Pharmaceutical composition and cosmetic composition

Abstract

Provided are a pharmaceutical composition or a pharmaceutical composition starting material comprising a supernatant of a culture medium after induced culture of pluripotent stem cells.

Description

[0001] This application is a divisional application of an application with an international application date of October 4, 2019, an international application number of PCT / JP2019 / 039396, an application number entering China of 201980073251.3, and an invention name of “Pharmaceutical Compositions and Cosmetic Compositions”. Technical Field

[0002] The present invention relates to a pharmaceutical composition and a cosmetic composition. Background Art

[0003] Embryonic stem cells (ES cells) are stem cells established from early human or mouse embryos. ES cells have the pluripotency to differentiate into all cells existing in the body. Human ES cells can now be used in cell transplantation therapies for many diseases such as Parkinson's disease, juvenile diabetes and leukemia. However, there are obstacles in the transplantation of ES cells. In particular, the transplantation of ES cells may cause the same immune rejection reaction as the rejection reaction caused by unsuccessful organ transplantation. In addition, there are many criticisms or objections to the use of ES cells established by destroying human embryos from an ethical point of view.

[0004] Under such circumstances, Professor Shinya Yamanaka of Kyoto University successfully established induced pluripotent stem cells (iPS cells) by introducing four genes: OCT3 / 4, KLF4, c-MYC and SOX2 into somatic cells. As a result, Professor Yamanaka won the 2012 Nobel Prize in Physiology or Medicine (see, for example, Patent Document 1). iPS cells are ideal pluripotent cells without rejection reactions and ethical issues. Therefore, it is expected that iPS cells will be used in cell transplantation therapy. On the other hand, there are reports that the culture medium used for iPS cell culture is reused in pharmaceutical compositions (see, for example, Patent Document 2).

[0005] Prior art literature Patent Literature Patent Document 1: Japanese Patent No. 4183742 Patent Document 2: Japanese Patent Application Publication No. 2016-128396 Summary of the invention

[0006] Technical issues However, the present inventors have verified that, in the culture method described in Patent Document 2, since iPS cells differentiate, the medium actually reused is not the medium after culturing iPS cells but the medium after culturing differentiated cells. One of the objects of the present invention is to provide a pharmaceutical composition and a cosmetic composition that effectively utilize the culture medium of iPS cells.

[0007] Technical Solution According to an embodiment of the present invention, there is provided a pharmaceutical composition or a pharmaceutical composition raw material, which includes a supernatant of a culture medium used for reprogramming somatic cells.

[0008] According to an embodiment of the present invention, there is provided an agent for preventing or ameliorating the formation of any one of spots, wrinkles, and sagging of the skin, comprising the above-mentioned pharmaceutical composition or pharmaceutical composition raw material.

[0009] According to an embodiment of the present invention, a cosmetic composition or a cosmetic composition raw material including a supernatant of a culture medium used when reprogramming somatic cells is provided.

[0010] According to an embodiment of the present invention, there is provided an agent for preventing or improving the formation of any one of spots, wrinkles, and sagging of the skin, comprising the above-mentioned cosmetic composition or cosmetic composition raw material.

[0011] According to an embodiment of the present invention, a collagen production promoter or a collagen production promoter raw material comprising a supernatant of a culture medium used when reprogramming somatic cells is provided.

[0012] According to an embodiment of the present invention, a hair growth agent, a hair growth agent raw material, a hair growth agent, or a hair growth agent raw material comprising a supernatant of a culture medium used for reprogramming somatic cells is provided.

[0013] According to an embodiment of the present invention, there is provided an activator for hair papilla cells or a raw material for an activator for hair papilla cells comprising a supernatant of a culture medium used when reprogramming somatic cells.

[0014] According to an embodiment of the present invention, there is provided an agent for promoting the production of a fibroblast growth factor family or a raw material for promoting the production of a fibroblast growth factor family comprising a supernatant of a culture medium used when reprogramming somatic cells.

[0015] According to an embodiment of the present invention, a vascular endothelial cell growth factor production-promoting agent or a vascular endothelial cell growth factor production-promoting agent raw material comprising a supernatant of a culture medium used when reprogramming somatic cells is provided.

[0016] According to an embodiment of the present invention, a wound treating agent or a wound treating agent raw material comprising a supernatant of a culture medium used for reprogramming somatic cells is provided.

[0017] According to an embodiment of the present invention, an epidermal cell proliferation promoting agent or an epidermal cell proliferation promoting agent raw material comprising a supernatant of a culture medium used when reprogramming somatic cells is provided.

[0018] According to an embodiment of the present invention, a pharmaceutical composition or a pharmaceutical composition raw material including a stem cell extract is provided.

[0019] In the above-mentioned pharmaceutical composition or pharmaceutical composition raw material, the stem cell extract may be in a paste form, or the stem cell extract may be freeze-dried.

[0020] According to an embodiment of the present invention, there is provided an agent for preventing or ameliorating the formation of any one of spots, wrinkles, and sagging of the skin, comprising the above-mentioned pharmaceutical composition or pharmaceutical composition raw material.

[0021] According to an embodiment of the present invention, a cosmetic composition or a cosmetic composition raw material including an extract of stem cells is provided.

[0022] In the above-mentioned cosmetic composition or cosmetic composition raw material, the stem cell extract may be in a paste form, or the stem cell extract may be freeze-dried.

[0023] According to an embodiment of the present invention, there is provided an agent for preventing or improving the formation of any one of spots, wrinkles, and sagging of the skin, comprising the above-mentioned cosmetic composition or cosmetic composition raw material.

[0024] According to an embodiment of the present invention, a collagen production promoter or a collagen production promoter raw material including an extract of stem cells is provided.

[0025] In the above-mentioned collagen production promoter or collagen production promoter raw material, the stem cell extract may be in a paste state, or the stem cell extract may be freeze-dried.

[0026] According to an embodiment of the present invention, a hair growth agent, a hair growth agent raw material, a hair growth agent, or a hair growth agent raw material comprising an extract of stem cells is provided.

[0027] In the above-mentioned hair growth agent, hair growth agent raw material, hair growth agent or hair growth agent raw material, the stem cell extract may be in a paste form, or the stem cell extract may be freeze-dried.

[0028] According to an embodiment of the present invention, an activator for hair papilla cells or a raw material for an activator for hair papilla cells including an extract of stem cells is provided.

[0029] In the above-mentioned hair papilla cell activator or hair papilla cell activator raw material, the stem cell extract may be in a paste form, or the stem cell extract may be freeze-dried.

[0030] According to an embodiment of the present invention, a fibroblast growth factor family production promoter or a fibroblast growth factor family production promoter raw material including a stem cell extract is provided.

[0031] In the above-mentioned fibroblast growth factor family production promoter or fibroblast growth factor family production promoter raw material, the stem cell extract may be in a paste form, or the stem cell extract may be freeze-dried.

[0032] According to an embodiment of the present invention, a vascular endothelial cell growth factor production promoter or a vascular endothelial cell growth factor production promoter raw material including a stem cell extract is provided.

[0033] In the above-mentioned vascular endothelial cell proliferation factor production promoter or vascular endothelial cell proliferation factor production promoter raw material, the stem cell extract may be in a paste form, or the stem cell extract may be freeze-dried.

[0034] According to an embodiment of the present invention, a wound therapeutic agent or a wound therapeutic agent raw material including a stem cell extract is provided.

[0035] In the above-mentioned wound treatment agent or wound treatment agent raw material, the stem cell extract may be in a paste form, or the stem cell extract may be freeze-dried.

[0036] According to an embodiment of the present invention, an epidermal cell proliferation promoter or an epidermal cell proliferation promoter raw material including an extract of stem cells is provided.

[0037] In the above-mentioned epidermal cell proliferation promoter or epidermal cell proliferation promoter raw material, the stem cell extract may be in a paste form, or the stem cell extract may be freeze-dried.

[0038] According to an embodiment of the present invention, a method for screening UV-resistant substances is provided, which includes: preparing skin cells obtained by differentiation induction from pluripotent stem cells, wherein the pluripotent stem cells are made from somatic cells derived from patients with aging diseases or skin diseases; irradiating the skin cells with ultraviolet rays; culturing the skin cells irradiated with ultraviolet rays in each of a plurality of different solutions; and selecting a culture medium with less damage to the skin cells caused by ultraviolet rays or a solution in which the skin cells exposed to ultraviolet rays repair quickly.

[0039] According to an embodiment of the present invention, a method for screening anti-drying substances is provided, which includes: preparing skin cells differentiated and induced from pluripotent stem cells, wherein the pluripotent stem cells are made from somatic cells derived from patients with aging diseases or skin diseases; drying the skin cells; culturing the dried skin cells in each of a plurality of different solutions; and selecting a solution with a high survival rate of the skin cells.

[0040] According to an embodiment of the present invention, a method for screening anti-drying substances is provided, which includes: preparing skin cells differentiated and induced from pluripotent stem cells, wherein the pluripotent stem cells are made from somatic cells derived from patients with aging diseases or skin diseases; drying the skin cells; culturing the dried skin cells in each of a plurality of different solutions; and selecting a solution in which the tight junctions of the skin cells are less damaged.

[0041] In the above method, at least one of occludin and claudin in tight junctions may be analyzed.

[0042] According to an embodiment of the present invention, a method for screening antioxidant stress substances is provided, which includes: preparing skin cells obtained by differentiation induction from pluripotent stem cells, wherein the pluripotent stem cells are made from somatic cells derived from patients with aging diseases or skin diseases; giving oxidative stress to the skin cells; culturing the skin cells given oxidative stress in each of a plurality of different solutions; and selecting a solution with a high survival rate of the skin cells.

[0043] According to an embodiment of the present invention, a method for screening a moisturizing promoting substance is provided, which comprises: preparing skin cells obtained by differentiation induction from pluripotent stem cells, wherein the pluripotent stem cells are made from somatic cells derived from patients with aging diseases or skin diseases; culturing the skin cells in each of a plurality of different solutions; and selecting a solution having a high amount of a natural moisturizing factor derived from the skin cells from the plurality of different solutions.

[0044] In the above method, the natural moisturizing factor may be at least one of ceramide and filaggrin.

[0045] According to an embodiment of the present invention, there is provided a method for testing skin's ultraviolet tolerance, comprising: preparing skin cells differentiated and induced from pluripotent stem cells produced from somatic cells derived from a subject; and testing for damage to the skin cells caused by ultraviolet rays.

[0046] According to an embodiment of the present invention, a method for testing the dryness tolerance of the skin is provided, comprising: preparing skin cells differentiated and induced from pluripotent stem cells produced from somatic cells derived from a subject; drying the skin cells; and testing the survival rate of the skin cells.

[0047] According to an embodiment of the present invention, a method for examining the dryness tolerance of the skin is provided, comprising: preparing skin cells differentiated and induced from pluripotent stem cells produced from somatic cells derived from a subject; drying the skin cells; and examining damage to tight junctions of the skin cells.

[0048] In the above method, in the examination of damage to tight junctions of skin cells, at least one of occludin and claudin in tight junctions may be analyzed.

[0049] According to an embodiment of the present invention, a method for examining skin's tolerance to oxidative stress is provided, comprising: preparing skin cells differentiated and induced from pluripotent stem cells produced from somatic cells derived from a subject; applying oxidative stress to the skin cells; and examining the survival rate of the skin cells.

[0050] According to an embodiment of the present invention, a method for testing the moisturizing ability of the skin is provided, which comprises: preparing skin cells differentiated and induced from pluripotent stem cells, wherein the pluripotent stem cells are produced from somatic cells derived from a subject; culturing the skin cells; and testing the amount of natural moisturizing factors derived from the skin cells.

[0051] Effects of the Invention According to the present invention, a pharmaceutical composition and a cosmetic composition which effectively utilize a culture medium of iPS cells can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a graph showing the results of the hyaluronic acid production test using fibroblasts in Example 2.

[0053] Figure 2 This is a graph showing the results of the type I collagen production test by fibroblasts in Example 4.

[0054] Figure 3 This is a table showing the results of the type I collagen production test by fibroblasts in Example 4.

[0055] Figure 4 This is a graph showing the results of the proliferation test of hair papilla cells in Example 5.

[0056] Figure 5 This is a table showing the results of the proliferation test of hair papilla cells in Example 5.

[0057] Figure 6 This is a graph showing the results of the VEGF production test using hair papilla cells in Example 6.

[0058] Figure 7 This is a table showing the results of the VEGF production test based on hair papilla cells in Example 6.

[0059] Figure 8 This is a graph showing the results of the FGF-7 production test based on hair papilla cells in Example 7.

[0060] Fig. 9This is a table showing the results of the FGF-7 production test based on hair papilla cells in Example 7.

[0061] Fig.10 These are photographs showing the results of the fibroblast migration test in Example 8.

[0062] Fig.11 These are photographs showing the results of the fibroblast migration test in Example 8.

[0063] Fig.12 This is a graph showing the results of the UV irradiation test on skin fibroblasts in Example 10.

[0064] Fig.13 This is a graph showing the results of the dryness stimulation test on skin fibroblasts of Example 11.

[0065] Fig.14 This is a graph showing the results of the oxidative stress test on skin fibroblasts in Example 12.

[0066] Fig.15 This is a graph showing the results of the oxidative stress test on skin fibroblasts in Example 12.

[0067] Fig.16 This is a microscope photograph of cells of a reference example.

[0068] Fig.17 This is a microscope photograph of cells of a reference example.

[0069] Fig.18 It is a distribution diagram showing the flow cytometry results of the cells of the reference example. DETAILED DESCRIPTION

[0070] The following is a detailed description of the embodiments of the present invention. It should be noted that the embodiments shown below illustrate the device and / or method for implementing the technical idea of ​​the present invention, and the combination of components of the technical idea of ​​the present invention is not specific to the following content. The technical idea of ​​the present invention can be modified in various ways in the claims.

[0071] (First embodiment) Each pharmaceutical composition, pharmaceutical composition raw material, cosmetic composition, and cosmetic composition raw material of the first embodiment includes the supernatant of the culture medium used when reprogramming somatic cells into pluripotent stem cells such as iPS cells. Each pharmaceutical composition, pharmaceutical composition raw material, cosmetic composition, and cosmetic composition raw material of the first embodiment may include a freeze-dried product of the supernatant of the culture medium used when reprogramming somatic cells into pluripotent stem cells such as iPS cells. Each pharmaceutical composition, pharmaceutical composition raw material, cosmetic composition, and cosmetic composition raw material of the first embodiment may also include the supernatant of the culture medium used when reprogramming somatic cells into pluripotent stem cells such as iPS cells in a capsule such as a nanocapsule or an emulsion such as a nanoemulsion.

[0072] iPS cells are induced by introducing initialization factors such as OCT3 / 4, KLF4, c-MYC and SOX2 into somatic cells such as differentiated cells such as blood cells and fibroblasts. Sometimes induction into iPS cells is called reprogramming, initialization, transformation, transdifferentiation (Transdifferentiation or Lineage reprogramming: transdifferentiation or lineage reprogramming) and cell fate reprogramming. Pluripotent stem cells can be induced while performing three-dimensional culture such as suspension culture. In three-dimensional culture, gel culture medium can be used or liquid culture medium can be used. Pluripotent stem cells are, for example, TRA1-60, OCT3 / 4, SSEA3, SSEA4, TRA1-81 and NANOG, and the positive rate of any one of them is 30% or more, 50% or more, and preferably 80% or more. The gel culture medium may not contain feeder cells.

[0073] As a medium for induction culture, for example, a human ES / iPS medium such as TeSR2 (STEMCELL Technologies) can be used. However, the medium is not limited thereto, and various stem cell media can be used. For example, Primate ES Cell Medium, Reprostem, ReproFF, ReproFF2, ReproXF (Reprocell), mTeSR1, TeSRE8, ReproTeSR (Stem Cell Technologies, Inc.), PluriSTEM (registered trademark) Human ES / iPS Medium (Merck), NutriStem (registered trademark) XF / FF Culture Medium for Human iPS and ES Cells, Pluriton reprogramming medium (Stemgent), PluriSTEM (registered trademark), Stemfit AK02N, Stemfit AK03 (Ajinomoto), ESC-Sure (registered trademark) Serum and Feeder-free Medium for hESC / iPS (Applied Stemcell), L7 (registered trademark) hPSC Culture medium system (LONZA) and primate ES cell culture medium (ReproCELL), etc.

[0074] The gel culture medium is prepared, for example, by adding deacylated gellan gum to the above-mentioned culture medium so that the final concentration is 0.5 wt % to 0.001 wt %, 0.1 wt % to 0.005 wt %, or 0.05 wt % to 0.01 wt %.

[0075] The gel culture medium may contain at least one polymer compound selected from the group consisting of gellan gum, hyaluronic acid, Rhamsan gum, diutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparan sulfate, keratin sulfate, chondroitin sulfate, dermatan sulfate, rhamnose sulfate, and salts thereof. In addition, the gel culture medium may also contain methylcellulose. By containing methylcellulose, aggregation between cells can be further suppressed.

[0076] Alternatively, the gel medium may also include poly(glycerolmonomethacrylate) (PGMA), poly(2-hydroxypropylmethacrylate) (PHPMA), poly(N-isopropylacrylamide) (PNIPAM), amine terminated, carboxylic acid terminated, maleimide terminated, N-hydroxysuccinimide ester terminated, triethoxysilane terminated, poly(N-isopropylacrylamide-co-acrylamide), poly(N-isopropylacrylamide-co-acrylic acid) and poly(N-isopropylacrylamide-co-acrylic acid). A small amount of temperature-sensitive gel selected from poly(N-isopropylacrylamide-co-butylacrylate), poly(N-isopropylacrylamide-co-methacrylic acid), poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate) and N-isopropylacrylamide.

[0077] Alternatively, each pharmaceutical composition, pharmaceutical composition raw material, cosmetic composition and cosmetic composition raw material of the first embodiment includes an extract of stem cells. The extract may be a liquid. That is, the extract may be an extract solution. Stem cells include pluripotent stem cells such as iPS cells, and embryonic stem cells (ES cells). Stem cells are, for example, TRA1-60 or OCT3 / 4 positive rates of 30% or more, 50% or more, and preferably 80% or more. When the stem cells are maintained in an undifferentiated state, the culture medium contains b-FGF of 10 ng / mL or more, preferably 40 ng / mL or more. In each pharmaceutical composition, pharmaceutical composition raw material, cosmetic composition and cosmetic composition raw material, the extract of stem cells may be a stem cell paste. Stem cell paste is obtained by grinding iPS cells. The extract of stem cells may also be a freeze-dried product. In addition, the extract of stem cells may also be a powder. Or the extract of stem cells may also be a lysate of stem cells.

[0078] The pharmaceutical composition of the first embodiment can be a skin coating composition. The pharmaceutical composition of the first embodiment can also be a skin disease therapeutic agent. As examples of diseases that can be treated with the skin disease therapeutic agent of the first embodiment, common acne, common psoriasis, keloid, seborrheic dermatitis, contact dermatitis, atopic dermatitis, atopic dry dermatitis, skin laxity (dermatoporosis), photoelastosis, solar keratosis, ptosis, clustered alopecia, hair loss, eyelash sparseness, chloasma, senile plaque, prickly heat, freckles, delayed bilateral Ota mother's spots, seborrheic keratosis, skin diseases caused by premature aging, and herpes simplex, etc. can be listed.

[0079] Examples of conditions that can be improved or eliminated by the cosmetic composition of the first embodiment include spots, freckles, wrinkles, sagging, dryness, reduced skin elasticity, dullness, sensitive skin, dry skin, and thinning hair. Effects of the cosmetic composition of the first embodiment include conditioning the skin, regulating skin texture, maintaining healthy skin, preventing rough skin, tightening skin, moisturizing skin, replenishing and maintaining moisture and oil in the skin, maintaining the softness of the skin, protecting the skin, preventing dry skin, making the skin soft, giving skin elasticity, giving skin luster, making the skin smooth, giving elasticity to the skin, fading spots, inhibiting wrinkles, and making the skin bright. Moreover, effects of the cosmetic composition of the first embodiment related to the scalp or hair include maintaining healthy scalp, growing hair, preventing thinning hair, preventing itching, preventing hair loss, promoting hair growth, promoting hair growth, preventing hair loss after illness or after childbirth, and nourishing hair.

[0080] The pharmaceutical composition of the first embodiment may be a wound treatment agent, an epidermal cell proliferation promoter, an epidermal regeneration promoter, a hair growth agent, a hair growth agent, and a treatment agent for thinning eyelashes. The pharmaceutical composition and the cosmetic composition of the first embodiment may also be a collagen production promoter, a hyaluronic acid production promoter, a hair growth agent, a fibroblast growth factor (FGF) family production promoter, and a vascular endothelial cell growth factor (VEGF) production promoter.

[0081] During hair growth, the hair matrix cells at the hair root divide, and the cells produced by them continue to form hair. On the other hand, during hair growth, there is a cycle called the hair cycle, which repeats the growth phase, regression phase, and resting phase. The hair papilla cells control the hair cycle by affecting the proliferation and differentiation of hair follicle epithelial stem cells through the production and release of growth factors. It can be said that the activation of hair papilla cells and hair matrix cells contributes to the mechanism of hair growth. In addition, corresponding to the hair cycle, blood vessel remodeling is actively carried out in the hair follicles, but if there is a problem with angiogenesis at this time, the supply of nutrients and oxygen for hair formation will become insufficient. It can be said that insufficient blood flow from the hair follicle vascular network is related to the condition of male pattern baldness (AGA).

[0082] The following is known about the genes of hair papilla cells and hair germination and hair growth. That is, FGF-7 and IGF-1 are known as growth factors secreted by hair papilla cells to hair matrix cells. These factors have the effect of maintaining hair follicle growth. Vascular endothelial growth factor (VEGF) is secreted by hair papilla cells and is related to the proliferation of hair follicle blood vessels. It also has the effect of autocrine proliferation of hair papilla cells, but the expression level decreases as the growth phase shifts to the regression phase. The expression level of VEGF gene decreases in the hair tissue of AGA (male pattern baldness). VEGFB competes with VEGFR-1, the receptor on which VEGF acts. Although VEGFB maintains the proliferation and permeability hyperactivity of vascular endothelial cells, its effect on hair follicles is not yet clear.

[0083] The pharmaceutical composition and the cosmetic composition of the first embodiment have the following effects: by directly acting on the hair papilla, the production of FGF-7, which is a hair growth promoting factor, is increased to promote hair growth, thereby extending the growth phase of the hair cycle and nurturing fine and soft hair to thick and strong hair, and the effect of increasing vascular endothelial growth factor (VEGF), which is secreted by hair papilla cells and is associated with hair follicle blood vessel proliferation, and the effect of promoting the proliferation of hair papilla cells by autocrine secretion. Therefore, the pharmaceutical composition and the cosmetic composition of the first embodiment can be used as an activator of hair papilla cells.

[0084] When used as a hair growth agent or a hair tonic, the pharmaceutical composition and the cosmetic composition of the first embodiment may contain other active ingredients such as minoxidil, dapoxetine, pantothenyl ethyl ether, tocopherol acetate, dipotassium glycyrrhizinate, and adenosine.

[0085] Examples of wounds that can be treated with the wound treatment agent of the first embodiment include burns, abrasions, lacerations, contusions, suture wounds, bedsores, and skin defect wounds.

[0086] The pharmaceutical composition and cosmetic composition of the first embodiment include an effective amount of the supernatant of the culture medium used when reprogramming somatic cells. Alternatively, the pharmaceutical composition and cosmetic composition of the first embodiment include an effective amount of stem cell extract. Here, the effective amount refers to an amount that can be used as a pharmaceutical composition or a cosmetic composition. The effective amount is appropriately set according to the patient's age, the target disease, the presence or absence of other successful ingredients, and the amount of other compounds.

[0087] The pharmaceutical composition and cosmetic composition of the first embodiment may include a carrier, excipient, disintegrant, buffer, emulsifier, suspending agent, pain-relieving agent, stabilizer, preservative, preservative, and physiological saline, etc., which are permitted in the formulation. Examples of excipients include lactose, starch, sorbitol, D-mannitol, and white sugar. Examples of disintegrants include carboxymethyl cellulose and calcium carbonate. Examples of buffers include phosphates, citrates, and acetates. Examples of emulsifiers include gum arabic, sodium alginate, and tragacanth.

[0088] Examples of suspending agents include glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of pain-relieving agents include benzyl alcohol, chlorobutanol, and sorbitol. Examples of preservatives include propylene alcohol and ascorbic acid. Examples of stabilizers include phenol, chlorobenzenesulfonic acid, benzyl alcohol, chlorobutanol, and toluene. Examples of preservatives include chlorobenzoic acid, p-hydroxybenzoic acid, and chlorobutanol.

[0089] In addition, within the scope of achieving the purpose of the pharmaceutical composition and the cosmetic composition of the first embodiment, the following ingredients can be added to the pharmaceutical composition and the cosmetic composition of the first embodiment, and the ingredients include: water, ethanol, surfactants (cationic, anionic, nonionic and amphoteric surfactants, etc.), moisturizing agents (glycerin, 1,3-butylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, pentylene glycol, polyquaternium salts, amino acids, urea, pyrrolidone carboxylates, nucleic acids, monosaccharides and oligosaccharides, and their derivatives, etc.), thickeners (polysaccharides, polyacrylates, carboxyvinyl polymers, polyvinyl pyrrolidone, polyvinyl alcohol, chitin, chitosan, alginic acid, carrageenan, xanthan gum and methylcellulose, and their derivatives, waxes, petrolatum, hydrocarbon saturated fatty acids, unsaturated fatty acids and silicone oils, and their derivatives, tri(caprylic / capric)glyceryl, and triglycerides such as tricaprylic acid glyceride, ester oils such as isopropyl stearate, natural oils (olive oil, camellia oil, avocado oil, almond oil, cocoa butter, evening primrose oil, grapeseed oil, etc.), Seed oil, macadamia oil, eucalyptus oil, rosehip oil, squalane, neroli oil, lanolin and ceramide, etc.), preservatives (hydroxybenzoic acid derivatives, dehydroacetate, photosensitizers, sorbic acid and phenoxyethanol, etc., and their derivatives, etc.), fungicides (sulfur, trichloromethane, salicylic acid, zinc pyrithione and hinokitiol, etc., and their derivatives, etc.), UV absorbers (p-aminobenzoic acid and methoxycinnamic acid, etc., and their derivatives, etc.), anti-inflammatory drugs (allantoin, bisabolol, ε-aminocaproic acid, acetylbenzoylcysteine ​​and glycyrrhizic acid, etc., and their derivatives, etc.), antioxidants (tocopherol, BHA, BHT and astaxanthin, etc., and their derivatives, etc. ), chelating agents (ethylenediaminetetraacetic acid and hydroxyethane diphosphonic acid, etc., and their derivatives, etc.), animal and plant extracts (hachijo grass, aloe, Yingshi, scutellaria, phellodendron, seaweed, papaya, chamomile, licorice, kiwi, cucumber, mulberry, birch, angelica, garlic, peony, hops, horse chestnut, lavender, rosemary, eucalyptus, milk, various peptides, placenta, royal jelly, euglena extract, hydrolyzed euglena extract and euglena oil, etc., and their components purified or fermented, etc.), pH adjusters (inorganic acids, inorganic acid salts, organic acids and organic acid salts, etc., and their derivatives, etc.), vitamins (vitamin A, vitamin B, vitamin C, vitamin D, ubiquinone and niacinamide, etc., and their derivatives, etc.), fermentation broth of yeast, Aspergillus and lactic acid bacteria, galactase culture broth, whitening agents (tranexamic acid, tranexamic acid cetyl ester hydrochloride, 4-n-butylresorcinol, arbutin, kojic acid, ellagic acid, glycyrrhizic flavonoids, niacinamide, and vitamin C derivatives, etc.), ceryl / ceramide derivatives, anti-wrinkle agents (retinol and retinal, and their derivatives, niacinamide and oligopeptides, etc., and their derivatives, etc., neutrophil elastase inhibition, and certain natural and synthetic ingredients with MMP-1 and MMP-2 inhibitory effects, etc.), titanium oxide, talc, mica, silicon dioxide, zinc oxide, iron oxide, silicon, and powders obtained by processing them, etc.

[0090] It should be noted that the ingredients that can be added to the pharmaceutical composition and the cosmetic composition of the first embodiment are not limited to the above contents, and any ingredients that can be used in pharmaceutical compositions and cosmetic compositions can be freely selected. When the pharmaceutical composition and the cosmetic composition of the first embodiment are used as wet compresses, in addition to the above ingredients, a base (kaolin and bentonite, etc.) and a gelling agent (polyacrylic acid salt and polyvinyl alcohol, etc.) can be added within the range to achieve the purpose. When the pharmaceutical composition and the cosmetic composition of the first embodiment are used as a shower gel, sulfates, bicarbonates, borates, pigments and moisturizers can be appropriately added within the range to achieve the purpose and prepared into a powder form or a liquid form.

[0091] The pharmaceutical composition and cosmetic composition of the first embodiment can be produced by conventional methods well known in the art.

[0092] (Second embodiment) The second embodiment of the screening method of the anti-ultraviolet material includes: preparing skin cells differentiated from iPS cells, wherein the iPS cells are prepared from somatic cells derived from patients with aging diseases such as progeria patients and skin disease patients or patients with skin diseases; irradiating the skin cells induced by any differentiation with ultraviolet rays; culturing the skin cells irradiated with ultraviolet rays in each of a plurality of different solutions; and selecting a solution in which the skin cells are less damaged by ultraviolet rays or a solution in which the skin cells exposed to ultraviolet rays are repaired quickly. It should be noted that the patient is not limited to humans, but also includes non-human animals.

[0093] Although Werner syndrome, xeroderma pigmentosum and Cockayne syndrome are listed as examples of progeria, there is no particular limitation. Although the somatic cells derived from progeria patients are, for example, fibroblasts, blood cells, epithelial cells, adult stem cells, keratinocytes, hair papilla cells and dental pulp stem cells, there is no particular limitation. iPS cells are induced by introducing reprogramming factors such as OCT3 / 4, KLF4, c-MYC and SOX2 into somatic cells derived from progeria patients. When inducing skin cells from iPS cells, for example, iPS cells are inoculated in a low-adhesion culture dish to which a culture medium without bFGF is added. Then, the culture medium is replaced every 2 days, and the embryoid bodies (EBs) formed after 8 days are inoculated into the culture dish, so that the cells adhere to the culture dish, and the cells are cultured with 10% FBS culture medium. Even if the cells reach confluence, the cells are detached from the culture dish with trypsin and subcultured. The subculture is repeated in the same way for 1 month. Then, fibroblast markers such as CD13 are used to confirm that the cells have differentiated into fibroblasts. Alternatively, it is possible to confirm whether undifferentiated iPS cells remain in the cells using iPS cell markers such as TRA 1-60. The skin cells to be induced to differentiate are, for example, skin fibroblasts, but are not particularly limited.

[0094] The skin cells induced to differentiate are irradiated with ultraviolet light (UV) after being appropriately cultured. The intensity, wavelength range and irradiation time of the ultraviolet light are appropriately set according to the purpose, usage and dosage of the anti-ultraviolet substance to be screened. The skin cells irradiated with UV are cultured in each of a plurality of different solutions containing different substances to be screened. The solution may be a culture medium. The screening target substance in the solution and the culture time are appropriately set according to the purpose, usage and dosage of the anti-ultraviolet substance to be screened.

[0095] Then, a solution with less damage to skin cells or a solution with faster skin cell repair is selected as the solution containing the anti-ultraviolet substance. For example, skin cells cultured in each of the multiple solutions are analyzed, and one or a group of solutions used for culturing skin cells with less damage are selected, and one or a group of solutions used for culturing skin cells with more damage are excluded. Alternatively, skin cells cultured in each of the multiple solutions are analyzed, and a solution or a group of solutions used for culturing skin cells that are quickly repaired are selected, and one or a group of solutions used for culturing skin cells that are slowly repaired or skin cells that are not well repaired are excluded.

[0096] According to the present inventors' concept, skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases or skin diseases such as progeria patients and skin disease patients tend to have no resistance to UV irradiation compared to skin cells of healthy individuals. Therefore, by using skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases or skin diseases such as progeria patients and skin disease patients, effective anti-ultraviolet substances can be screened.

[0097] (Third Embodiment) The third embodiment of the method for testing skin ultraviolet resistance includes: preparing skin cells differentiated from pluripotent stem cells prepared from somatic cells derived from a subject; irradiating the skin cells with ultraviolet rays; and testing for damage to the skin cells caused by the ultraviolet rays.

[0098] The subject may be a disease patient or a healthy person. The somatic cells derived from the subject are obtained from the subject in advance, and the method may not include the step of obtaining the somatic cells from the subject. If the damage to the skin cells caused by ultraviolet rays is large, it can be determined that the skin cells of the subject do not have ultraviolet resistance. If the damage to the skin cells caused by ultraviolet rays is small, it can be determined that the skin cells of the subject have ultraviolet resistance. According to this method, it is possible to check whether the skin cells of the subject have ultraviolet resistance based on the test results of the damage to the skin cells caused by ultraviolet rays.

[0099] (Fourth Embodiment) The fourth embodiment of the screening method for anti-drying substances includes: preparing skin cells obtained by differentiation induction from iPS cells, wherein the iPS cells are made from somatic cells derived from patients with aging diseases or skin diseases such as progeria patients and skin disease patients; drying the skin cells; culturing the dried skin cells in each of a plurality of different solutions; and selecting a solution with a high survival rate of the skin cells.

[0100] The skin cells obtained by differentiation induction in the same manner as in the second embodiment are dried after appropriate culture. When drying the skin cells, for example, the culture medium surrounding the skin cells is removed. In addition, air flow can be applied to the skin cells, or the skin cells can be dried using a desiccant or the like. The drying method and drying time of the skin cells are appropriately set according to the purpose, usage and dosage of the anti-drying substance screened. The dried skin cells are cultured in each of a plurality of different solutions containing different substances that are the screening objects. The solution can be a culture medium. The screening object substance in the solution and the culture time, etc. are appropriately set according to the purpose, usage and dosage of the anti-drying substance screened.

[0101] Then, the survival rate of the skin cells cultured in each of the plurality of solutions is detected, and a solution with a high survival rate of the skin cells is selected as a solution including an anti-desiccation substance. For example, the survival rate of the skin cells cultured in each of the plurality of solutions is detected, one or a group of solutions used for culturing skin cells with a high survival rate are selected, and one or a group of solutions used for culturing skin cells with a low survival rate are excluded.

[0102] It should be noted that, instead of selecting a solution with a high survival rate of skin cells, a solution with less damage to the tight junctions of skin cells may be selected. In this case, at least one of occludin and claudin in the tight junctions may be analyzed, and a solution with a large amount of at least one of occludin and claudin may be selected. Generally, if skin cells are dry, the tight junctions are damaged, and the amounts of occludin and claudin decrease. Therefore, for example, the tight junctions of skin cells cultured in each of a plurality of solutions are analyzed, and one or a group of solutions used for culturing skin cells with less damage to the tight junctions are selected, and one or a group of solutions used for culturing skin cells with greater damage to the tight junctions are excluded.

[0103] According to the present inventors' view, skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases or skin diseases such as progeria patients and skin disease patients tend to have no resistance to desiccation compared to skin cells of healthy people. Therefore, by using skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases or skin diseases such as progeria patients and skin disease patients, effective anti-desiccation substances can be screened.

[0104] (Fifth Embodiment) The method for testing the dryness tolerance of the skin according to the fifth embodiment includes: preparing skin cells differentiated and induced from pluripotent stem cells prepared from somatic cells derived from a subject; drying the skin cells; and testing the survival rate of the skin cells.

[0105] The subject may be a disease patient or a healthy person. The somatic cells derived from the subject are obtained from the subject in advance, and the method may not include the step of obtaining the somatic cells from the subject. If the survival rate of the skin cells is high, it can be determined that the skin cells of the subject have dryness tolerance. If the survival rate of the skin cells is low, it can be determined that the skin cells of the subject do not have dryness tolerance. According to this method, it is possible to check whether the skin cells of the subject have dryness tolerance based on the test results of the survival rate of the skin cells.

[0106] It should be noted that damage to the tight junctions of skin cells may be examined instead of examining the survival rate of skin cells. If the damage to the tight junctions is small, it can be determined that the skin cells of the subject have dryness tolerance, and if the damage to the tight junctions is large, it can be determined that the skin cells of the subject do not have dryness tolerance.

[0107] (Sixth Embodiment) The sixth embodiment of the screening method for antioxidant stress substances includes: preparing skin cells obtained by differentiation induction from iPS cells, wherein the iPS cells are made from somatic cells derived from patients with aging diseases or skin diseases such as progeria patients and skin disease patients; applying oxidative stress to the skin cells; culturing the skin cells to which oxidative stress has been applied in each of a plurality of different solutions; and selecting a solution with a high survival rate of the skin cells.

[0108] Skin cells induced to differentiate in the same manner as in the second embodiment are subjected to oxidative stress after appropriate culture. The method of applying oxidative stress to skin cells is not particularly limited, but includes, for example, adding oxidizing substances such as hydrogen peroxide to the culture medium in which the skin cells are being cultured. The method of applying oxidative stress to skin cells and the time of applying oxidative stress to skin cells are appropriately set according to the purpose, usage and dosage of the anti-oxidative stress substances screened. After removing the substance that applies oxidative stress, the skin cells subjected to oxidative stress are cultured in each of a plurality of different solutions containing different substances that are the objects of screening. The solution may be a culture medium. The screening object substance in the solution and the culture time are appropriately set according to the purpose, usage and dosage of the anti-oxidative stress substances screened.

[0109] Then, the survival rate of the skin cells cultured in each of the multiple solutions is detected, and a solution with a high survival rate of the skin cells is selected as a solution containing an antioxidant stress-resistant substance. For example, the survival rate of the skin cells cultured in each of the multiple solutions is detected, and one or a group of solutions used for culturing skin cells with a high survival rate are selected, and one or a group of solutions used for culturing skin cells with a low survival rate are excluded.

[0110] According to the present inventors' concept, skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases or skin diseases such as progeria patients and skin disease patients tend to have no resistance to oxidative stress compared to skin cells of healthy individuals. Therefore, by using skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases or skin diseases such as progeria patients and skin disease patients, effective anti-oxidative stress substances can be screened.

[0111] (Seventh Embodiment) The seventh embodiment of the method for examining skin resistance to oxidative stress comprises: preparing skin cells differentiated from pluripotent stem cells prepared from somatic cells derived from a subject; applying oxidative stress to the skin cells; and examining the survival rate of the skin cells.

[0112] The subject may be a disease patient or a healthy person. Somatic cells derived from the subject are obtained from the subject in advance, and the method may not include the step of obtaining somatic cells from the subject. If the damage to the skin cells caused by oxidative stress is large, it can be determined that the skin cells of the subject do not have oxidative stress tolerance. If the damage to the skin cells caused by oxidative stress is small, it can be determined that the skin cells of the subject have oxidative stress tolerance. According to this method, it is possible to check whether the skin cells of the subject have oxidative stress tolerance based on the test results of the damage to the skin cells caused by oxidative stress.

[0113] (Eighth Implementation Method) The eighth embodiment of the screening method for moisturizing promoting substances includes: preparing skin cells obtained by differentiation induction from pluripotent stem cells, wherein the pluripotent stem cells are made from somatic cells derived from patients with aging diseases such as progeria patients and skin disease patients or patients with skin diseases; culturing the skin cells in each of a plurality of different solutions; and selecting a solution containing a large amount of natural moisturizing factors derived from skin cells from the plurality of different solutions.

[0114] The skin cells differentiated and induced in the same manner as in the second embodiment are cultured in each of a plurality of different solutions containing different substances to be screened. The screening substances in the solutions and the culture time are appropriately set according to the purpose, usage and dosage of the moisturizing promoting substances to be screened.

[0115] Then, the amount of natural moisturizing factor derived from skin cells in each of the multiple solutions is detected. As natural moisturizing factors, although not particularly limited, ceramide and filaggrin can be cited. A solution with a large amount of natural moisturizing factor is selected as a solution containing a moisturizing promoting substance. For example, the amount of natural moisturizing factor in each of the multiple solutions is detected, and one or a group of solutions with a large amount of natural moisturizing factor are selected, and one or a group of solutions with a small amount of natural moisturizing factor are excluded.

[0116] According to the present inventors, the expression level of moisturizing promoting substances in skin cells tends to decrease as aging and / or skin diseases progress. Therefore, effective moisturizing promoting substances can be screened by using skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases or skin diseases such as progeria patients and skin disease patients.

[0117] (Ninth embodiment) The ninth embodiment of the method for testing the moisturizing ability of skin comprises: preparing skin cells differentiated from pluripotent stem cells prepared from somatic cells derived from a subject; culturing the skin cells; and testing the amount of natural moisturizing factor derived from the skin cells.

[0118] The subject may be a disease patient or a healthy person. The somatic cells derived from the subject are obtained from the subject in advance, and the method may not include the step of obtaining the somatic cells from the subject. If the amount of the natural moisturizing factor derived from the skin cells is large, it can be determined that the moisturizing ability of the skin cells of the subject is high. If the amount of the natural moisturizing factor derived from the skin cells is small, it can be determined that the moisturizing ability of the skin cells of the subject is low. According to this method, it is possible to check whether the skin cells of the subject have moisturizing ability based on the amount of the natural moisturizing factor derived from the skin cells.

[0119] Example (Example 1: Preparation of a culture medium for simultaneous culture of blood cells reprogrammed into iPS cells) Hematopoietic cell culture medium was prepared by adding growth factors to a serum-free hematopoietic cell culture medium with animal-derived components (Stemspan ACF, Stem Cell Technologies, Inc.). 2 × 10 5 Blood cells (peripheral blood mononuclear cells) were added to each well, and the blood cells were suspended in the hematopoietic system cell gel culture medium. Then, the 12-well culture dish was placed in a CO2 incubator at 37°C to culture the cells in suspension.

[0120] Three days after the start of cell culture, hematopoietic cell gel medium was added appropriately to each well. Six days after the start of cell culture, Sendai virus vector kit for iPS cell production (CytoTune 2.0 Reprogramming Kit, registered trademark, Thermo Fisher Scientific) was added to the culture medium to introduce genes in each well so that the virus titer (Titer) was 1 to 20, or episomal plasmid (Thermo Fisher Scientific) was electroporated to introduce genes, and then gel medium was added to each well to culture cells.

[0121] Gellan gum was added to the hES medium to a final concentration of 0.02% to prepare a stem cell gel medium. Starting from two days after infection, 2 mL of stem cell gel medium was added to each well every two days. After 14 days of infection, it was confirmed that iPS cell clusters were induced and the gel medium supernatant was recovered. The recovered gel medium supernatant was filtered and sterilized with a filter, and the gel medium supernatant that passed through the filter was used as the supernatant of the reprogramming medium of Example 1.

[0122] (Reference Example 1: Preparation of a culture medium for culturing iPS cells while maintaining them in an undifferentiated state) Human iPS cells were cultured by adhesion maintenance using mTeSR1 (registered trademark, Stem Cell Technologies, Inc.) or StemFit (registered trademark, Ajinomoto Co., Ltd.) on adhesion culture dishes coated with Matrigel (registered trademark, Corning Incorporated) or laminin 511. Human iPS cells were passaged every week. During passage, ES cell dissociation medium (TrypLE Select, registered trademark, Thermo Fisher Scientific Inc.) was used for treatment.

[0123] The human iPS cells maintained and cultured as described above were detached from the adherent culture dish using ES cell dissociation solution (TrypLE Select, registered trademark, Thermo Fisher Scientific) and divided into single cells. Then, human iPS cells were inoculated in a stem cell medium gelled by adding gellan gum and 10 μmol / L ROCK inhibitor (Selleck), and the human iPS cells were suspended and cultured for 14 days. In the case of 14 days of suspension culture, the gelled stem cell medium was replenished in the culture vessel every two days.

[0124] Then, the gelatinized stem cell medium in which human iPS cells are suspended is filtered through a mesh filter to remove cell aggregates. Furthermore, the filtered gelatinized stem cell medium is centrifuged at 1500 g for 5 minutes to precipitate cells and gel, and the supernatant of the centrifuged stem cell medium is recovered again, centrifuged at 3000 rpm for 3 minutes, and the supernatant of the centrifuged stem cell medium is filtered through a 0.22 μm filter. The supernatant of the filtered stem cell medium is used as the supernatant of the medium after the maintenance and culture of iPS cells in Reference Example 1.

[0125] In addition, it was confirmed that the iPS cells obtained by maintaining culture were positive for NANOG, OCT3 / 4, and TRA1-60, which are undifferentiation markers.

[0126] (Example 2: Hyaluronic acid production test based on fibroblasts) DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin was prepared as growth medium A. Then, normal human fibroblasts derived from adults (KF-4109, strain No. 01035, Kurabo Industries, Ltd.) were cultured at a concentration of 5×10 3 The cells were suspended in proliferation medium A at 0.1 mL / well and seeded into a 96-well plate and cultured in a CO2 incubator (5% CO2, 37°C) for 1 day.

[0127] DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin was prepared as test medium A. Then, the supernatant solutions of Example 1 and Reference Example 1 were respectively mixed with the test medium A to obtain the supernatant-supplemented medium A of Example 1 and Reference Example 1 at a concentration of 10%. The proliferation medium A in some wells where fibroblasts were being cultured was replaced with the supernatant-supplemented medium A of Example 1 and Reference Example 1, respectively. In addition, the proliferation medium A in some wells was replaced with a DMEM medium not supplemented with FBS, penicillin-streptomycin and supernatant (no supplemented test medium A) as a negative control.

[0128] After replacing the culture medium, the fibroblasts were cultured for 3 days, and the supernatant of the culture medium was collected. The hyaluronan concentration in the supernatant of the culture medium was detected using DueSet Hyaluronan (DueSet Hyaluronan Detection Kit) (Cat. No.: DY3614, R&D Systems). Figure 1 It was confirmed that fibroblasts cultured in a medium supplemented with the supernatant of the reprogramming medium of Example 1 produced more than double the amount of hyaluronic acid compared to fibroblasts cultured in a medium supplemented with the supernatant of the medium for maintaining and culturing iPS cells of Reference Example 1.

[0129] (Example 3: Preparation of iPS cell extract) iPS cells were cultured on matrigel-coated and laminin-coated substrates using Teser1, Teser2, StemFit, Essential8, Teser-E8, and Nutri Stem. When the iPS cells reached 80% confluence, the iPS cells were detached from the culture vessel using Tryple Select (cell dissociation solution), and the solution containing the detached iPS cells was centrifuged at 200g for 5 minutes to collect the iPS cells in a 1.5 mL test tube. Then, the iPS cell pellet was ground with a pestle (Pestle in G-Tube, Thermo Fisher Scientific), and the iPS cell extract containing the ground iPS cell paste was flash-frozen with liquid nitrogen. When using the iPS cell extract, the iPS cell extract was suspended in 5 mL of culture medium and incubated overnight at 4°C. The next day, the suspension was centrifuged at 1500g for 10 minutes to remove cell debris from the solution. Then, the solution was filtered with a filter, and the solution that passed through the filter was used as the iPS cell extract.

[0130] (Example 4: Fibroblast-based Type I collagen production test) Normal human fibroblasts derived from an adult were cultured using the proliferation medium A in the same manner as in Example 2. In addition, a test medium A was prepared in the same manner as in Example 2. Then, the extract of the iPS cells of Example 3 was mixed with the test medium A to obtain an extract-added medium A containing the extract of the iPS cells of Example 3 at a concentration of 50.0 v / v% or 100.0 v / v%. The proliferation medium A in some wells where fibroblasts were being cultured was replaced with the extract-added medium A. In addition, the proliferation medium A in some wells was replaced with a DMEM medium to which FBS, penicillin-streptomycin, and the iPS cell extract were not added (no test medium A added) as a negative control.

[0131] After replacing the culture medium, the fibroblasts were cultured for 3 days, and the supernatant of the culture medium was recovered and stored at -80°C. Then, the supernatant of the culture medium was thawed and the concentration of type I collagen in the supernatant of the culture medium was detected using a human type I collagen enzyme-linked immunosorbent assay (ELISA) kit (model: EC1-E105). The results were recorded in Figure 2 and Figure 3 It was confirmed that fibroblasts cultured in a medium supplemented with an iPS cell extract produced a larger amount of collagen than fibroblasts cultured in a medium not supplemented with an iPS cell extract.

[0132] (Example 5: Proliferation test of hair papilla cells) A culture medium for dermal papilla cells (model: TMTPGM-250, Toyobo) supplemented with special additives (fetal bovine serum, insulin-transferrin-triiodothyronine mixture, bovine pituitary extract, cyproterone acetate) was prepared as proliferation medium B. Next, normal human dermal papilla cells (model: CA60205a, lot number (Lot. No.): 2868, Toyobo) were incubated at a concentration of 1.2×10 4 The cells were suspended in proliferation medium B at 0.3 mL / well and seeded in a 48-well plate coated with type I collagen and cultured in a CO2 incubator (5% CO2, 37°C) for 1 day.

[0133] The extract of the iPS cells of Example 3 and the culture medium for hair papilla cells without additives (no-additive test culture medium B) were mixed to obtain the extract-added culture medium B containing 50.0 v / v% or 100.0 v / v% of the extract of the iPS cells of Example 3. The proliferation culture medium B in some wells was replaced with the culture medium for hair papilla cells without additives and the extract of iPS cells (no-additive test culture medium B) as a negative control.

[0134] The dermal papilla cells were cultured in the replaced medium for 3 days and the number of viable cells was determined using the WST-8 method. Figure 4 and Figure 5 It was confirmed that the proliferation of hair papilla cells was better in the case of using the non-additive test medium B containing the iPS cell extract of Example 3 than in the case of using the non-additive test medium B without the iPS cell extract. Therefore, it is suggested that the iPS cell extract has the effect of treating thinning hair, preventing hair loss, promoting hair growth, and promoting hair growth.

[0135] (Example 6: VEGF production test based on hair papilla cells) Normal human dermal papilla cells were cultured for 1 day in the proliferation medium B in the same manner as in Example 5. Then, the supernatant of the reprogramming medium of Example 1 was added to the proliferation medium B in some wells at a concentration of 10.0 v / v% and 20.0 v / v%. In addition, the extract of iPS cells of Example 3 was added to the proliferation medium B in some wells at a concentration of 50.0 v / v% and 100.0 v / v%.

[0136] The proliferation medium B in some wells was replaced with the non-additive test medium B as a negative control. In addition, the proliferation medium B in some wells was replaced with an adenosine-supplemented medium obtained by adding 100 μmol / L adenosine to the culture medium for hair papilla cells and a minoxidil-supplemented medium obtained by adding 30 μmol / L minoxidil to the culture medium for hair papilla cells as reference controls. In addition, the proliferation medium B in some wells was replaced with a DMSO-supplemented medium obtained by adding 0.1% DMSO to the culture medium for hair papilla cells as a vehicle control for minoxidil.

[0137] After replacing the culture medium, the dermal papilla cells were cultured for 3 days, and the supernatant of the culture medium was collected and stored at -80°C. Then, the supernatant of the culture medium was thawed, and the concentration of vascular endothelial growth factor (VEGF) in the supernatant of the culture medium was detected using a human ELISA kit (model: ab100519, Abcam). Figure 6 and Figure 7 It was shown that the supernatant of the culture medium in which iPS cells were induced and cultured and the extract of iPS cells promoted the production of VEGF by hair papilla cells. Therefore, it is suggested that the supernatant of the culture medium in which iPS cells were induced and cultured and the extract of iPS cells are effective for hair growth, hair growth and hair maintenance.

[0138] (Example 7: FGF-7 production test based on hair papilla cells) Normal human dermal papilla cells were cultured for 1 day in the proliferation medium B in the same manner as in Example 5. Then, the supernatant of the reprogramming medium of Example 1 was added to the proliferation medium B in some wells at a concentration of 10.0 v / v% and 20.0 v / v%. In addition, the extract of iPS cells of Example 3 was added to the proliferation medium B in some wells at a concentration of 50.0 v / v% and 100.0 v / v%.

[0139] The proliferation medium B in some wells was replaced with the non-additive test medium B as a negative control. In addition, the proliferation medium B in some wells was replaced with an adenosine-supplemented medium obtained by adding 100 μmol / L adenosine to the culture medium for hair papilla cells and a minoxidil-supplemented medium obtained by adding 30 μmol / L minoxidil to the culture medium for hair papilla cells as a reference control. In addition, the proliferation medium B in some wells was replaced with a DMSO-supplemented medium obtained by adding 0.1% DMSO to the culture medium for hair papilla cells as a vehicle control for minoxidil.

[0140] After replacing the culture medium, the dermal papilla cells were cultured for 3 days, and the supernatant of the culture medium was recovered and stored at -80°C. Then, the supernatant of the culture medium was thawed, and the concentration of fibroblast growth factor 7 (FGF-7) in the supernatant of the culture medium was detected using the FGF-7 human ELISA kit (model: ab100519, Abcam). Figure 8 and Fig. 9 It was shown that the supernatant of the culture medium in which iPS cells were induced and cultured and the extract of iPS cells promoted the production of FGF-7 by hair papilla cells. Therefore, it is suggested that the supernatant of the culture medium in which iPS cells were induced and cultured and the extract of iPS cells are effective for hair growth, hair growth and hair maintenance.

[0141] (Example 8: Migration test of fibroblasts) Normal human fibroblasts derived from adults were cultured at a concentration of 1×10 5 ~2×10 5 The cells were suspended in 10% FBS medium in a cell / well format and seeded in a plate of a kit for detecting migration ability (Radius Cell Migration Assay, registered trademark). Next, human fibroblasts were treated with 10 μg / mL of mitomycin C (model: 20898-21, Nacalai tesque) for 2 hours to stop the cell division of human fibroblasts. Then, human fibroblasts were cultured in a CO2 incubator (5% CO2, 37°C) for 1 day.

[0142] The medium in some plates was replaced with a medium obtained by adding the iPS cell extract of Example 3 to a 10% FBS medium at a concentration of 10 v / v%. In addition, the medium in some plates was replaced with a medium obtained by adding the supernatant of the reprogramming medium described in Example 1 to a 10% FBS medium at a concentration of 10.0 v / v% and 20.0 v / v%.

[0143] The proliferation medium B on a portion of the plates was replaced with epidermal cell medium without the addition of a proliferation additive as a negative control (no-additive test medium B).

[0144] The human fibroblasts were treated according to the experimental protocol to perform the migration test. In the process of wound healing, human fibroblasts migrate to the wound and the wound shrinks. In this example, a microplate reader was used to analyze whether the human fibroblasts migrated to the part that was plugged before the migration test and did not adhere to the human fibroblasts. Specifically, 23 hours after the replacement of the culture medium, the epidermal cells were stained with Hechest and observed.

[0145] The results are Fig.10 and Fig.11It was confirmed that the iPS cell extract and the supernatant of the medium in which iPS cells were induced and cultured promote the migration ability of human fibroblasts. Therefore, it was shown that the iPS cell extract and the supernatant of the medium in which iPS cells were induced and cultured are effective for wound healing.

[0146] (Example 9: Preparation of cells derived from progeria patients) Fibroblasts from patients with Werner syndrome (AG04110), patients with xeroderma pigmentosum (GM16684 and GM16687), and patients with Cockayne syndrome (GM01098) were purchased from the Coriell Institute for Medical Research. Fibroblasts from these progeria patients were induced into iPS cells. iPS cells were further differentiated into skin fibroblasts.

[0147] (Example 10: UV irradiation test of skin fibroblasts) Normal human skin fibroblasts derived from adults and skin fibroblasts induced from somatic cells of progeria patients prepared in Example 9 were mixed at a concentration of 2×10 5 The cells / well were suspended in assay medium A, seeded in 6-well plates, and cultured in a CO2 incubator (5% CO2, 37°C) for 1 day.

[0148] On the second day, each skin fibroblast in the well was irradiated with 302 nm ultraviolet light for 15 minutes using a UV irradiator. Next, the supernatant solution of Reference Example 1 and the test medium A were mixed at a volume ratio of 10.00:90.00 to obtain the supernatant-added medium A of Reference Example 1 at a concentration of 10.00 v / v%. The test medium A in some wells was replaced with the supernatant-added medium A of Reference Example 1. On the second day, all cells were detached from the wells using trypsin, and the cells were stained with 7-aminoactinomycin D (7-AAD), and the cell death rate was detected using a flow cytometer.

[0149] As a result, if Fig.12As shown, for any type of skin fibroblasts, the cell death rate of cells irradiated with UV increased compared to cells not irradiated with UV. In addition, compared with normal skin fibroblasts, the mortality rate caused by UV irradiation of skin fibroblasts induced from somatic cells of patients with progeria was higher. However, compared with skin fibroblasts in a culture medium to which the supernatant of the culture medium for maintaining iPS cells was not added, the survival rate of skin fibroblasts in a culture medium to which the supernatant of the culture medium for maintaining iPS cells was added was higher. Therefore, it is suggested that skin fibroblasts induced from somatic cells of patients with progeria are sensitive to UV irradiation and are suitable for screening of anti-UV substances.

[0150] (Example 11: Drying stimulation test of skin fibroblasts) In the same manner as in Example 10, normal human skin fibroblasts derived from adults and skin fibroblasts induced from somatic cells of progeria patients prepared in Example 9 were cultured for 1 day using test medium A. On the next day, each well was dried in the vent of the clean bench for 40 seconds. Next, the test medium A in some wells was replaced with the supernatant-added medium A of Reference Example 1. In addition, the supernatant solution of Example 1 and the test medium A were mixed in a volume ratio of 10.00:90.00 to obtain the supernatant-added medium A of Example 1 at a concentration of 10.00 v / v%. The test medium A in some wells was replaced with the supernatant-added medium A of Example 1. On the next day, all cells were detached from the wells using trypsin, and the cells were stained with 7-aminoactinomycin D (7-AAD), and the cell death rate was detected using a flow cytometer.

[0151] As a result, if Fig.13 As shown, the mortality rate of skin fibroblasts induced from somatic cells of patients with progeria due to dryness stimulation is higher than that of normal skin fibroblasts. However, compared with skin fibroblasts in a culture medium to which the supernatant of the culture medium for maintaining and culturing iPS cells is not added, the survival rate of skin fibroblasts in a culture medium to which the supernatant of the culture medium for maintaining and culturing iPS cells is added is higher. In addition, compared with skin fibroblasts in a culture medium to which the supernatant of the culture medium for maintaining and culturing iPS cells is added, the survival rate of skin fibroblasts in a culture medium to which the supernatant of the culture medium for induced and culturing blood cells into iPS cells is added is higher. Therefore, it is suggested that skin fibroblasts induced from somatic cells of patients with progeria are sensitive to dryness stimulation and are suitable for screening of anti-dryness stimulation substances.

[0152] (Example 12: Oxidative stress test of skin fibroblasts) In the same manner as in Example 10, normal human skin fibroblasts derived from adults and skin fibroblasts induced from somatic cells of progeria patients prepared in Example 9 were cultured for 1 day using test medium A. On the second day, hydrogen peroxide was added to the test medium A in each well in such a manner that the concentration became 0.03%. After 10 minutes, the culture medium in some wells was returned to the test medium A not containing hydrogen peroxide. In addition, the culture medium in some wells was replaced with the supernatant-added medium A of Example 1 and Reference Example 1, respectively. Furthermore, the culture medium in some wells was replaced with a culture medium containing the extract of iPS cells of Example 3. On the second day, all cells were detached from the wells using trypsin, and the cells were stained with 7-aminoactinomycin D (7-AAD), and the cell death rate was detected using a flow cytometer.

[0153] As a result, if Fig.14 and Fig.15 As shown, compared with normal skin fibroblasts, the mortality rate of skin fibroblasts induced by somatic cells of patients with xeroderma pigmentosum due to dryness stimulation is higher. Fig.15 It represents the cell death rate of skin fibroblasts induced from somatic cells of patients with xeroderma pigmentosum. Fig.15 As shown, the survival rate of skin fibroblasts in the medium supplemented with the supernatant of the medium when iPS cells were maintained, the medium supplemented with the supernatant of the medium when blood cells were induced to become iPS cells, and the medium supplemented with the extract of iPS cells was higher than that of skin fibroblasts in the unsupplemented test medium A. Therefore, it is suggested that skin fibroblasts induced from somatic cells of xeroderma pigmentosum patients are sensitive to oxidative stress and are suitable for screening of anti-oxidative stress substances.

[0154] (Reference example) Human iPS cells were cultured according to the examples described in Japanese Patent Application Laid-Open No. 2016-128396. That is, human iPS cells were cultured on feeder cells on an adhesion culture dish using the same stem cell culture medium as in Example 1. Human iPS cells were passaged weekly. During passage, human iPS cells were treated with a stripping solution containing 0.25% trypsin, 0.1 mg / mL collagenase IV, 1 mmol / LCaCl2, and 20% KSR.

[0155] The human iPS cells cultured as described above were peeled off from the adherent culture dish using ES cell dissociation solution (TrypLE Select, registered trademark, Thermo Fisher Scientific). The peeled human iPS cells were suspended and cultured for 1 week in the non-gelled human iPS cells added to the non-adhesive culture dish. As a result, embryoid bodies (EBs) were formed. The formed embryoid bodies were inoculated on the adherent culture dish and grown (outgrowth) in DMEM containing 10% FBS and 1% Anchiant (registered trademark, antifungal agent) for 1 week.

[0156] Next, the cells were detached from the adhesion culture dish using a 0.05% trypsin-EDTA solution, and the cells separated into single cells were seeded on a new adhesion culture dish, and then the cells were cultured for 1 week using DMEM containing 10% FBS as a culture medium.

[0157] After confirming that the cells have reached 70% to 80% confluence, the cells were observed. Fig.16 (a). Usually, the morphology of undifferentiated iPS cells is as follows Fig.16 As shown in the photograph (b) of FIG. Therefore, it was observed morphologically that the cells cultured in this reference example did not maintain an undifferentiated state. In addition, after culturing the cells of this reference example for 21 days, the cells were treated with anti-OCT3 / 4 antibodies labeled with fluorescent reagents and anti-NANOG antibodies labeled with fluorescent reagents, and the cells were observed under a microscope. The results are shown in FIG. Fig.17 Shown. Fig.17 (a) shows a photograph of cells observed without using excitation light. Fig.17 (b) shows a photograph of cells observed using excitation light corresponding to a fluorescent reagent bound to an anti-OCT3 / 4 antibody. Fig.17 (c) shows a photograph of cells observed using excitation light corresponding to a fluorescent reagent bound to an anti-NANOG antibody. Fig.17 (b) and Fig.17 In (c), no fluorescence was observed, indicating that the cells were OCT3 / 4 negative and NANOG negative. Furthermore, after examining the cells using a flow cytometer, it was confirmed that the cultured cells were negative for NANOG, OCT3 / 4, and TRA 1-60, which are undifferentiated markers. Thus, it was confirmed that the cells did not maintain an undifferentiated state but were differentiated.

Claims

1. An agent for preventing and improving the formation of any one of spots, wrinkles and sagging of the skin, characterized in that: Contains extracts of pluripotent stem cells.

2. A collagen production promoter, characterized in that Contains extracts of pluripotent stem cells.

3. A hair growth agent or hair growth agent, characterized in that: Contains extracts of pluripotent stem cells.

4. An activator for hair papilla cells, characterized in that: Contains extracts of pluripotent stem cells.

5. A fibroblast growth factor family production promoter, characterized in that: Contains extracts of pluripotent stem cells.

6. A vascular endothelial cell proliferation factor production promoter, characterized in that: Contains extracts of pluripotent stem cells.

7. A wound treatment agent, characterized in that: Contains extracts of pluripotent stem cells.

8. An epidermal cell proliferation promoter, characterized in that Contains extracts of pluripotent stem cells.

9. A secretion activator of IGF-1, FGF-7 or VEGF of hair papilla cells, characterized in that: Contains the supernatant of the culture medium used when reprogramming somatic cells.

10. A hair follicle growth maintaining agent, characterized in that: Contains the supernatant of the culture medium used when reprogramming somatic cells.

11. A hair papilla cell proliferation agent, characterized in that: Contains the supernatant of the culture medium used when reprogramming somatic cells.

12. A hyaluronic acid production promoter, characterized in that Contains the supernatant of the culture medium used when reprogramming somatic cells.

13. A secretion activator of IGF-1, FGF-7 or VEGF of hair papilla cells, characterized in that: Contains extracts of pluripotent stem cells.

14. A hair follicle growth maintaining agent, characterized in that: Contains extracts of pluripotent stem cells.

15. A hair papilla cell proliferation agent, characterized in that: Contains extracts of pluripotent stem cells.

16. A hyaluronic acid production promoter, characterized in that Contains extracts of pluripotent stem cells.

17. Use of a supernatant of a culture medium in which pluripotent stem cells have been induced and cultured for preparing a pharmaceutical composition for preventing the formation and improving any one of spots, wrinkles and sagging of the skin.

18. Use of a supernatant of a culture medium in which pluripotent stem cells have been induced and cultured for preparing a cosmetic composition for preventing the formation and improving any one of spots, wrinkles and sagging of the skin.

19. Use of the supernatant of a culture medium in which pluripotent stem cells are induced and cultured in the preparation of a drug for hair growth, hair development and hair maintenance.

20. Use of the supernatant of a culture medium for inducing and culturing pluripotent stem cells in the preparation of a wound treatment drug.

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