highly likely to be involved in wound healing and tissue repair. It also contributes to hair growth by regulating Wnt signaling.

[0079] Elastin (ELN) is a protein that is a component of elastic fibers. In the dermis of the skin, it works together with collagen fibers to give the skin firmness and elasticity. The presence of elastin helps maintain skin firmness and elasticity, leading to improved wrinkle reduction and increased skin hydration. It also helps keep blood vessels flexible and contributes to maintaining the elasticity of ligaments.

[0080] Collagen (COL) is a protein that makes up the dermis, ligaments, tendons, bones, and cartilage, and is a major component of the extracellular matrix. The presence of collagen improves skin wrinkles and enhances the skin's ability to retain moisture.

[0081] CLEC11A (C-Type Lectin Domain Family 11 member A) is a protein secreted by mesenchymal stem cells (MSCs) and, in cooperation with other MSC-secreted proteins (CTGF, SERPINE1, HTRA1, THBS1, DKK3, MYDDGF, etc.), may exert broad therapeutic effects against osteochondral damage, nerve damage, cardiovascular damage, and myocardial infarction. For example, it is thought to be useful in the treatment of osteoporosis because it promotes bone formation by accelerating the differentiation of mesenchymal progenitor cells into mature osteoblasts, thereby contributing to bone repair and maintenance.

[0082] INHBA (Inhibin Subunit Beta A) is one of the proteins secreted by MSCs, and in cooperation with other MSC-secreted proteins, it may exert a wide range of therapeutic effects on osteochondral damage, nerve damage, cardiovascular damage, myocardial infarction, and other conditions.

[0083] SCG2 (Secretogranin II) is a member of the chromogranin / secretogranin family of neuroendocrine proteins and is suggested to be involved in the packaging and sorting of peptide hormones and neuropeptides into secretory vesicles. It is also known to have anti-inflammatory effects.

[0084] Granulin Precursors (GRNs) are regulatory factors that control protein transport to lysosomes and regulate the activity of lysosomal enzymes. They are also secreted proteins that act as growth factors involved in inflammation, wound healing, and cell proliferation. They directly act on dermal fibroblasts and endothelial cells, functioning as wound-associated growth factors that promote cell division, migration, and the formation of capillary-like structures. They have antioxidant effects by inhibiting TNF-mediated neutrophil activation and preventing the release of oxidants and proteases, and are expected to suppress age spots and wrinkles by promoting epithelial cell proliferation. Furthermore, they play a role in regulating nerve cell inflammation by maintaining nerve cell survival, axonal elongation, and nerve cell integrity.

[0085] HDGF (Hepatoma-Derived Growth Factor) is a heparin-binding growth factor involved in cell proliferation, cell differentiation, angiogenesis, and apoptosis. It is also involved in epithelial-mesenchymal transition and cell migration, and may have wound-healing effects, leading to expectations of skin "rejuvenation."

[0086] GMFB (Glia Maturation Factor Beta) is thought to be useful in neurological diseases because it stimulates brain cell differentiation and nerve regeneration. In recent years, this protein has been found to be expressed in tissues other than the nervous system and is involved in regulating cytoskeletal rearrangement, immune responses, and oxidative stress responses, and is associated with several inflammatory diseases and tumors. Therefore, new applications such as tissue regeneration and cosmetic surgery are expected.

[0087] EFEMP1 (EGF-containing fibrin-like extracellular matrix protein 1) is a growth factor that binds to EGFR, the EGF receptor, and induces EGFR autophosphorylation and activation of downstream signaling pathways. Because it functions as a negative regulator of cell adhesion, migration, and chondrocyte differentiation, it is thought to be useful for improving skin health and rejuvenation through wound healing.

[0088] MIA (Melanoma-derived Growth Regulatory Protein) is a secreted cytokine belonging to the MIA / OTOR family. It is a growth-regulating autocrine protein secreted from chondrocytes and other cells. It is expected to play a role in wound healing by competitively binding to fibronectin and laminin within cells, thereby detaching cells from the cell matrix, promoting cell migration, and contributing to wound healing.

[0089] CXCL12 / SDF-1 (CXC Motif Chemokine Ligand 12 / SDF-1) is a type of CXC chemokine. It is known to induce lymphocytes, mesenchymal stem cells, and vascular endothelial progenitor cells, and is expected to promote angiogenesis at the site of inflammation and be involved in wound healing.

[0090] MANF (Mesencephalic Astrocyte-derived Neurotrophic Factor) is a neurotrophic factor that is induced by endoplasmic reticulum stress. Because it promotes the survival and growth of nerve cells, it is considered useful in the treatment of neurodegenerative diseases.

[0091] Pleiotrophin (PTN) is a secreted growth factor that promotes cell division in fibroblasts, epithelial cells, and endothelial cells, inducing nerve axon extension. This cell division helps regulate skin turnover, leading to improved skin condition. It is also thought to be useful in wound healing.

[0092] JAG1 (Jagged-1 protein) is a protein found on the surface of keratinocytes and contributes to the shedding of senescent cells. As JAG1 expression decreases with age, epidermal turnover slows down, and senescent cells accumulate in the epidermis. By regulating skin turnover through JAG1 expression, improvement in skin condition can be expected.

[0093] NRG1 (Neuregulin-1) is a member of the epidermal growth factor superfamily and, along with its receptor, is widely distributed in the brain. In the nervous system, it plays a role as a neurotrophic factor in the differentiation and development of GABAergic neurons and glial cells, and in regulating the expression of glutamate receptors. In recent years, it has also attracted attention as a gene associated with schizophrenia. NRG1 is also a cardiac growth factor released from endothelial cells and is necessary for cardiac development, structural maintenance, and functional integrity. NRG1 and its receptor, the ErbB family, may play a beneficial role in the treatment of chronic heart failure (CHF) by promoting cardiomyocyte survival, improving sarcomere structure, maintaining Ca2+ homeostasis, and enhancing pump function. Downstream effectors of NRG1 / ErbB include cardiac-specific myosin light chain kinase (cMYLK), PP1, SERCA2, and FAK, suggesting that NRG1 has potential as a therapeutic agent for CHF. It is also considered useful in the treatment of neurological diseases such as ALS.

[0094] Macrophage Migration Inhibitory Factor (MIF) is a cytokine involved in immune responses and inflammation. It controls macrophage migration, causing macrophages to accumulate at the site of inflammation and triggering inflammation and immune responses. It is also an essential factor for T lymphocyte activation. MIF is also involved in cell proliferation, and is particularly expected to promote tumor growth and angiogenesis, thus contributing to wound healing.

[0095] EDN1 (Endothelin-1) is a cyclic peptide primarily produced by vascular endothelial cells, possessing potent vasoconstrictive properties. Due to its vasodilatory and vasoconstrictive effects, EDN1 is considered useful in wound healing and the treatment of ischemia or blood flow disorders. It is also thought to be involved in promoting blood circulation in the skin.

[0096] CXCL1 (CXC Motif Chemokine Ligand 1) is a type of CXC chemokine. It attracts immune system cells and other non-hematopoietic cells to injury or infection sites and plays a role in regulating immune and inflammatory responses. It is expressed at high levels during inflammatory responses and is expected to improve wound healing and immune function.

[0097] WNT5A / WNT5B are signaling molecules that induce differentiation from pluripotent stem cells into mesoderm and endoderm progenitor cells. They are also involved in the proliferation, growth, and migration of embryonic stem cells. Because they play a crucial role in stem cell proliferation and regeneration, they are highly likely to be involved in wound healing and tissue repair. Furthermore, since they are involved in hair follicle development, they are expected to contribute to hair growth.

[0098] Fibronectin 1 (FN1) is a cell adhesion molecule. It promotes cell adhesion and extension, thereby inducing cell migration. Furthermore, as part of the extracellular matrix, it provides a cellular environment and is involved in the regulation of differentiation and cell proliferation, thus playing an important role in wound healing. It is also one of the proteins secreted by mesenchymal stem cells (MSCs), and when used in the culture supernatant of MSCs, it may work in conjunction with other secreted proteins of MSCs to exert a wide range of therapeutic effects on osteochondral damage, nerve damage, cardiovascular damage, myocardial infarction, and other conditions.

[0099] SPARC (Secreted Protein Acidic and Rich in Cysteine) promotes cell proliferation, differentiation, and migration. It is present in the epidermis and dermis, and its levels increase during wound healing, suggesting its effectiveness in wound healing.

[0100] KRT1 (Keratin 1), KRT2 (Keratin 2), KRT9 (Keratin 9), and KRT10 (Keratin 10) are proteins that make up the cytoskeleton and form the intermediate filaments of epithelial cells. They play a role in improving skin hydration by retaining moisture in the stratum corneum and preventing moisture evaporation. They also act as a barrier against external stimuli.

[0101] THBS1 (Thrombospondin-1), along with THBS2, is known as an angiogenesis inhibitor. Because THBS1 binds to fibrinogen, fibronectin, laminin, collagen types V and VII, and integrin α-V / β-1, it is thought to be involved in platelet aggregation, angiogenesis, and tumor formation, and is therefore useful in wound healing, ischemia, blood flow disorders, and infarction. THBS1, along with THBS2, has been shown to significantly improve UV-induced skin damage and wrinkles, reduce irritant skin reactions, and repair skin damage by regulating the levels of angiogenesis-promoting and angiogenesis-inhibiting factors. Furthermore, data science has reported that THBS1 is an effective factor against skin aging.

[0102] THBS2 (Thrombospondin-2), along with THBS1, is an extracellular matrix protein that mediates intercellular and cell-matrix interactions. THBS2, together with THBS1, has been shown to significantly improve UV-induced skin damage, reduce wrinkles, alleviate irritant skin reactions, and repair skin damage by regulating the levels of pro-angiogenic and anti-angiogenic factors. Improvements in wrinkles through extracellular matrix and cellular remodeling are anticipated.

[0103] IGFBP5 (Insulin-like Growth Factor Binding Protein 5) is an insulin-like growth factor binding protein primarily expressed in the prostate, mammary glands, skin, and hair follicles. In particular, in human hair follicles, it is expressed in the dermal papilla and root sheath and is known to be important for the remodeling of the extracellular matrix, potentially playing a role in hair growth and development. It is also thought to be useful in the treatment of liver diseases by enhancing lipid metabolism and insulin sensitivity.

[0104] PCOLCE (Procollagen C-endopeptidase Enhancer 1 / PCPE-1) is a secreted extracellular matrix glycoprotein expressed in the skin. PCOLCE promotes enzymatic cleavage of type I procollagen by binding to it, thereby increasing C-proteinase activity. Because PCOLCE expression regulates skin cell turnover, it is expected to improve skin condition.

[0105] IGFBP7 (Insulin-like Growth Factor Binding Protein 7) binds to insulin-like growth factor (IGF) with low affinity, regulating the amount of IGF available in tissues, and also binds to IGF receptors to regulate downstream signaling pathways. It controls protein synthesis, cell proliferation, and survival in tissue regeneration and wound healing. Therefore, IGFBP7 is thought to play a role in normalizing cell turnover in tissue regeneration and wound healing. It is known to have anti-fibrotic activity, and is expected to have an effect in suppressing fibrosis caused by inflammation.

[0106] VCAN (Versican) is an extracellular matrix-type chondroitin sulfate / dermatan sulfate proteoglycan. Increased VCAN production has been shown to increase collagen and elastin production from fibroblasts, resulting in thicker, firmer collagen fibers and improved elasticity and moisture retention. Therefore, VCAN is expected to regenerate weakened dermis and improve sagging. It is considered useful for skin regeneration by increasing collagen and elastin production from fibroblasts.

[0107] LGALS3BP (Galectin-3-binding Protein / Lectin Galactoside-binding Soluble 3 Binding Protein) is a member of the β-galactoside-binding protein family and is involved in biological processes such as cell adhesion, interaction with the extracellular matrix, cell activation, angiogenesis, chemotaxis, cell growth, cell differentiation, cell cycle, metastasis, and apoptosis. It is also involved in cancer, inflammation, fibrosis, heart disease, and stroke.

[0108] PTX3 (Pentraxin 3) is an inflammatory protein produced in response to inflammation in the body. It plays a role in inflammation, tissue remodeling and repair, and wound healing, and is therefore expected to promote innate immunity.

[0109] Agrin (AGRN) is a large heparan sulfate proteoglycan. It is involved in the connection between nerve and muscle cells and is thought to be useful in Alzheimer's disease and other conditions. During development, AGRN is secreted from developing motor nerve terminals and plays a role in the aggregation of acetylcholine receptors, thereby forming motor nerve terminals. In addition to the motor nervous system, it has also been found in the central nervous system, where it binds to the α3 site of Na+ / K+-ATPase and performs activity-dependent regulation. Therefore, Agrin may have an effect on the formation and regulation of both the motor nervous system and the central nervous system.

[0110] LOXL2 (Lysyl Oxidase Homolog 2) is thought to be useful in wound healing because it cross-links type IV collagen and influences the sprouting of new blood vessels.

[0111] FSTL1 (Follistatin-like 1) is a secreted extracellular matrix glycoprotein. Because FSTL1 is involved in many biological processes such as cell proliferation, apoptosis, and tissue differentiation, it is expected to be useful in wound healing and cosmetic applications requiring tissue regeneration and repair. It regulates the action of growth factors involved in cell proliferation and differentiation. It is considered useful for tissue regeneration, such as in skin.

[0112] NID2 (Nidogen 2) is involved in the formation and maintenance of the basement membrane by binding to the laminin γ1 chain and linking laminin to type IV collagen. Therefore, NID2 is thought to provide a scaffold necessary for cell migration and proliferation during the synthesis and proliferation phases of the wound healing process.

[0113] In addition to its role in glycolysis, ENO1 (enolase 1) is involved in various processes such as growth regulation, hypoxia tolerance, and allergic reactions. In particular, it has multifaceted functions, including potentially functioning in the intravascular and pericellular fibrinolytic systems and acting as a plasminogen receptor and activator on the cell surface of various cell types, including leukocytes and neurons. Therefore, ENO1 may play a role in regulating cell growth during the synthesis and proliferation phases of wound healing.

[0114] HGF (hepatocyte growth factor) is a multifunctional cytokine that primarily acts on epithelial-derived cells, influencing various cell types. For example, it affects inflammation, tissue repair, morphogenesis, angiogenesis, tumor propagation, immunomodulation of viral infections, and cardiovascular metabolic activity, and is therefore expected to be useful in treating wounds, ischemia, blood flow disorders, infarction, immune diseases, and neurological diseases. Furthermore, as a factor derived from the dermis, HGF not only regenerates and maintains skin tissue but also promotes wound healing through the promotion of cutaneous angiogenesis and also stimulates hair follicle development.

[0115] NELL (PKC-binding protein, Neural Epidermal Growth Factor-like) is a secreted protein that binds to protein kinase C (PKC), which is involved in intracellular signaling. Through this binding, it regulates neuronal differentiation, growth, cell adhesion, and migration in bone formation and especially in the development of the nervous system, playing a crucial role in the growth of axons and dendrites.

[0116] PTPRZ1 (Tyrosine-Specific Protein Phosphatase Receptor Z1) functions as a receptor protein phosphatase that regulates neuronal differentiation and synapse formation. Because it is involved in synapse formation and the maintenance of neural stem cells, it is considered useful in the treatment of neurological diseases.

[0117] SEMA3A (semaphorin 3A) is involved in intercellular signaling and plays an important role in various tissues, including the nervous system, immune system, skin, and bone. Because it is involved in nerve axon guidance and participates in the formation of neural circuits and the regulation of immune cells, it is considered useful in the treatment of neurological diseases.

[0118] NTF3 (Neurotrophin-3 / NT-3) is a neurotrophic factor. Because it is necessary for the survival, development, and function of nerve cells, it is considered useful in the treatment of neurological diseases.

[0119] NCAM1 (Neural Cell Adhesion Molecule 1) is a cell adhesion molecule found on the surface of nerve cells, glial cells, skeletal muscle cells, and other cells. Because NCAM1 plays a crucial role in the development and function of the nervous system, including neurite outgrowth, nerve cell migration, synapse formation and plasticity, learning, and memory, it is considered useful in the treatment of neurological diseases.

[0120] DCC (Deleted in Colorectal Cancer) is a receptor for the axon guidance molecule netrin. It plays a major role in a process called "axon guidance," which directs nerve cell axons to reach the correct location. DCC functions as a receptor for a molecule called netrin-1, which is present on the cell membrane. By binding to netrin-1, DCC transmits signals within the cell that determine the direction in which axons grow.

[0121] Interleukins (ILs) are a group of cytokines with various functions depending on the type. Because they are involved in many functions of the immune system, they are considered useful in the treatment of immune diseases and neurological disorders.

[0122] NTN1 (Netrin-1) is an axon guidance molecule that primarily plays a role in the development of the nervous system, guiding the proper growth of nerve cell axons towards their destinations. Because it is involved in many other functions, including neural circuit development, axonal branching, and synapse formation, it is considered useful in the treatment of neurological disorders.

[0123] IL6ST (Interleukin 6 Cytokine Family Signal Transducer) is a signal transducer shared by many cytokines, including interleukin 6 (IL-6), to transmit signals within cells. It is also known as gp130. Because it is involved in the survival and differentiation of nerve cells and neuroinflammation, it is considered useful in the treatment of immune and neurological diseases.

[0124] Furthermore, the pharmaceutical and cosmetic compositions according to the examples preferably contain a portion of the cell culture supernatant, or various proteins and exosomes isolated from the cell culture supernatant. The action of the various proteins contained in the cell culture supernatant suppresses inflammation of the skin, including the epidermis and dermis, and further enhances its regenerative and repair capabilities. Such proteins are preferably at least one protein selected from the group consisting of PDGF, VGF, TGF-α, TGF-β, CSF, MMP, TIMP, VEGF, FGF, KGF, IGF, NTF, GDF, ANGPT, BMP, SFRP, ELN, COL, CLEC, INHBA, DKK, SCG, GRN, HDGF, GMFB, EFEMP, FN, SPARC, KRT, THBS, IGFBP, PCOLCE, VCAN, LGALS3BP, HGF, SDF-1NTF3, and IL.

[0125] In addition to the above, the culture supernatant obtained by culturing iPS cells according to the present invention preferably contains at least one of the following proteins as cytokines and growth factors. If it contains TNFSF11 (TNF superfamily member 11) and / or CCL28 (CC motif chemokine ligand 28), it has immunomodulatory and anti-inflammatory effects. Furthermore, if it contains at least one protein selected from the group consisting of GDF3 (growth differentiation factor 3), VAV3 (vav guanine nucleotide exchange factor 3), FGF2 (fibroblast growth factor 2), PDGFB (platelet-derived growth factor subunit B), TGFA (transforming growth factor alpha), CXCL3 (CXC motif chemokine ligand 3), BMP3 (bone morphogenetic protein 3), PDGFA (platelet-derived growth factor subunit A), and BMP6 (bone morphogenetic protein 6), it acts on wound healing and cell proliferation. Furthermore, if it contains CXCL5 (CXC motif chemokine ligand 5) and / or ANGPT4 (angiopoietin 4), it induces angiogenesis and vascular protection, making it useful for ischemia, blood flow disorders, and infarction. In addition, it may contain at least one protein selected from the group consisting of TDGF1 (teratocarcinoma-derived growth factor 1), VGF (VGF nerve growth factor inducible), NODAL (nodal growth differentiation factor), LEFTY1 (left-right determination factor 1), and INSL3 (insulin-like 3).

[0126] In the present invention, the culture supernatant obtained by culturing iPS cells preferably contains at least one protein selected from the group consisting of VGF, FGF, PDGF, BMP, and TGF-α, and more preferably contains VGF, FGF, PDGF, BMP, and TGF-α.

[0127] In addition to the above, the culture supernatant obtained by culturing iPS cells according to the present invention preferably contains at least one of the following proteins as an extracellular matrix: If it contains NMU (neuromedin U) and / or RARRES2 (retinoic acid receptor responder 2), it has immunomodulatory and anti-inflammatory effects. If it contains at least one protein selected from the group consisting of CNMD (chondromodulin), MMP17 (matrix metallopeptidase 17), VASH2 (vasohibin 2), TMPRSS2 (transmembrane serine protease 2), and PRSS8 (serine protease 8), it acts on wound healing and cell proliferation. If it contains COL26A1 (collagen type XXVI alpha 1 chain), it induces angiogenesis and vascular protection, and is therefore useful for ischemia, blood flow disorders, and infarction. In addition, it may contain at least one protein selected from the group consisting of PTPRZ1 (protein tyrosine phosphatase receptor type Z1), SCG3 (secretogranin III), SFRP2 (secreted frizzled related-protein 2), NELL2 (neural EGFL-like 2), NTS (neurotensin), NPTX2 (neuronal pentraxin 2), ADGRB1 (adhesion G protein-coupled receptor B1), DRAXIN (dorsal inhibitory axon guidance protein), SORL1 (sortilin-related receptor 1), APELA (apelin receptor early endogenous ligand), BTBD17 (BTB domain containing 17), and GAL (galanin and GMAP prepropeptide).

[0128] In addition to the above, the culture supernatant (pre-purified cardiomyocyte) obtained by culturing cardiomyocytes obtained by differentiating iPS cells according to the present invention preferably contains at least one of the following proteins as cytokines and growth factors: If it contains at least one protein selected from the group consisting of IL33 (interleukin 33), IL32 (interleukin 32), and CCL3 (CC motif chemokine ligand 3), it has immunomodulatory and anti-inflammatory effects. If it contains at least one protein selected from the group consisting of BMP5 (bone morphogenetic protein 5), IGF2 (insulin-like growth factor 2), CCL21 (CC motif chemokine ligand 21), WNT2 (Wnt family member 2), AREG (amphiregulin), CXCL1 (CXC motif chemokine ligand 1), and / or GDF10 (growth differentiation factor 10), it acts on wound healing and cell proliferation. If the product contains at least one protein selected from the group consisting of ANGPTL3 (angiopoietin-like protein-3), AGT (angiotensinogen), HGF (hepatocyte growth factor), SPP1 (secreted phosphoprotein 1), GDF2 (growth differentiation factor 2), CXCL17 (CXC motif chemokine ligand 17), and ANGPT1 (angiopoietin 1), it induces angiogenesis and vascular protection, making it useful for ischemia, blood flow disorders, and infarction. If it contains DKK, a skin whitening effect is obtained. In addition, it may contain at least one protein selected from the group consisting of FAM3B (FAM3 metabolism regulating signaling molecule B) and / or CMTM5 (CKLF-like MARVEL transmembrane domain containing 5).

[0129] In addition to the above, the culture supernatant (pre-purified cardiomyocyte) obtained by culturing cardiomyocytes obtained by differentiating iPS cells according to the present invention preferably contains at least one of the following proteins as an extracellular matrix: If it contains at least one protein selected from the group consisting of APOA4 (apolipoprotein A4), APOB (apolipoprotein B), APOA2 (apolipoprotein A2), APOC3 (apolipoprotein C3), APOA1 (apolipoprotein A1), and RBP4 (retinol binding protein 4), it has immunomodulatory and anti-inflammatory effects. If it contains at least one protein selected from the group consisting of FGA (fibrinogen alpha chain), FGB (fibrinogen beta chain), FGG (fibrinogen gamma chain), and SERPINF2 (serpin family F member 2), it acts on wound healing and cell proliferation. If it contains VTN (vitronectin), it induces angiogenesis and vascular protection, and is therefore useful for ischemia, blood flow disorders, and infarction. In addition, it may contain at least one protein selected from the group consisting of AFP (alpha fetoprotein), SERPINA1 (serpin family A member 1), AHSG (alpha-2-HS-glycoprotein), TTR (transthyretin), TF (transferrin), ALB (albumin), ITIH2 (inter-alpha-trypsin inhibitor heavy chain 2), CHI3L1 (chitinase 3-like-1), and SERPINA7 (serpin family A member 7).

[0130] In addition to the above, the culture supernatant (purified cardiomyocyte) obtained by culturing cardiomyocytes obtained by differentiating iPS cells according to the present invention after purification through a purification process preferably contains at least one of the following proteins as cytokines and growth factors. If it contains JAG2 (jagged canonical Notch ligand 2), it has immunomodulatory and anti-inflammatory effects. If it contains at least one protein selected from the group consisting of BMP5, BMP7 (bone morphogenetic protein 7), FGF1 (fibroblast growth factor 1), PDGFD (platelet-derived growth factor D), BMP2 (bone morphogenetic protein 2), FGF18, FGF13 (fibroblast growth factor 13), FGF10 (fibroblast growth factor 10), FGF9 (fibroblast growth factor 9), and FGF7, it acts on wound healing and cell proliferation. If the protein contains at least one protein selected from the group consisting of GDF2, BMP10 (bone morphogenetic protein 10), AGT, and ANGPT1, it is useful for ischemia, blood flow disorders, and infarction because it induces angiogenesis and vascular protection. In addition, it may also contain at least one protein selected from the group consisting of LEFTY2 (left-right determination factor 2), CMTM5, NRTN (neurturin), NRG2 (neuregulin 2), and CSPG5 (chondroitin sulfate proteoglycan 5).

[0131] In addition to the above, the culture supernatant (purified cardiomyocyte) obtained by culturing cardiomyocytes obtained by differentiating iPS cells according to the present invention after purification through a purification process preferably contains at least one of the following proteins as an extracellular matrix: If it contains at least one protein selected from the group consisting of MASP1 (MBL associated serine protease 1), NCAM1 (neural cell adhesion molecule 1), and C7 (complement C7), it has immunomodulatory and anti-inflammatory effects. If it contains at least one protein selected from the group consisting of A2M (alpha-2-macroglobulin), BRINP3 (BMP / retinoic acid inducible neural specific 3), NPNT (nephronectin), EMILIN2 (elastin microfibril interfacer 2), ADAMTSL5 (ADAMTS-like 5), and WNT11 (Wnt family member 11), it acts on wound healing and cell proliferation. If the product contains at least one protein selected from the group consisting of COL9A1 (collagen type IX alpha 1 chain), COL2A1 (collagen type II alpha 1 chain), LAMA2 (laminin subunit alpha 2), FREM1 (FRAS1-related extracellular matrix 1), and COL21A1 (collagen type XXI alpha 1 chain), it is useful for ischemia, blood flow disorders, and infarction because it induces angiogenesis and vascular protection. In addition, it may also contain at least one protein selected from the group consisting of NPPA (natriuretic peptide A), NPPB (natriuretic peptide B), TRH (thyrotropin releasing hormone), RSPO3 (R-spondin 3), PRSS35 (serine protease 35), and RELN (reelin).

[0132] In addition to the above, the culture supernatant obtained by culturing mesenchymal stem cells obtained by differentiating iPS cells according to the present invention preferably contains at least one of the following proteins as cytokines and growth factors: If it contains at least one protein selected from the group consisting of CXCL8 (CXC motif chemokine ligand 8), IL1B (interleukin 1 beta), CXCL1, IL11 (interleukin 11), IL6 (interleukin 6), CCL2 (CC motif chemokine ligand 2), CXCL2 (CXC motif chemokine ligand 2), CLCF1 (cardiotrophin-like cytokine factor 1), and IL1A (interleukin 1 alpha), it has immunomodulatory and anti-inflammatory effects. If it contains at least one protein selected from the group consisting of GDF5 (growth differentiation factor 5), INHBA (inhibin subunit beta A), PDGFC (platelet-derived growth factor C), NGF (nerve growth factor), and EREG (epiregulin), it acts on wound healing and cell proliferation. In addition, it may contain at least one protein selected from the group consisting of LIF (interleukin 6 family cytokine), FGF5 (fibroblast growth factor 5), GDNF (glial cell line-derived neurotrophic factor), ESM1 (endothelial cell specific molecule 1), SCG2 (secretogranin II), and SPRED3 (sprouty-related EVH1 domain containing 3).

[0133] In addition to the above, the culture supernatant obtained by culturing mesenchymal stem cells obtained by differentiating iPS cells according to the present invention preferably contains at least one of the following proteins as an extracellular matrix. If CFH is included, it has immunomodulatory and anti-inflammatory effects. If at least one protein selected from the group consisting of MMP1 (matrix metallopeptidase 1), FAP (fibroblast activation protein alpha), TGFBI (transforming growth factor beta induced), CPA4 (carboxypeptidase A4), ADAM12 (ADAM metallopeptidase domain 12), ABI3BP (ABI family member 3 binding protein), PTX3 (pentraxin 3), PAMR1 (peptidase domain containing associated with muscle regeneration 1), LOX (lysyl oxidase), PLAU (plasminogen activator, urokinase), SNED1 (sushi, nidogen and EGF-like domains 1), SERPINE1 (serpin family E member 1), and BGN (biglycan) is included, it acts on wound healing and cell proliferation. When the formula contains at least one protein selected from the group consisting of SRGN (serglycin), COL6A3 (collagen type VI alpha 3 chain), COL5A3 (collagen type V alpha 3 chain), FN1 (fibronectin 1), COL1A1 (collagen type I alpha 1 chain), and LTBP2 (latent transforming growth factor beta binding protein 2), it induces angiogenesis and vascular protection, and is therefore useful for ischemia, blood flow disorders, and infarction.

[0134] In the present invention, it is preferable that the culture supernatant obtained when mesenchymal stem cells obtained by differentiating iPS cells are cultured contains at least one protein selected from the group consisting of HGF, FGF, SDF-1, PDGF, and DKK, and it is even more preferable that it contains HGF, FGF, SDF-1, PDGF, and DKK.

[0135] 4. Cosmetic Composition of the Present Invention The cosmetic composition containing the culture supernatant of the present invention provides cosmetic effects to the skin and hair. Skin includes all skin on the face, arms, and body. Hair includes all hair on the body, such as scalp hair, eyebrows, and eyelashes. The cosmetic effects obtained by the cosmetic composition of the present invention include a reduction in blemishes and wrinkles formed on the skin, and the provision of firmness and moisture to the skin. It is also effective in improving sagging and depressions caused by age-related changes, and is useful as a composition for improving wrinkles, sagging, or depressions. In addition, it is effective for anti-aging as it is also effective for dry eyes, dry mouth, and dry vagina, which are often problems after menopause in women. It is also useful for skin whitening, and is effective for dullness and pigmentation in the VIO area of ​​women, for example. For hair, it has a hair growth effect that keeps the scalp clean and provides an environment in which hair can grow thicker, longer, and stronger, and a hair regrowth effect that activates hair follicles and grows new hair on the scalp where hair has fallen out. Thus, the cosmetic composition according to the present invention is useful for both hair growth and hair restoration.

[0136] The amounts of iPS cells and the culture supernatant of cells obtained by differentiating iPS cells in the cosmetic composition of the present invention are not particularly limited and can be appropriately set according to the required storage stability and intended use.

[0137] The form of the cosmetic composition of the present invention is not particularly limited, but is preferably a form suitable for application to the skin or hair. For example, it may be a liquid or a solid. Liquid compositions may be in the form of lotions, foundations, serums, essences, sprays, creams, packs, pastes, gels, emulsions, powders, frozen products, or freeze-dried products.

[0138] In the cosmetic method of the present invention, the steps for applying the cosmetic composition to a target include applying the cosmetic composition to the target object, intradermal injection, subcutaneous injection, intravenous injection, intravenous drip infusion, or intranasal administration, but are not limited to these, and any general method of administering the cosmetic composition can be used.

[0139] The cosmetic composition of the present invention may contain any component in addition to the culture supernatant used in the present invention. Examples of optional components include preservatives, additives, emulsifiers, gelling agents, humectants, excipients, moisture inhibitors, strengtheners, thickeners, antioxidants, sweeteners, acidulants, seasonings, colorants, fragrances, whitening agents, moisturizers, oily components, UV absorbers, surfactants, alcohols, powder components, colorants, aqueous components, water, and skin nutrients. These may be used individually or in combination of two or more.

[0140] In particular, when the cosmetic composition of the present invention is used as a hair growth agent (for hair growth) and / or a hair regrowth agent (for hair regrowth), other ingredients to enhance the hair growth effect (such as finasteride, minoxidil, t-flavanone, ginseng extract, ginseng extract, swertia japonica extract, 6-benzylaminopurine, adenosine, ethinylestradiol, forskolin, cephalatin, pantothenyl ethyl ether, etc.) may be included, as well as diphenhydramine hydrochloride, glycyrrhetinic acid, hinokitiol, tocopherol acetate, dipotassium glycyrrhizinate, pyridoxine hydrochloride, resorcinol, menthol, 1,3-butylene glycol, ethanol, fragrance, and purified water.

[0141] When the cosmetic composition of the present invention is used as a hair growth agent and / or hair restoration agent, it is particularly preferable that the cells obtained by differentiating iPS cells are cardiomyocytes and / or mesenchymal stem cells.

[0142] 5. Pharmaceutical Composition of the Present Invention The pharmaceutical composition containing the culture supernatant of the present invention contains growth factors related to angiogenesis and wound healing, and therefore has the effect of inducing angiogenesis and healing wounds. In addition, it contains a large amount of molecules and hormones with anti-inflammatory effects and has an anti-inflammatory effect. Furthermore, it contains molecules that promote hair growth and is effective in treating thinning hair and hair loss (AGA: Androgenetic alopecia, male hormone type baldness).

[0143] The pharmaceutical composition containing the culture supernatant of the present invention is effective against metabolic diseases such as diabetes and non-alcoholic fatty liver disease (NASH) because it increases cell viability, promotes proliferation and repair, suppresses cell death, inhibits fatty acid uptake, and promotes the breakdown of lipid droplets. For the use of the pharmaceutical composition of the present invention in the treatment of metabolic diseases, it is particularly preferable that the differentiated cells derived from iPS cells are cardiomyocytes.

[0144] The pharmaceutical composition of the present invention is not particularly limited and may contain other active ingredients that can be included in pharmaceuticals, depending on the intended use.

[0145] The amounts of iPS cells and the culture supernatant of cells obtained by differentiating iPS cells in the pharmaceutical composition of the present invention are not particularly limited and can be appropriately set according to the required storage stability and intended use.

[0146] The administration method of the pharmaceutical composition of the present invention is not particularly limited, but examples include topical application, subcutaneous injection, and oral administration. The pharmaceutical composition of the present invention may be a liquid or a solid. The optimal form for forming the final product, such as a topical skin preparation, a beverage, or other pharmaceutical or quasi-drug or cosmetic product, can be arbitrarily selected. For example, it may be in the form of a liquid, suspension, emulsion, cream, ointment, gel, liniment, powder, granule, pill, lotion, or poultice and drink.

[0147] In the treatment of wounds, ischemia, blood flow disorders, and infarctions of the present invention, the steps for applying the culture supernatant of the present invention to the target include, but are not limited to, topical application, intradermal injection, subcutaneous injection, intravenous injection, intravenous infusion, and intranasal administration of the culture supernatant of the present invention to the target object. In general, methods of administering the pharmaceutical composition can be used.

[0148] When the pharmaceutical composition of the present invention is used to treat wounds, ischemia, blood flow disorders, or infarction, it is particularly preferable that the differentiated cells derived from iPS cells are cardiomyocytes and / or mesenchymal stem cells.

[0149] The pharmaceutical composition of the present invention may further contain other components, as long as they do not interfere with the above method. These other components are not particularly limited, but include, for example, carbohydrates such as glucose, maltose, sucrose, lactose, raffinose, trehalose, mannitol, hydroxyethyl starch, and pullulan; organic acids such as gluconic acid, lactic acid, acetic acid, propionic acid, β-hydroxybutyric acid, and citric acid; electrolytes such as sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and potassium carbonate; vitamins such as L-ascorbic acid and vitamin E; glycine, glutamic acid, and other nutrients. Examples of such substances include amino acids such as nic acid and lysine, hormones such as antidiuretic hormone and insulin, anticoagulants such as citric acid, citrate, heparin and sodium edetate, antihypertensive agents such as calcium channel blockers, adrenergic β-receptor antagonists and angiotensin-converting enzyme inhibitors, nucleic acid bases such as adenosine triphosphate, antifreeze agents such as freeze-proof proteins, reactive oxygen species scavengers, cell activators, antibiotics, antiplatelet factors, liver damage inhibitors, excipients, binders, disintegrants, dispersants, viscosity agents, reabsorption enhancers, surfactants, solubilizers, preservatives, antimicrobials, emulsifiers, isotonic agents, stabilizers, buffers, and pH adjusters, and at least one of these may be added as needed.

[0150] In particular, when the pharmaceutical composition of the present invention is used as a hair growth agent (for hair growth) and / or a hair regrowth agent (for hair regrowth), other ingredients to enhance the hair growth effect (such as finasteride, minoxidil, t-flavanone, ginseng extract, ginseng extract, swertia japonica extract, 6-benzylaminopurine, adenosine, ethinylestradiol, forskolin, cephalatin, pantothenyl ethyl ether, etc.) may be included, as well as diphenhydramine hydrochloride, glycyrrhetinic acid, hinokitiol, tocopherol acetate, dipotassium glycyrrhizinate, pyridoxine hydrochloride, resorcinol, menthol, 1,3-butylene glycol, ethanol, fragrance, and purified water.

[0151] In this invention, a wound refers to physical damage to the body surface tissue caused by external or internal factors, and includes abrasions, cuts, lacerations, splits, crush injuries, contusions, or puncture wounds. It also includes both acute and chronic wounds.

[0152] In this invention, ischemia refers to a condition in which blood flow becomes difficult, resulting in a decrease in blood flow from arteries to tissues and organs. Ischemic heart disease refers to a condition in which blood flow to the heart muscle is interrupted due to narrowing or blockage of the coronary arteries. Examples include, but are not limited to, angina pectoris and myocardial infarction.

[0153] In this invention, blood flow disorder refers to a condition in which blood flow is stagnant, and is synonymous with circulatory disorder or poor blood circulation. In blood flow disorder, blood does not flow properly within the blood vessels, resulting in insufficient supply of oxygen and nutrients to tissues and organs. Such blood flow disorders include, but are not limited to, arteriosclerosis, thrombosis, or angina pectoris.

[0154] In this invention, infarction refers to a disease in which a terminal artery or terminal vein is blocked, resulting in necrosis of the tissue supplying it. Examples of occlusion accompanied by tissue necrosis include, but are not limited to, myocardial infarction, cerebral infarction, ulcer, or necrosis.

[0155] In this invention, immune diseases include inflammatory diseases. The culture supernatant according to this invention contains proteins involved in anti-inflammatory effects and immune tolerance, and is therefore expected to have therapeutic effects against immune diseases and inflammatory diseases.

[0156] In this invention, immune tolerance refers to a state of absence or suppression of a specific immune response to a particular antigen, and means the suppression of immune responses such as allergies, autoimmune diseases, or immune responses due to transplantation, etc.

[0157] In this invention, periodontal tissue refers to the tissue surrounding the tooth that supports the function of the tooth, and includes the gingiva, cementum, periodontal ligament, and alveolar bone.

[0158] In this invention, metabolic diseases refer to any disease in which there is an abnormality in the body's metabolic function, such as type 2 diabetes and non-alcoholic fatty liver disease (NASH).

[0159] There are no particular limitations on the uses of the pharmaceutical and cosmetic compositions of the present invention, and they can be appropriately selected according to the purpose. For example, a wide range of uses are possible, such as pharmaceuticals, quasi-drugs, cosmetics, and food and beverages. For example, when a composition containing cell culture supernatant according to the present invention is used as a hair growth agent and / or hair regrowth agent, the hair growth agent and / or hair regrowth agent may be used in any of the following applications: pharmaceuticals, quasi-drugs, cosmetics, and food and beverages.

[0160] The culture supernatant according to this invention is not directly derived from the human body, but rather from cells cultured from human iPS cells; therefore, it is subject to appropriate scientific control and is highly safe.

[0161] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited in any way to the following examples.

[0162] Experiment 1. Preparation of Culture Supernatant (Example 1) Culture Supernatant of Human iPS Cells The culture supernatant of undifferentiated human iPS cells was prepared as follows. Human iPS cells (QHJI14s04) were seeded in a 10 cm dish and cultured at 37°C under 5% CO2 conditions. The day of seeding was designated as Day 0, and after culturing in StemFit medium for two days, the culture supernatant was collected and centrifuged, and the culture supernatant after centrifugation was stored at -30°C. Hereinafter, the culture supernatant obtained in this manner will be referred to as "iPSC Supernatant" (Example 1).

[0163] (Examples 2 and 3) Human iPS cell-derived cardiomyocytes (iPS-CMs) differentiated from human iPS cell lines were prepared as follows. iPS-CMs were differentiated according to Matsuura K, et al., Creation of human cardiac cell sheets using pluripotent stem cells. Biochem Biophys Res Commun, 2012, 425(2), pp.321-7. Briefly, human iPS cells (QHJI14s04) were seeded in a spheroid-forming culture vessel and cultured at 37°C under 5% CO2 conditions. As shown in Figure 1, the day of seeding was designated as Day 0, and differentiation into mesoderm was induced for 4 days in StemFit medium. Next, from Day 4 to Day 7, cardiac specification was initiated using differentiation induction medium supplemented with HSA, VEGF, SB431542, Dorsomorphin, and IWP-3. From Day 8 to Day 15, cardiomyocyte differentiation was induced using differentiation induction medium supplemented with HSA, VEGF, and bFGF. Finally, from Day 16 to Day 22, purified culture was performed to concentrate cells that had completed differentiation into cardiomyocytes using DMEM (glucose-free) medium supplemented with HSA, insulin, and lactate, and from Day 23 to Day 25, alpha-modified Eagle medium (α-MEM medium) supplemented with HSA and insulin. Medium changes were performed every two days from Day 8 to Day 15, and once a day from Day 16 to Day 25. The culture supernatant was collected during medium changes on Day 16 and Day 25. The collected culture supernatant was filtered using a 0.22 μm mesh filter and stored at -30°C. In this specification, the culture supernatants from Day 16 and Day 25 will be referred to as "iPSCM_Dd16" (Example 2; pre-purification myocardium) and "iPSCM_Pd9" (Example 3; post-purification myocardium), respectively. The myocardium from Day 4 to Day 7 will be referred to as "differentiated myocardium," the myocardium from Day 8 to Day 16 as "pre-purification myocardium," and the myocardium from Day 17 to Day 25 as "post-purification myocardium."

[0164] (Example 4) Human mesenchymal stem cells (iPS-MSCs) differentiated from human iPS cell lines were prepared as follows. First, iPS-MSCs were differentiated from human iPS cell lines (QHJI14s04) according to Qingguo Z, et al., MSCs derived from iPSCs with a modified protocol are tumor-tropic but have much less potential to promote tumors than bone marrow MSCs. Proc Natl Acad Sci, 2014, 112 (2), 530-535. Next, the mesenchymal stem cell expansion culture was carried out as follows. Frozen mesenchymal stem cells were thawed and placed in plastic culture vessels coated with gelatin solution (Nitta Gelatin, #GLS-250) in a 1.33 × 10⁶ container. 4 cells / cm 2 Seeds were seeded at the following density. The culture medium was changed with fresh medium the day after seeding. For the expansion culture of mesenchymal stem cells, α-MEM medium containing 10% fetal bovine serum and 2 ng / mL recombinant human basic fibroblast growth factor (bFGF) was used as the culture medium. The culture supernatant was collected from mesenchymal stem cells as follows: Mesenchymal cells after 3-4 days of expansion culture were washed with Ca, Mg-free Dulbecco's phosphate-buffered saline (D-PBS(-)) and detached from the culture vessel using recombinant cell dissociation enzyme CTS TrypLE Select Enzyme (ThermoFisher Scientific, #A1285901). The detached mesenchymal stem cells were collected by centrifugation and placed in a plastic culture vessel coated with gelatin solution at a concentration of 1.33 × 10⁶. 4 cells / cm 2 The seeds were sown at this density. The total culture medium volume was approximately 0.2 mL / cm³. 2 The culture medium was adjusted to achieve the following. Two days after seeding, the medium was replaced with fresh medium, and the culture supernatant was collected two to seven days later. Contaminants were removed from the cultured cells collected by centrifugation, and the supernatant was stored at -30°C. Hereinafter, the culture supernatant obtained in this manner will be referred to as "iPS-MSC supernatant" (Example 4).

[0165] <Bioassay> Experiment 2. Hair Growth Evaluation Experiment 2-1. Evaluation of iPS-CM culture supernatant on the proliferation of human dermal papilla cells Human dermal papilla cells were seeded in 24-well plates and cultured for 1 day at 37°C under 5% CO2. After removing the medium, the culture supernatant from Example 2 or 3 was diluted 2.5 times with α-MEM, or the medium of the positive or negative control was used for the medium change. As the positive control, the culture supernatant of MSCs derived from umbilical cord blood was used. After culturing for 3 days at 37°C under 5% CO2, the cells were fixed and stained with DAPI (4',6-diamidino-2-phenylindole). The number of stained cells in each well was then measured using a fluorescence microscope. The results are shown in Figure 2.

[0166] Compared to the positive and negative controls, the culture supernatants of Examples 2 and 3 showed significant proliferation of dermal papilla cells. The umbilical cord blood-derived MSC culture supernatant used as the positive control is already used in hair growth treatment at clinics. Compared to existing culture supernatants, the culture supernatant according to the present invention has a high proliferative effect on dermal papilla cells, and it has become clear that it has a superior effect on hair growth and / or hair restoration treatment compared to existing culture supernatants.

[0167] Experiment 2-2. In vivo hair growth test using mice Experiment 2-2-1. Test using myocardial culture supernatant The hair growth effect of the iPS-CM culture supernatant of the present invention was verified using 7-week-old mice (C57BL / 6, male). First, the hair on the back of the mice was removed using clippers or depilatory cream. Starting 1 day after depilation, the culture supernatants of Examples 2 and 3 were administered intradermally twice a week, with 50 μL of culture supernatant per injection at two locations per mouse. As a negative control, α-MEM medium was administered intradermally in the same manner as in the examples. As a positive control, 5% minoxidil was applied to the epidermis five times a week. The flowchart of the experiment is shown in Figure 3. Intradermal administration is considered to be a method similar to the papule method, one of the AGA treatment methods that injects the active ingredient between the epidermis and dermis. In addition, it is said that the active ingredient can be precisely targeted and injected into the desired area with a syringe, resulting in a high probability and certainty of effectiveness. The results are shown in Figure 4.

[0168] On the 10th day of administration, significant hair growth was observed in Example 3. Further, on the 14th day of administration, in both Example 2 and Example 3, good hair growth equivalent to or better than that of the positive control was observed. When the proteins contained in Example 2 and 3 were examined, surprisingly, predominant expression of KGF, IGF, and VEGF was observed. It is considered that the expression of KGF assisted in the production of keratin necessary for hair, supported hair follicles and hair roots, etc., and prepared an environment conducive to hair growth. IGF is thought to act on hair matrix cells and promote the division of hair matrix cells. It is thought that the expression of VEGF branched the blood vessels in the scalp that carry nutrients necessary for hair growth and / or hair nourishment, forming new blood vessels. Also, it is thought that VEGF improved blood flow. The fact that these three factors were contained in the culture supernatant is considered to support the observation of predominant hair growth in the mice administered with Example 2 and 3.

[0169] Experiment 2-2-2. Test Using MSC Culture Supernatant Using 7-week-old mice (C3H / He, male), the hair growth effect of the culture supernatant of iPS-MSC of the present invention was verified. Since the hair cycle of C3H / He mice is relatively clear, it has high versatility in research related to hair nourishment and / or hair growth. Also, since 7 weeks old corresponds to the telogen phase of the hair cycle, these mice are suitable for hair growth tests.

[0170] First, the hair on the back of the mice was removed using a clipper or depilatory cream. Starting from 1 day after hair removal, the culture supernatant of Example 4 was applied to the entire hair removal area (25mm x 4mm = 10cm 2 )5 times / week. The administration site is indicated by an arrow in Fig. 5 (A). As a negative control, Ringer's solution (Otsuka Pharmaceutical Factory) was applied to the entire hair removal area in the same manner as Example 4. As a positive control, 5% minoxidil was applied to the epidermis 5 times / week in the same manner as Example 4. The results are shown in Fig. 5.

[0171] On the 13th day of administration, significant hair growth was observed in Example 4 compared to the negative control. Further, on the 16th day of administration, significant hair growth equivalent to that of the positive control was observed in Example 4.

[0172] Experiment 2-3. Human Hair Growth Test Using the Culture Supernatant of iPS-CM

[0173] The culture supernatants of iPS cell-derived cardiomyocytes—differentiated cardiomyocytes (cardiomyocytes from Day 4 to Day 7), pre-purification cardiomyocytes (cardiomyocytes from Day 8 to Day 16), and post-purification cardiomyocytes (cardiomyocytes from Day 17 to Day 25)—were all mixed and concentrated 10-fold by ultrafiltration (Cytiva, Hollow fiber 10kD) (hereinafter referred to as "mixed culture supernatant 1") to obtain a test sample. A total of 1 mL of the test sample was injected into multiple locations on the scalp of the subject (human) for each treatment. The treatment was performed on the subject every three weeks. Before treatment, at 3 weeks (approximately 3 weeks after the first treatment), at 6 weeks (approximately 3 weeks after the second treatment), and at 9 weeks (approximately 3 weeks after the third treatment), the degree of hair growth was examined in four different locations (Frontal 1 left, Frontal 2 left, Temporal 1 right, and Temporal 2 right) on the same subject. As a representative result, the measurement results for "Frontal (frontal region) 1 left" are shown in Figures 6(A) to (C). In addition, the results for all four measured areas are shown in Figures 6(D) to (F).

[0174] Significant hair growth was observed at 9 weeks compared to before treatment (Figure 6(A)). From Figures 6(B) and (C), it was found that the number of vellus hairs / small terminals (lower values ​​in the bar graph) decreased, while the hair width and number of hairs in large, medium, and small terminals (upper values ​​in the bar graph) increased significantly. A trend of increased hair growth was observed in all measurement sites (Figures 6(D) to (F)).

[0175] Experiment 3. Evaluation of Metabolic Diseases Experiment 3-1. Evaluation of iPS-CM Culture Supernatant on Human Immortalized Hepatocyte Line (HepG2) Example 5, obtained by concentrating Example 2 (pre-purification cardiomyocyte) 10-fold by ultrafiltration (Milipore, Amicon Ultra-15 3kD), Example 6, obtained by concentrating Example 3 (purification cardiomyocyte) 10-fold by ultrafiltration (same method), and a mixture of Examples 5 and 6 (hereinafter referred to as "mixed culture supernatant 2") were used to evaluate the effect of iPS cell-derived cardiomyocyte culture supernatant on intracellular lipid accumulation. Ringer's solution (Otsuka Pharmaceutical Co., Ltd.) was used as a negative control. The tests were carried out according to 3-1-1 to 3-1-3 below.

[0176] 3-1-1. Pre-culture and seeding of intracellular fat accumulation-inducing human immortalized hepatocyte line HepG2 (obtained from Tohoku University, Medical Cell Resource Center) into 24-well plates (2.5 × 10⁻¹⁰ 5 Cells were cultured at 37°C under 5% CO2 conditions for 24 hours. 300 μL of Ringer's solution or culture supernatant (Examples 5, 6, or mixed culture supernatant 2) and 700 μL of RPMI1640 + 10% FBS were mixed and used as culture medium. The following day, the old medium was removed from each well, and 700 μL of PA / OA Mix (palmitic acid and oleic acid diluted with RPMI1640 + 1% hAlb + 10% FBS to a final concentration of 250 μM palmitic acid and 500 μM oleic acid, respectively) was added for sensitization culture (37°C, 5% CO2, 24 hours). 3-1-2. Staining of intracellular lipid droplets The PA / OA Mix was completely removed from the sensitized wells, 1 mL of 4% PFA was added, and the wells were allowed to stand at room temperature for 1 hour. The 4% PFA was removed, and the wells were washed with 1 mL of distilled water. 1 mL of oil red o staining solution was added to each well, and after gentle shaking, the mixture was allowed to react at room temperature for 30 minutes. The staining solution was removed and the mixture was washed three times with distilled water. If nuclear staining was to be performed afterward, 1 mL of hematoxylin solution was added to each well, and after standing at room temperature for 3 minutes, the mixture was washed three times with distilled water. 3-1-3. Elution of staining solution and measurement of absorbance Distilled water was completely removed from the wells after oil red o staining of the lipid droplets. 500 μL of 2-propanol was added to each well and allowed to stand for 5 minutes to elute the staining solution. The eluate was collected, thoroughly mixed, and then centrifuged (500xg, 3 min, 22°C). 200 μL of the supernatant was transferred to a 96-well plate and the absorbance at a wavelength of 490 nm was measured (Multiskan Sky™, thermoscientific).

[0177] The results are shown in Figure 7. Compared to the negative control, intracellular lipid accumulation was significantly suppressed when cultured using Example 5 and mixed culture supernatant 2 (Dunnett test). *** p<0.001, p<0.1) (Figure 7(A)). Micrographs of the wells after oil red staining of lipid droplets showed that when Examples 5, 6 and mixed culture supernatant 2 were added, the size and number of stained lipid droplets were suppressed compared to Ringer's solution (Figure 7(B)). This suggests that the culture supernatant of the present invention is useful for the treatment of non-alcoholic fatty liver disease (NASH). Possible mechanisms for suppressing intracellular lipid accumulation include the culture supernatant improving the viability of hepatocytes, promoting proliferation and repair, suppressing cell death, inhibiting fatty acid uptake, and promoting the degradation of lipid droplets.

[0178] Experiment 3-2. Evaluation of iPS-MSC culture supernatant in a mouse diabetes model. The effect of the culture supernatant of the present invention on diabetes was verified using 6-week-old db / db mice (male). These mice are available as a type 2 diabetes model mouse and are commonly used as a diabetic model animal for drug efficacy evaluation.

[0179] The flow of the experiment is shown in Figure 8(A). Mice were fasted overnight starting the day before administration. The blood glucose levels of the mice were measured the following morning, and they were divided into groups (n=6) so that the average blood glucose levels were uniform across all groups. The culture supernatants of differentiating myocardium (myocardium from Day 4 to Day 7), pre-purification myocardium (myocardium from Day 8 to 16), and post-purification myocardium (myocardium from Day 17 to 25) were all mixed and concentrated 10-fold by ultrafiltration (Cytiva, Hollow fiber 10kD) (same as "Mixed Culture Supernatant 1" in Experiment 2-3) and administered intravenously once a week at a dose of 200 μL each time for 8 weeks (total of 8 administrations). When administering the culture supernatant or positive control, the mice were fasted overnight starting the day before administration. The following morning, after administering the culture supernatant or positive control to the mice, blood was collected from the tail vein of the mice and blood glucose levels were measured. As a negative control, Ringer's solution (Otsuka Pharmaceutical Co., Ltd.) was administered in the same manner as in the example. As a positive control, dulaglutide (Trulicity, which lowers blood glucose levels by binding to the GLP-1 receptor), Eli Lilly, 1.5 mg / mL was administered subcutaneously twice a week at a dose of 200 μL each time. Body weight and food intake were also measured once a week. Eight weeks after the start of the study (the week following the eighth administration of culture supernatant), a glucose tolerance test and pathological specimen collection were performed as the final evaluation. The study was conducted according to sections 3-2-1 and 3-2-2 below.

[0180] 3-2-1. Measurement of Fasting Blood Glucose Blood was collected from the tail vein of mice that had been fasted overnight from the day before administration. The collected blood was subjected to an Accu-Chek ST meter (Roche DC Japan Co., Ltd.) fitted with a test strip (Accu-Chek ST strip) to measure blood glucose levels. Since the measurement range for blood glucose levels is 10 to 600 mg / dL, the measured value was set to 10 mg / dL if it was 10 mg / dL or less, and to 600 mg / dL if it was 600 mg / dL or more.

[0181] 3-2-2. Oral Glucose Tolerance Test (OGTT) Mice were fasted overnight starting the day before the glucose tolerance test, and their blood glucose levels were measured the following morning. This fasting blood glucose level was defined as the pre-glucose value. After measuring the pre-glucose value, 10% glucose was administered orally at a dose of 10 mL / kg (1 g / kg), and blood glucose levels were measured 0.5, 1, 2, 4, and 6 hours after administration. After measuring the blood glucose level 6 hours after administration, water and food were provided.

[0182] The results are shown in Figure 8. The time course of fasting blood glucose is shown in Figure 8(B). When subjected to Dunnet's test (n=6, *** p<0.001, ** p<0.01, * p<0.05), it was found that mixed culture supernatant 1 significantly suppressed fasting blood glucose levels compared to Ringer's solution at 5 weeks after administration (Figure 8(B)). In addition, in the oral glucose tolerance test, 3 out of 5 animals in the group administered Ringer's solution showed blood glucose levels above the detection limit (600 mg / dL) up to 4 hours from the start of the test. Compared to the group administered Ringer's solution, the group administered mixed culture supernatant 1 showed a significantly lower blood glucose level 2 hours after the start of the test (Figure 8(C)). These results suggest that the culture supernatant of the present invention is useful for the treatment and / or prevention of diabetes.

[0183] The results of experiments 3-1 and 3-2 clearly demonstrate that the culture supernatant of the present invention is useful for the treatment and / or prevention of metabolic diseases, particularly NASH and diabetes. This can be understood from the fact that IGF and other compounds are expressed in the culture supernatant of iPS-CM (Tables 4 and 7, etc.).

[0184] Experiment 4. Measurement of Exosome Amount in iPS-MSC Supernatant The amount of exosomes in the culture supernatant of iPS cells, umbilical cord blood, adipose tissue, and dental pulp-derived MSCs was measured. ELISA was used for detection, and CD63, an exosome marker, was used as the marker. The results are shown in Figure 9.

[0185] Compared to MSCs derived from umbilical cord blood, fat, and dental pulp, iPS-MSCs released significantly more exosomes from their culture supernatant. This result clearly indicates that iPS cell-derived MSCs have properties different from those of MSCs derived from other body tissues. In fact, differences in cell morphology during culture and the phenomenon of iPS-MSCs having a higher proliferation rate compared to other MSCs have been observed. Based on these experimental results, it is presumed that the properties of the iPS-MSCs of the present invention, obtained by differentiation induction from iPS cells for about 2 to 4 weeks, are closer to those of MSCs present in the fetal stage than to those present in the living bodies of adults and children. Since a large number of growth factors and exosomes involved in tissue formation are thought to be secreted during the fetal stage, it is thought that iPS cell-derived MSCs and other MSCs differ completely not only in cell properties but also in the properties of their culture supernatant and their usefulness when used for pharmaceutical and / or cosmetic purposes. In addition to differences in the properties of the culture supernatant itself, iPS-MSC cell supernatant is more effective for pharmaceutical and cosmetic applications due to its high exosome release rate, making it more advantageous than using culture supernatants derived from other cell types.

[0186] From the above results, it can be inferred that the fact that cells differentiated from iPS cells have different properties from other cells not differentiated from iPS cells is not limited to MSCs, but is also true for cardiomyocytes. The culture supernatant of iPS-CM and the culture supernatant of other cardiomyocytes have different properties, and the culture supernatant of iPS-CM contains a variety of useful substances, making it highly useful for use in pharmaceutical and / or cosmetic compositions.

[0187] Experiment 5. Evaluation of iPS-CM culture supernatant on human fibroblast proliferation. In the present invention, fibroblast proliferation ability means that a culture supernatant containing at least one protein selected from the group consisting of PDGF, TGF-β, CSF, MMP, TIMP, VEGF, FGF, KGF, IGF, GDF, ANGPT, and HGF promotes fibroblast proliferation through its fibroblast proliferation effect, thereby enhancing skin firmness and elasticity and mitigating damage such as epidermal damage. Furthermore, the culture supernatant of the present invention can be used in all applications where exhibiting fibroblast proliferation effect is significant, in addition to these applications.

[0188] To investigate the effects of the culture supernatant of human iPS cell-derived cardiomyocytes, in vitro cell proliferation tests were performed using Examples 2 and 3, the culture supernatant of Example 2 concentrated 10-fold by ultrafiltration (Milipore, Amicon Ultra-15 3kD) (Example 5), and the culture supernatant of Example 3 concentrated 10-fold by ultrafiltration (same method) (Example 6). α-MEM and DMEM were used as negative control groups. A medium suitable for fibroblast proliferation (PromoCell, Fibroblast Media 2) was used as a positive control group.

[0189] Human normal dermal fibroblasts (NHDF) were seeded in a 96-well plate and stored at 37°C in 5% CO2. ２ Cells were cultured as shown below. Day 1 was defined as one day after seeding, and on Day 1, the culture medium was changed with the supernatant from Examples 2, 3, 5, and 6, or with the positive and negative control media, respectively. Four similar plates were prepared, and one plate was collected each day to measure the number of cells in each well over four days. A kit (CyQUANT Cell Proliferation Assays) that indirectly measures cell number by measuring the amount of DNA in the wells was used for measurement. The number of times the cell number increased at each sampling point, with the number of cells on Day 1 set to 1, was plotted against the number of culture days (Figure 10).

[0190] Figure 10 shows that when Examples 2 and 3 were added, significantly higher fibroblast proliferation was observed compared to when the negative control medium was added. Surprisingly, this level was equivalent to or greater than that of Fibroblast media 2, a medium suitable for fibroblast proliferation. When the expression of FGF in Examples 2 and 3 was examined at this time (Figure 11), it was revealed that FGF was present in Example 2 (D16) and Example 3 (P9). It is thought that the FGF contained in the culture supernatant promoted fibroblast proliferation. Furthermore, as shown in Examples 5 and 6 in Figure 10, it was revealed that the degree of fibroblast proliferation was further increased by concentrating the culture supernatant. The reason why the activity increased in the concentrated culture supernatant is presumed to be that factors useful for fibroblast proliferation contained in the culture supernatant were concentrated, while unnecessary substances such as waste products were removed.

[0191] Experiment 6. Evaluation of iPS-MSC culture supernatant on human fibroblast proliferation. To investigate the effect of culture supernatants of human iPS cell-derived MSCs and somatic cell-derived MSCs on cell proliferation, in vitro cell proliferation tests were performed using the culture supernatants of umbilical cord blood-derived MSCs and dental pulp-derived MSCs from Example 4. DMEM was used as a negative control. A medium suitable for fibroblast proliferation (PromoCell, Fibroblast Media 2) was used as a positive control. Human normal dermal fibroblasts (NHDF) were seeded in 96-well plates and cultured at 37°C and 5% CO2. ２ Cells were cultured as shown below. Day 1 was defined as one day after seeding. On Day 1, the culture medium was changed with iPS cells, umbilical cord blood, supernatant of dental pulp-derived MSCs, or a positive control (Fibroblast Media 2) or negative control (DMEM). One identical plate was prepared, and each plate was collected on Day 1 and Day 4. To measure the number of cells in each well, a kit that indirectly measures cell count by measuring the amount of DNA in the well (CyQUANT Cell Proliferation Assays) was used. The graph shows how many times the cell number increased at each sampling time, with the cell number on Day 1 set to 1 (Figure 12).

[0192] Figure 12 shows that when MSC supernatant was added, significantly higher fibroblast proliferation was observed compared to when the negative control medium was added. Surprisingly, the proliferation was significantly higher when iPS-MSC culture supernatant was added compared to when umbilical cord blood and dental pulp-derived MSC culture supernatant was added. The reason for the significantly higher proliferation observed in iPS-MSC culture supernatant is presumed to be the presence of factors useful for fibroblast proliferation in iPS-MSC culture supernatant, as shown in Tables 9 and 10, and the high exosome content, as shown in Figure 9.

[0193] Experiment 7-1. Evaluation of iPS-CM culture supernatant against the migratory activity of human fibroblasts (wound-healing assay) A wound-healing assay was performed on Examples 2 and 3 to investigate the possibility of wound healing. Human normal dermal fibroblasts (NHDF) were seeded in a 48-well plate, and the next day the medium was changed to mitomycin C-containing medium and incubated for 2 hours to stop growth. Then, a scratch was created on the culture surface by scraping it with a pipette tip of a micropipette P1000. The medium was changed with the culture supernatant of Example 5 or 6 (a 10-fold concentrated version of the culture supernatant from Example 2 or 3), and cell migration was observed for 2 days. For measurement, time-lapse imaging was performed using a microscope (Keyence BZ-X800), and the cell occlusion rate was calculated from the obtained images. α-MEM (iPSCM_Pd9; control for Example 3) and DMEM (iPSCM_Dd16; control for Example 2) were used as negative controls, and Fibroblast media 2 was used as a positive control. The results are shown in Figure 13.

[0194] When the culture supernatants from Examples 5 and 6 were used, the increase in occlusion rate was significantly faster compared to the negative control. The occlusion rate of the positive control was measured on a separate day, and the occlusion rate of the positive control after 2 days was approximately 20% (data unavailable). Therefore, it was found that Examples 5 and 6 exhibited higher cell migration activity than the positive control.

[0195] Experiment 7-2. Evaluation of iPS-CM culture supernatant against the migratory activity of human keratinocyte cell lines (wound-healing assay) Enhanced migratory and proliferative capacity of keratinocytes (keratinocytes) helps maintain the skin's turnover cycle and plays a crucial role in maintaining the skin's barrier function. Keratinocytes, along with hair matrix cells, are known to be involved in hair growth. Furthermore, keratinocytes are known to be involved in immunity. Their main role is to produce and secrete various cytokines, thereby promoting the activation of immune cells.

[0196] The epidermis is composed of various types of cells, but keratinocytes make up the majority. As these keratinocytes proliferate and migrate to the upper layers, they differentiate and ultimately form the stratum corneum. Epidermal cell adhesion plays a crucial role in the normal differentiation of the epidermis and the maintenance of the stratum corneum. The migratory properties of keratinocytes influence epidermal cell adhesion. Furthermore, keratinocyte migration is important in the wound healing process, particularly in terms of enhanced cell migration.

[0197] In this invention, the migratory and proliferative ability of keratinocytes means promoting the migration and proliferation of keratinocytes, enhancing epidermal metabolism, accelerating epidermal wound healing, and having a hair growth effect. Furthermore, the keratinocyte migration and proliferation promoting agent of this invention can be used in all applications where exhibiting keratinocyte migration and proliferation activity is significant, in addition to the applications mentioned above.

[0198] A wound-healing assay was performed using the keratinocyte line (PHK16-0b) in the same manner as in Experiment 7-1. The procedure was the same as in Experiment 7-1, except that the culture supernatant from Example 2 or 3, and the culture supernatant from Example 7 (obtained by concentrating the culture supernatant from Example 2 20-fold through an ultrafiltration membrane (Milipore, Amicon Ultra-15 3kD) or the culture supernatant from Example 8 (obtained by concentrating the culture supernatant from Example 3 20-fold through an ultrafiltration membrane) were used. A medium containing 100 ng / mL of EGF in α-MEM was used as a positive control. The results are shown in Figure 14.

[0199] Figure 14(A) shows the results using the culture supernatant of unpurified cardiomyocytes (iPSCM-Dd16). Figure 14(B) shows the results using the culture supernatant of purified cardiomyocytes (iPSCM-Pd9). These results clearly show that the culture supernatant of iPS-CMs has migratory activity toward human keratinocytes. Furthermore, since Examples 7 and 8 (shown as dark squares in Figure 14) showed higher activity than Examples 2 and 3 (shown as dark circles in Figure 14), it was found that this activity can be concentrated. Comparing the results of Examples 2 and 3, the culture supernatant of purified cardiomyocytes (Figure 14(B)) showed higher migratory activity without concentration of the culture supernatant.

[0200] The results above show that when human fibroblasts and human keratinocyte cell lines are treated with the culture supernatant of iPS-CM differentiated from human iPS cells, their migratory activity is significantly improved. Furthermore, it was found that concentrating the culture supernatant further enhances migratory activity. This improvement in migratory activity indicates that this culture supernatant has excellent efficacy in wound healing.

[0201] Experiment 8. Evaluation of angiogenesis using culture supernatants of iPS-CM and iPS-MSCs. 125 μL of pre-thawed Matrigel (Corning) was added to a 48-well plate and allowed to stand at 37°C for at least 30 minutes to solidify the gel. Then, 125 μL each of the culture supernatants of HuMedia-EG2 (positive control, Kurabo), Ringer's solution (negative control, Otsuka Pharmaceutical Co., Ltd.), differentiated cardiomyocytes (cardiomyocytes from Day 4 to Day 7), pre-purification cardiomyocytes (cardiomyocytes from Day 8 to Day 16), and purified cardiomyocytes (cardiomyocytes from Day 17 to Day 25) were mixed and concentrated 10-fold by ultrafiltration (Cytiva, Hollow fiber 10kD) (same as "Mixed Culture Supernatant 1" in Experiment 3-2) and added. Human umbilical vein endothelial cells (HUVEC) (Kurabo) were seeded (5.0 × 10⁶) onto the sample and Matrigel. 4Cells were cultured at 37°C under 5% CO2 conditions for 24 hours. The following day, the culture medium was removed from the wells, 250 μL of staining solution (calcein AM, Dojin Chemical) was added, and the mixture was incubated at 37°C for 30 minutes. After incubation, the staining solution was removed, and 250 μL of fresh culture medium was added. Images were taken using a fluorescence microscope (Keyence BZ-X800), and the cell area ratio was calculated from the obtained images.

[0202] Angiogenesis was observed in all groups to which the culture supernatant was added (Figure 15(A)). The cell area ratio in the groups to which the culture supernatant was added was also higher than that of the negative control, indicating that there were cells equal to or greater than those in the positive control (Figure 15(B)). These results confirm that the culture supernatant of the present invention has an angiogenic effect.

[0203] A pharmaceutical or cosmetic composition containing the culture supernatant of iPS cells according to the present invention preferably contains at least one protein selected from the group consisting of VGF, FGF, PDGF, BMP, and TGF-α. It will be understood that these proteins are selected based on the results of gene expression analysis of the iPSC culture supernatant described later. A pharmaceutical or cosmetic composition containing the culture supernatant of cardiomyocytes obtained by differentiation induction from iPS cells according to the present invention preferably contains at least one protein selected from the group consisting of PDGF, TGF-β, CSF, MMP, TIMP, VEGF, FGF, KGF, IGF, GDF, ANGPT, BMP, SFRP, ELN, COL, IL, CLEC, INHBA, DKK, SCG, GRN, HDGF, GMFB, EFEMP, FN, SPARC, KRT, THBS, IGFBP, PCOLCE, VCAN, LGALS3BP, SDF-1, and NTF3, and more preferably contains at least one protein selected from the group consisting of FGF, KGF, IGF, VEGF, and DKK. It will be understood that these proteins are selected based on the hair growth promotion and cell migration activation demonstrated in the present examples, the pharmacological effects on cardiomyocytes (no data available), and the results of the proteomic analysis of the culture supernatant of iPS-CM described later. The pharmaceutical or cosmetic composition according to the present invention, which contains the culture supernatant of mesenchymal stem cells obtained by differentiation induction from iPS cells, preferably contains at least one protein selected from the group consisting of PDGF, TGF-β, CSF, MMP, TIMP, VEGF, HGF, FGF, IGF, GDF, ANGPT, BMP, SFRP, COL, IL, CLEC, INHBA, DKK, SCG, GRN, HDGF, PDGF, GMFB, EFEMP, FN, SPARC, KRT, THBS, IGFBP, PCOLCE, VCAN, LGALS3BP, and SDF-1, and more preferably contains at least one protein selected from the group consisting of HGF, FGF, SDF-1, PDGF, and DKK.The pharmaceutical or cosmetic composition according to the present invention, which contains the culture supernatant of nervous system cells obtained by differentiation induction from iPS cells, preferably contains at least one protein selected from the group consisting of FGF, PDGF, TIMP, MMP, GDF, NELL, PTPRZ1, SEMA3A, NCAM1, DCC, NTN1, and IL6ST, selected based on the characteristics of nervous system cells differentiated from iPS cells, and more preferably contains at least one protein selected from the group consisting of FGF, PDGF, TIMP, MMP, and GDF. It is also preferable to contain the factors detected in Tables 11 to 13 described later.

[0204] <Mechanism Analysis> <microRNA Seq Analysis> Experiment 9. Secretome Analysis in Extracellular Vesicles Extracellular vesicles (EVs) are vesicles with a diameter of about 100 nm secreted by various types of cells and are abundant in the culture supernatant. Surrounded by a lipid bilayer, they contain many signaling molecules such as mRNA, microRNA (miRNA), and proteins, and play a role in intercellular communication by binding to other cells and tissues. miRNA is a short, single-stranded RNA of about 22 nucleotides, and there are about 2500 types of miRNA in the human body, most of which regulate gene expression in the cytoplasm. Extracellular vesicles (EVs) were extracted from the culture supernatant of (1) iPSC supernatant, (2) differentiated cardiomyocyte (iPSCM_Dd16) and purified cardiomyocyte (iPSCM_Pd9), and (3) iPS-MSC supernatant prepared above, and miRNA expression analysis was performed by small RNA-seq. The results are shown in Table 1.

[0205]

[0206] As shown in Table 1, miRNAs were most specific to the culture supernatant of iPS cells (196 types), while some were common across cell types (53 types). Among these, 11 types specific to the culture supernatant of iPS-CM (iPS cell-derived cardiomyocytes) were selected and summarized in Table 2 below along with their functions. The iPS-CM according to the present invention is characterized by expressing these miRNAs.

[0207]

[0208] Next, Table 3 shows the top 30 miRNAs in the EVs contained in the culture supernatants of iPS cells (Example 1), iPS-CMs (Examples 2 and 3), and iPS-MSCs (Example 4). The culture supernatants of cells containing iPS cells or differentiated cells derived from iPS cells according to the present invention are characterized by expressing these miRNAs.

[0209]

[0210] <Gene Expression Analysis (RNA-seq)> Experiment 10. Expression Profiles of Humoral Factors and Extracellular Matrix-Related Molecules The top 20 genes in mRNA expression of molecules related to humoral factors and extracellular matrix were summarized in Tables 4 and 5 below for the iPSC supernatant, iPSCM_Dd16, iPSCM_Pd9, and iPS-MSC supernatant prepared above.

[0211]

[0212]

[0213] Experiment 11. GO analysis of humoral factors and extracellular matrix showing above-average expression (GO cluster analysis of the top 20 significantly correlated genes) Next, GO analysis (cluster analysis of the top 20 significantly correlated GOs) was performed on the above top 20 genes for humoral factors showing above-average expression and extracellular matrix-related genes showing above-average expression. The results are shown in Figures 16 and 17, respectively.

[0214] The results for humoral factors (Figure 16) showed significant enrichment of gene ontology, suggesting differences in pharmacological effects among the different cell types from which the culture supernatant originated. Different changes in immune system molecules were observed in MSCs and cardiomyocytes. In cardiomyocytes, molecules related to tissue formation and repair were predominantly elevated. No significant enrichment of gene ontology was detected in iPSCs. Regarding extracellular matrix-related genes (Figure 17), it was difficult to detect significant enrichment in cell types other than iPS-MSCs due to the high number of expressed genes.

[0215] Experiment 12. GO analysis of humoral factors and extracellular matrix showing above-average expression (PPI enrichment analysis) In the GO analysis of humoral factors showing above-average expression, an enrichment analysis of protein-protein interactions (PPIs) was performed. Table 6 shows the top three ontologities of the top three genes constituting the top three PPI networks. Expression of genes involved in cell proliferation and tissue repair was observed in Examples 2 and 3. It is thought that the effects shown in bioassay experiments 5-8 were obtained due to the presence of these factors. Expression of factors involved in organogenesis was also observed in Example 2. Expression of genes involved in immune tolerance, anti-inflammatory effects, extracellular matrix modeling, and tissue repair was observed in Example 4.

[0216]

[0217] <Proteome Analysis> Experiment 13. Proteome Analysis of iPS-CM Culture Supernatant The culture supernatant obtained during the differentiation induction process from iPS cells to iPS-CM was sampled in four stages, as shown in Figure 1, and proteome analysis was performed on each sample. Samples were prepared using the following procedure for proteome analysis. Step 13-1.) First, to remove albumin, 800 μL of Binding / Wash Buffer and 80 μL of ProMax Albumin Removal Particles were added to 200 μL of sample (using the ProMax Albumin Removal Kit (Polysciences)), and after stirring at room temperature for 20 minutes, the beads were washed three times with 500 μL of Binding / Wash Buffer. Then, 100 mM Tris pH 8.0, 4% SDS, 20 mM NaCl, and 10% ACN were added to the proteins bound to the beads, and after stirring at room temperature for 10 minutes, the supernatant was collected. The protein concentration was measured by a BCA assay and adjusted to a protein concentration of 0.2 μg / μL with 100 mM Tris pH 8.0, 4% SDS, 20 mM NaCl, and 10% ACN. 20 μL of SP3 beads (using Cytiva's Sera-Mag) were added to the protein lysis solution (20 μg of protein), and then three times the volume of 1-propanol was added. The mixture was then mixed at room temperature for 20 minutes. After washing the beads twice with 1-propanol and once with ethanol, 500 ng of Trypsin / Lys-C Mix (Promega) was added to fragment the protein into peptides, and the mixture was incubated overnight at 37°C. (Step 13-2.) To cleave the SS bonds of the protein and alkylate it, the digested sample was added to a final concentration of 10 mM TCEP and 40 mM 2-chloroacetamide, incubated at 80°C for 15 minutes, then 16 μL of 5% TFA was added and the mixture was stirred. After desalting using a reversed-phase spin column (GL-Tip SDB, GL Sciences), the peptide was dried using a centrifugal evaporator and then dissolved by adding 0.1% TFA-0.01% DMNG.The peptide concentration was adjusted to 100 ng / μL using 0.1% TFA-0.01% DMNG, and the proteins in the supernatant were detected and proteomic analysis was performed using the UltiMate 3000 RSLC nano LC System (Thermo Fisher Scientific).

[0218] Data was obtained for each sample (N=4). Clustering was performed on the proteins (4,684) whose expression was detected in at least one of the 16 samples, and a heatmap was created (Figure 18). From the results in Figure 18, it was considered that the proteins contained in the culture supernatant could be broadly divided into four groups. This also correlated with the number of differentiation induction stages being four, and it was determined that there are four groups in the culture supernatant samples.

[0219] Next, for the four populations obtained above, we identified growth factors, cytokines, cardiac differentiation, and factors contributing to the extracellular matrix (ECM) from among those expressed in multiple populations. The growth factors are shown in Table 7, and the cytokines, myocardial differentiation, and ECM are shown in Table 8. From these results, the culture supernatant of iPS-CM contained the following factors in addition to myocardial-specific factors. As shown in Tables 7-8, the culture supernatant contains factors that promote angiogenesis and is considered useful for wound healing. Furthermore, since many proteins involved in the ECM were present, cosmetic effects such as improving skin hydration and suppressing wrinkles can be obtained, making it effective for anti-aging. Surprisingly, it was also revealed that the culture supernatant of myocardial cells contains factors involved in hair growth. It is thought that the effects shown in bioassay experiments 2-3 were obtained due to the presence of these factors.

[0220]

[0221]

[0222] Experiment 14. Proteomic analysis of iPS-MSC culture supernatant. Culture supernatant obtained during the differentiation induction process from iPS cells to iPS-MSCs was sampled on day 2 of culture, and proteomic analysis of the sample was performed. Step 14-1.) The sample was subjected to the same procedure as in Step 13-1. of Experiment 13 to obtain protein peptide fragments. Step 14-2.) 20 μL of 5% TFA was added to the obtained peptide fragments, and after processing with a closed-type ultrasonic shredder, desalting was performed using a reverse-phase spin column, followed by drying in a centrifugal evaporator. 2% ACN-0.1% TFA was added to dissolve the peptides from the closed-type ultrasonic shredder. The peptide concentration was adjusted to 100 ng / μL using 0.1% TFA-0.01% DMNG, and proteins in the supernatant were detected and proteomic analysis was performed using the UltiMate 3000 RSLC nano LC System (Thermo Fisher Scientific). Data was obtained for each sample (N=4). The top 25 and 30 growth factors, including cytokines, and the top 30 total proteins, including ECM, were identified. The growth factors are shown in Table 9, and the total proteins are shown in Table 10.

[0223] As shown in Tables 9-10, the culture supernatant of iPS-MSCs contains factors that promote angiogenesis and are therefore considered useful for wound healing. Furthermore, the presence of numerous proteins involved in the extracellular matrix (ECM) suggests that it may have cosmetic effects such as improving skin hydration and suppressing wrinkles, making it effective for anti-aging. The presence of the skin whitening factor DKK1 also suggests that it may be useful for skin whitening.

[0224]

[0225]

[0226] Experiment 15. ELISA analysis of iPS-MSC culture supernatant. iPS-MSCs obtained by differentiation induction from iPS cells were seeded in a 6-well plate and cultured for 1 day at 37°C under 5% CO2, after which the culture medium was changed. Culture was then continued at 37°C under 5% CO2, and the culture supernatant was sampled every two days. The amount of HGF contained in the sample was measured using an ELISA kit (R&D systems, Human HGF ELISA Kit - Quantikine). The results are shown in Figure 19.

[0227] As is clear from Figure 19, HGF was found to be present in the culture supernatant of iPS-MSCs, and it was also found that the amount increased over time.

[0228] Experiment 16. Proteomic analysis of iPS-MSC culture supernatant Differentiated iPS cell-derived neural stem cells (iPS-NSCs) and neural progenitor cells (iPS-NSPCs), obtained during the differentiation induction of iPS cells into nervous system cells, were cultured in Neurobasal medium supplemented with N2B27. Sampling was performed on the second day of culture, and the collected culture supernatant was subjected to proteomic analysis of the sample. Step 16-1.) The sample was subjected to the same procedure as in Step 13-1. of Experiment 13 to obtain protein peptide fragments. Step 16-2.) 20 μL of 5% TFA was added to the obtained peptide fragments, and after processing with a closed-type ultrasonic shredder, desalting was performed using a reverse-phase spin column, followed by drying with a centrifugal evaporator. 2% ACN-0.1% TFA was added to dissolve the peptides from the closed-type ultrasonic shredder. The peptide concentration was adjusted to 100 ng / μL using 0.1% TFA-0.01% DMNG, and proteins in the supernatant were detected and proteomic analysis was performed using the UltiMate 3000 RSLC nano LC System (Thermo Fisher Scientific). Data was obtained for each sample (N=4). The top 25 and 30 factors for growth factors, including cytokines, and total protein, including ECM, were identified and are shown in Table 11 for growth factors and Table 12 for total protein. Factors related to nervous system function were also extracted and are shown in Table 13.

[0229] As shown in Tables 11-12, the culture supernatant of iPS cell-derived nervous system cells contained factors that promote angiogenesis, as well as MMPs and TIMPs. Therefore, it can be said that it has cosmetic effects such as improving skin hydration and suppressing wrinkles, making it effective for anti-aging. Furthermore, as shown in Table 13, many factors related to nerve development and neurological diseases were extracted from the supernatants of iPS-NSCs and iPS-NSPCs, suggesting their usefulness in treating these conditions. In particular, FGF13 (FGF13 is reduced in the brains of mouse models and patients with Alzheimer's disease, and overexpression of FGF13 is known to improve nerve cell damage and enhance learning and memory abilities), MANF (neurotrophic factor; MANF levels in the brain decrease with age, and MANF deficiency is known to impair spatial learning and memory abilities), and NCAM (known to be involved in nerve cell adhesion, axon growth, synaptic plasticity, learning, and memory) were extracted. Therefore, the culture supernatant of iPS cell-derived nervous system cells of the present invention is considered effective as an anti-aging composition for the brain and nervous system.

[0230]

[0231]

[0232]

Claims

1. A pharmaceutical or cosmetic composition comprising the culture supernatant of cells including iPS cells or differentiated cells derived from iPS cells.

2. The composition according to claim 1, wherein the differentiated cells are at least one cell selected from the group consisting of cardiomyocytes, nerve cells, mesenchymal stem cells, and hepatocytes.

3. The composition according to claim 1, wherein the iPS cells are of human origin.

4. The composition according to claim 1, used for the treatment of wounds, ischemia, blood flow disorders, infarction, osteoporosis, immune diseases, neurological diseases, metabolic diseases, periodontitis, or regeneration of periodontal tissue.

5. The composition according to claim 4, wherein the ischemia, the impaired blood flow, or the infarction is arteriosclerosis, thrombosis, angina pectoris, myocardial infarction, cerebral infarction, ulcer, or necrosis.

6. The composition according to claim 5, wherein the differentiated cells are cardiomyocytes and / or mesenchymal stem cells.

7. The composition according to claim 4, wherein the metabolic disease is diabetes mellitus or non-alcoholic fatty liver disease.

8. The composition according to claim 7, wherein the differentiated cells are cardiomyocytes.

9. The composition according to claim 1, which is used for anti-inflammatory and / or immune tolerance.

10. The composition according to claim 9, wherein the differentiated cells are mesenchymal stem cells.

11. The composition according to claim 1, which is used for skin whitening, improvement of blemishes, wrinkles, sagging or depressions, or for hair growth or hair regeneration.

12. The composition according to claim 11, wherein the differentiated cells are cardiomyocytes and / or mesenchymal stem cells, and are for hair growth or hair regeneration.

13. The composition according to claim 11, wherein the differentiated cells are mesenchymal stem cells and are for the improvement of wrinkles, sagging, or depressions.

14. The composition according to claim 1, wherein the differentiated cells are nerve cells and are for anti-aging of the brain and nervous system.

15. The composition according to claim 1, wherein the culture supernatant of the iPS cells contains at least one protein selected from the group consisting of VGF, FGF, PDGF, BMP, and TGF-α.

16. The composition according to claim 1, wherein the differentiated cells derived from iPS cells are cardiomyocytes, and the culture supernatant contains at least one protein selected from the group consisting of FGF, KGF, IGF, VEGF, and DKK.

17. The composition according to claim 1, wherein the differentiated cells are mesenchymal stem cells, and the culture supernatant contains at least one protein selected from the group consisting of HGF, FGF, SDF-1, PDGF, and DKK.

18. The composition according to claim 1, wherein the differentiated cells are nerve cells, and the culture supernatant contains at least one protein selected from the group consisting of FGF, PDGF, TIMP, MMP, and GDF.

19. The composition according to any one of claims 15 to 18, wherein the culture supernatant is concentrated with the protein.

20. The composition according to claim 1, wherein the culture supernatant of the iPS cells contains at least one microRNA selected from the group consisting of miR-21-5p, miR-182-5p, miR-148b-3p, miR-7-5p, and miR-378a-3p.

21. The composition according to claim 1, wherein the differentiated cells are cardiomyocytes, and the culture supernatant contains at least one microRNA selected from the group consisting of miR-1-3p, miR-143-3p, miR-21-5p, miR-218-5p, and miR-30a-5p.

22. The composition according to claim 1, wherein the differentiated cells are mesenchymal stem cells, and the culture supernatant contains at least one microRNA selected from the group consisting of miR-100-5p, miR-21-5p, miR-143-3p, miR-30a-5p, and miR-27a-3p.

23. The composition according to claim 1, wherein the culture supernatant of the iPS cells contains VGF, FGF, PDGF, BMP, TGF-α, miR-21-5p, miR-182-5p, miR-148b-3p, miR-7-5p, and miR-378a-3p.

24. The composition according to claim 1, wherein the differentiated cells are cardiomyocytes, and the culture supernatant contains FGF, KGF, IGF, VEGF, DKK, miR-1-3p, miR-143-3p, miR-21-5p, miR-218-5p, and miR-30a-5p.

25. The composition according to claim 1, wherein the differentiated cells are mesenchymal stem cells, and the culture supernatant contains HGF, FGF, SDF-1, PDGF, DKK, miR-100-5p, miR-21-5p, miR-143-3p, miR-30a-5p, and miR-27a-3p.

26. The composition according to claim 1, wherein the differentiated cells are nerve cells, and the culture supernatant contains FGF, PDGF, TIMP, MMP, and GDF.

27. The composition according to claim 1, wherein the culture supernatant is obtained by culturing cells in serum-free medium and / or does not contain any animal-derived components.

Images