HK40113318A - Pharmaceutical composition and cosmetic composition - Google Patents

Description

Pharmaceutical compositions and cosmetic compositions

This application is a divisional application of the application filed on October 4, 2019, with international application number PCT/JP2019/039396, Chinese application number 201980073251.3, and entitled "Pharmaceutical Compositions and Cosmetic Compositions".

Technical Field

This invention relates to pharmaceutical compositions and cosmetic compositions.

Background Technology

Embryonic stem cells (ES cells) are stem cells derived from early human or mouse embryos. ES cells possess pluripotency, capable of differentiating into all cell types present in the body. Currently, human ES cells are used in cell transplantation therapies for numerous diseases, including Parkinson's disease, juvenile diabetes, and leukemia. However, obstacles exist in ES cell transplantation. In particular, ES cell transplantation can trigger the same immune rejection response as that associated with unsuccessful organ transplantation. Furthermore, the use of ES cells derived from the destruction of human embryos faces considerable ethical criticism and opposition.

Against this backdrop, Professor Shinya Yamanaka of Kyoto University successfully created induced pluripotent stem cells (iPS cells) by introducing four genes—OCT3/4, KLF4, c-MYC, and SOX2—into somatic cells. For this achievement, Professor Yamanaka was awarded the 2012 Nobel Prize in Physiology or Medicine (see, for example, Patent Document 1). iPS cells are ideal pluripotent cells that do not present rejection reactions or ethical issues. Therefore, the application of iPS cells in cell transplantation therapy is anticipated. On the other hand, there are reports of reusing the culture medium used for iPS cell culture in pharmaceutical compositions (see, for example, Patent Document 2).

Existing technical documents

Patent documents

Patent Document 1: Japanese Patent No. 4183742

Patent Document 2: Japanese Patent Application Publication No. 2016-128396

Summary of the Invention

Technical issues

However, the inventors have verified that in the culture method described in Patent Document 2, due to iPS cell differentiation, the culture medium actually reused is not the culture medium after culturing iPS cells, but rather the culture medium after culturing differentiated cells. One of the objectives of this invention is to provide pharmaceutical and cosmetic compositions that effectively utilize the culture medium of iPS cells.

Technical solution

According to an embodiment of the present invention, a pharmaceutical composition or pharmaceutical composition raw material is provided, which includes the supernatant of the culture medium used when reprogramming somatic cells.

According to embodiments of the present invention, an agent for preventing and improving the formation of any one of spots, wrinkles, and sagging skin, comprising the above-described pharmaceutical composition or pharmaceutical composition raw materials, is provided.

According to embodiments of the present invention, a cosmetic composition or cosmetic composition raw material comprising the supernatant of a culture medium used in reprogramming somatic cells is provided.

According to embodiments of the present invention, an agent for preventing and improving the formation of any one of spots, wrinkles, and sagging skin, comprising the above-described cosmetic composition or cosmetic composition raw materials, is provided.

According to embodiments of the present invention, a collagen production promoter or collagen production promoter raw material is provided, comprising the supernatant of the culture medium used in reprogramming somatic cells.

According to embodiments of the present invention, a hair growth agent, hair growth agent raw material, hair growth agent or hair growth agent raw material is provided, comprising the supernatant of the culture medium used when reprogramming somatic cells.

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

According to embodiments of the present invention, a fibroblast growth factor family production promoter or a fibroblast growth factor family production promoter raw material is provided, comprising the supernatant of the culture medium used when reprogramming somatic cells.

According to embodiments of the present invention, a vascular endothelial cell proliferation factor production promoter or a raw material for a vascular endothelial cell proliferation factor production promoter is provided, comprising the supernatant of the culture medium used when reprogramming somatic cells.

According to embodiments of the present invention, a wound treatment agent or wound treatment agent raw material comprising the supernatant of the culture medium used when reprogramming somatic cells is provided.

According to embodiments of the present invention, an epidermal cell proliferation promoter or epidermal cell proliferation promoter raw material is provided, comprising the supernatant of the culture medium used in reprogramming somatic cells.

According to embodiments of the present invention, a pharmaceutical composition or pharmaceutical composition raw material comprising an extract of stem cells is provided.

In the above-mentioned pharmaceutical composition or pharmaceutical composition raw materials, the stem cell extract may be in paste form, or the stem cell extract may be obtained by freeze-drying.

According to embodiments of the present invention, an agent for preventing and improving the formation of any one of spots, wrinkles, and sagging skin, comprising the above-described pharmaceutical composition or pharmaceutical composition raw materials, is provided.

According to embodiments of the present invention, cosmetic compositions or cosmetic composition ingredients comprising stem cell extracts are provided.

In the above-mentioned cosmetic composition or cosmetic composition raw materials, the stem cell extract may be in paste form, or the stem cell extract may be obtained by freeze-drying.

According to embodiments of the present invention, an agent for preventing and improving the formation of any one of the following skin conditions—spots, wrinkles, and sagging—is provided, comprising the above-described cosmetic composition or cosmetic composition raw materials.

According to embodiments of the present invention, a collagen production promoter or collagen production promoter raw material comprising an extract of stem cells is provided.

In the aforementioned collagen production promoters or collagen production promoter raw materials, the stem cell extract can be in paste form, or the stem cell extract can be obtained by freeze-drying.

According to embodiments of the present invention, a hair growth agent, hair growth agent raw material, hair growth product, or hair growth agent raw material comprising an extract of stem cells is provided.

In the aforementioned hair growth agents, hair growth agent raw materials, hair growth products, or hair growth agent raw materials, the stem cell extract may be in paste form, or the stem cell extract may be freeze-dried.

According to embodiments of the present invention, an activator of hair papilla cells or a raw material for activating hair papilla cells comprising extracts of stem cells is provided.

In the above-mentioned dermal papilla cell activators or dermal papilla cell activator raw materials, the stem cell extract can be in paste form, or the stem cell extract can be obtained by freeze-drying.

According to embodiments of the present invention, a fibroblast growth factor family production promoter or a fibroblast growth factor family production promoter raw material comprising an extract of stem cells is provided.

In the aforementioned fibroblast growth factor family production promoters or fibroblast growth factor family production promoter raw materials, the stem cell extract can be in paste form, or the stem cell extract can be obtained by freeze-drying.

According to embodiments of the present invention, a vascular endothelial cell proliferation factor production promoter or a raw material for a vascular endothelial cell proliferation factor production promoter comprising an extract of stem cells is provided.

In the above-mentioned vascular endothelial cell proliferation factor production promoters or raw materials for vascular endothelial cell proliferation factor production promoters, the stem cell extract can be in paste form, or the stem cell extract can be obtained by freeze-drying.

According to embodiments of the present invention, a wound treatment agent or wound treatment agent raw material comprising an extract of stem cells is provided.

In the aforementioned wound treatment agents or raw materials for wound treatment agents, the stem cell extract may be in paste form, or the stem cell extract may be obtained by freeze-drying.

According to embodiments of the present invention, an epidermal cell proliferation promoter or an epidermal cell proliferation promoter raw material comprising an extract of stem cells is provided.

In the above-mentioned epidermal cell proliferation promoters or epidermal cell proliferation promoter raw materials, the stem cell extract can be in paste form, or the stem cell extract can be obtained by freeze-drying.

According to an embodiment of the present invention, a method for screening anti-ultraviolet substances is provided, comprising: preparing skin cells differentiated and induced from pluripotent stem cells, the pluripotent stem cells being derived from somatic cells derived from patients with aging diseases or skin diseases; irradiating the skin cells with ultraviolet light; culturing the ultraviolet-irradiated skin cells in each of a plurality of different solutions; and selecting a culture medium that causes less damage to the skin cells due to ultraviolet light or a solution that allows the skin cells exposed to ultraviolet light to repair quickly.

According to an embodiment of the present invention, a method for screening anti-drying substances is provided, comprising: preparing skin cells differentiated and induced from pluripotent stem cells derived 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 solutions with high survival rates of the skin cells.

According to an embodiment of the present invention, a method for screening anti-drying substances is provided, comprising: preparing skin cells differentiated and induced from pluripotent stem cells derived 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 solutions with minimal damage to the tight junctions of the skin cells.

The above method allows for the analysis of at least one of the closing proteins and tight proteins in tight junctions.

According to an embodiment of the present invention, a method for screening antioxidants is provided, comprising: preparing skin cells differentiated and induced from pluripotent stem cells derived from somatic cells derived from patients with aging diseases or skin diseases; administering oxidative stress to the skin cells; culturing the oxidatively stressed skin cells in each of a plurality of different solutions; and selecting solutions with high survival rates of the skin cells.

According to an embodiment of the present invention, a method for screening moisturizing promoting substances is provided, comprising: preparing skin cells differentiated and induced from pluripotent stem cells, the pluripotent stem cells being derived 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 from the plurality of different solutions the solution containing the highest amount of natural moisturizing factors derived from the skin cells.

In the above methods, the natural moisturizing factor can be at least one of ceramide and filaggrin.

According to an embodiment of the present invention, a method for testing the ultraviolet tolerance of skin is provided, comprising: preparing skin cells induced from the differentiation of pluripotent stem cells, which are derived from somatic cells derived from a subject; and examining skin cell damage caused by ultraviolet radiation.

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

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

In the above method, when examining damage to tight junctions in skin cells, at least one of the closing proteins and tight proteins in the tight junctions can be analyzed.

According to an embodiment of the present invention, a method for testing the oxidative stress tolerance of skin is provided, comprising: preparing skin cells differentiated and induced from pluripotent stem cells, the pluripotent stem cells being derived from somatic cells derived from a subject; administering oxidative stress to the skin cells; and examining the survival rate of the skin cells.

According to an embodiment of the present invention, a method for examining the moisturizing ability of skin is provided, comprising: preparing skin cells differentiated and induced from pluripotent stem cells, the pluripotent stem cells being derived from somatic cells derived from a subject; culturing the skin cells; and examining the amount of natural moisturizing factors derived from the skin cells.

Invention Effects

According to the present invention, it is possible to provide pharmaceutical compositions and cosmetic compositions that effectively utilize culture media containing iPS cells.

Attached Figure Description

Figure 1 is a graph showing the results of the hyaluronic acid production experiment based on fibroblasts in Example 2.

Figure 2 is a graph showing the results of the fibroblast-based type I collagen production assay of Example 4.

Figure 3 is a table showing the results of the fibroblast-based type I collagen production assay of Example 4.

Figure 4 is a graph showing the results of the proliferative assay of dermal papilla cells in Example 5.

Figure 5 is a table showing the results of the proliferative assay of dermal papilla cells in Example 5.

Figure 6 is a graph showing the results of the VEGF production experiment based on dermal papilla cells in Example 6.

Figure 7 is a table showing the results of the VEGF production assay based on dermal papilla cells in Example 6.

Figure 8 is a graph showing the results of the FGF-7 production experiment based on dermal papilla cells in Example 7.

Figure 9 is a table showing the results of the FGF-7 production experiment based on dermal papilla cells in Example 7.

Figure 10 is a photograph showing the results of the migration assay of fibroblasts in Example 8.

Figure 11 is a photograph showing the results of the migration assay of fibroblasts in Example 8.

Figure 12 is a graph showing the results of a UV irradiation experiment on skin fibroblasts in Example 10.

Figure 13 is a graph showing the results of the dry stimulation test on skin fibroblasts in Example 11.

Figure 14 is a graph showing the results of an oxidative stress test on skin fibroblasts in Example 12.

Figure 15 is a graph showing the results of an oxidative stress test on skin fibroblasts in Example 12.

Figure 16 is a microscope photograph of the cells in the reference example.

Figure 17 is a microscope photograph of the cells in the reference example.

Figure 18 is a distribution diagram showing the flow cytometry results of the reference example cells.

Detailed Implementation

The embodiments of the present invention will now be described in detail. It should be noted that the embodiments shown below exemplify apparatus and/or methods for embodying the technical concept of the present invention, and the combination of components of the technical concept of the present invention is not specific to the following description. Various modifications can be made to the technical concept of the present invention in the claims.

(First Implementation)

The pharmaceutical compositions, pharmaceutical composition raw materials, cosmetic compositions, and cosmetic composition raw materials of the first embodiment include the supernatant of the culture medium used when reprogramming somatic cells into pluripotent stem cells such as iPS cells. The pharmaceutical compositions, pharmaceutical composition raw materials, cosmetic compositions, and cosmetic composition raw materials 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. The pharmaceutical compositions, pharmaceutical composition raw materials, cosmetic compositions, and cosmetic composition raw materials of the first embodiment may also contain the supernatant of the culture medium used when reprogramming somatic cells into pluripotent stem cells such as iPS cells in capsules such as nanocapsules or emulsions such as nanoemulsions.

iPS cells are induced in somatic cells, such as blood cells and fibroblasts, by introducing initialization factors such as OCT3/4, KLF4, c-MYC, and SOX2. The induction into iPS cells is sometimes referred to as reprogramming, initialization, transformation, transdifferentiation (or lineage reprogramming), and cell fate reprogramming. Pluripotent stem cells can be induced simultaneously with three-dimensional culture, such as suspension culture. In three-dimensional culture, gel or liquid media can be used. Pluripotent stem cells are those with a positive rate of, for example, TRA1-60, OCT3/4, SSEA3, SSEA4, TRA1-81, and NANOG of ≥30%, ≥50%, and preferably ≥80%. The gel medium may not contain feeder cells.

As a culture medium for induction culture, human ES/iPS media such as TeSR2 (STEMCELL Technologies) can be used. However, the culture medium is not limited to this; various stem cell culture media can be used. Examples include Primate ES Cell Medium, Reprostem, ReproFF, ReproFF2, ReproXF (Reprocell), mTeSR1, TeSRE8, ReproTeSR (STEMCELL Technologies), PluriSTEM (registered trademark) human ES/iPS medium (Merck), and NutriStem XF/FF Culture Medium for Human iPS and ES cells. 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 medium system (LONZA), and primate ES cell medium (ReproCELL), etc.

The gel culture medium is prepared, for example, by adding deacylated gellan gum to the above-mentioned culture medium to a final concentration of 0.5% to 0.001% by weight, 0.1% to 0.005% by weight, or 0.05% to 0.01% by weight.

Gel culture media may contain at least one high molecular weight compound selected from the group consisting of gellan gum, hyaluronic acid, Rhamsan gum, diutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectin ester acid, heparan sulfate, heparin, heparin sulfate-like substances, keratin sulfate, chondroitin sulfate, dermatan sulfate, rhamnose sulfate, and their salts. Additionally, gel culture media may also contain methylcellulose. The presence of methylcellulose can further inhibit cell aggregation.

Alternatively, the gel culture medium may also contain poly(glycerolmonomethacrylate) (PGMA), poly(2-hydroxypropylmethacrylate) (PHPMA), poly(N-isopropylacrylamide) (PNIPAM), amine-terminated, carboxylic acid-terminated, maleimide-terminated, N-hydroxysuccinimide (NHS) ester-terminated, triethoxysilane-terminated, poly(N-isopropylacrylamide-copolymer-acrylamide) (PNIPAM), etc. A small amount of thermosensitive gel selected from pylacrylamide-co-acrylamide), poly(N-isopropylacrylamide-co-acrylic acid), poly(N-isopropylacrylamide-co-butyl acrylate), poly(N-isopropylacrylamide-co-methacrylic acid), poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-isopropylacrylamide.

Alternatively, the various pharmaceutical compositions, pharmaceutical composition raw materials, cosmetic compositions, and cosmetic composition raw materials of the first embodiment include stem cell extracts. 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). For example, the stem cells have a TRA1-60 or OCT3/4 positivity rate of 30% or more, 50% or more, preferably 80% or more. When stem cells are cultured in an undifferentiated state, the culture medium contains 10 ng/mL or more, preferably 40 ng/mL or more of β-FGF. In the various pharmaceutical compositions, pharmaceutical composition raw materials, cosmetic compositions, and cosmetic composition raw materials, the stem cell extract may be a stem cell paste. The stem cell paste is obtained by grinding iPS cells. The stem cell extract may also be a freeze-dried product. Alternatively, the stem cell extract may also be a powder. Or, the stem cell extract may also be a lysate of stem cells.

The pharmaceutical composition of the first embodiment can be a topical skin composition. The pharmaceutical composition of the first embodiment can also be a skin disease treatment agent. Examples of diseases that can be treated with the skin disease treatment agent of the first embodiment include acne vulgaris, psoriasis vulgaris, keloids, seborrheic dermatitis, contact dermatitis, atopic dermatitis, atopic xerosis, dermatoporosis, actinic elastinosis, actinic keratosis, ptosis, alopecia areata, hair loss, sparse eyelashes, melasma, age spots, prickly heat, freckles, late-onset bilateral nevus of Ota, seborrheic keratosis, skin diseases caused by progeria, and herpes simplex.

Examples of conditions that can be improved or eliminated by the cosmetic composition of the first embodiment include spots, freckles, wrinkles, sagging, dryness, decreased skin elasticity, dullness, sensitive skin, dry skin, and thinning hair. Effects of the cosmetic composition of the first embodiment include adjusting skin texture, maintaining skin health, preventing roughness, firming skin, moisturizing skin, replenishing and maintaining skin's moisture and oil, maintaining skin softness, protecting skin, preventing dryness, making skin supple, giving skin elasticity, giving skin radiance, making skin smooth, providing elasticity to the skin, fading spots, inhibiting wrinkles, and making skin luminous. Furthermore, effects related to the scalp or hair of the cosmetic composition of the first embodiment include maintaining scalp health, promoting hair growth, preventing thinning hair, preventing itching, preventing hair loss, promoting hair growth, preventing post-illness or postpartum hair loss, and nourishing hair.

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 regrowth agent, and a treatment for sparse eyelashes. The pharmaceutical composition and 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 proliferation factor (VEGF) production promoter.

In hair growth, the hair matrix cells in the hair root divide, and the resulting cells continuously form hair. On the other hand, hair growth involves a cycle called the hair cycle, which repeatedly consists of anagen (growth) phase, catagen (transitional) phase, and telogen (resting) phase. Dermal papilla cells influence the proliferation and differentiation of hair follicle epithelial stem cells through the production and release of proliferation factors, thus controlling the hair cycle. It can be said that the activation of dermal papilla cells and hair matrix cells contributes to the mechanism of hair growth. Furthermore, corresponding to the hair cycle, vascular remodeling actively occurs in the hair follicle; however, if angiogenesis is impaired at this time, the supply of nutrients and oxygen for hair formation becomes insufficient. It can be said that insufficient blood flow from the hair follicle vascular network is associated with male pattern baldness (AGA).

Regarding the genes of dermal papilla cells and their relationship to germination and hair growth, the following is known: FGF-7 and IGF-1 are known to be proliferative factors secreted by papilla cells from hair matrix cells. These factors play a role in maintaining hair follicle growth. Vascular endothelial growth factor (VEGF) is secreted by dermal papilla cells and is associated with hair follicle angiogenesis. It also has an autocrine effect on dermal papilla cell proliferation, but its expression decreases as it transitions from the anagen to catagen phase. VEGF gene expression is reduced in hair tissue in AGA (male pattern baldness). VEGFB competes with the receptor for VEGF, VEGFR-1. Although VEGFB maintains the proliferative and permeability-enhancing activity of vascular endothelial cells, its effect on hair follicles remains unclear.

The pharmaceutical and cosmetic compositions of the first embodiment have the following effects: by acting directly on the dermal papilla, they promote hair growth by increasing the production of FGF-7, a hair growth promoting factor, thereby prolonging the anagen phase of the hair cycle and transforming fine hair into thicker, stronger hair; they also increase the secretion of vascular endothelial growth factor (VEGF) by dermal papilla cells, which is associated with hair follicle angiogenesis, and further promote the proliferation of dermal papilla cells through autocrine secretion. Therefore, the pharmaceutical and cosmetic compositions of the first embodiment can be used as dermal papilla cell activators.

When used as a hair growth agent or hair regrowth agent, the pharmaceutical composition and cosmetic composition of the first embodiment may include other active ingredients such as minoxidil, thymol, pantothenic acid ethyl ether, tocopheryl acetate, dipotassium glycyrrhizate, and adenosine.

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.

The pharmaceutical and cosmetic compositions of the first embodiment include an effective amount of the supernatant of the culture medium used in reprogramming somatic cells. Alternatively, the pharmaceutical and cosmetic compositions of the first embodiment include an effective amount of stem cell extract. Here, an effective amount refers to an amount that can exert its efficacy as a pharmaceutical or cosmetic composition. The effective amount is appropriately set according to the patient's age, the target disease, the presence or absence of other active ingredients, and the amount of other complexes.

The pharmaceutical and cosmetic compositions of the first embodiment may include formulation-permitted carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, analgesics, stabilizers, preservatives, antiseptics, and physiological saline, etc. 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 rubber arabic, sodium alginate, and astragalus gum.

Examples of suspending agents include glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, carboxymethylcellulose, and sodium lauryl sulfate. Examples of analgesics include benzyl alcohol, chlorobutanol, and sorbitol. Examples of preservatives include allyl alcohol and ascorbic acid. Examples of stabilizers include phenol, chlorobenzenesulfonate, benzyl alcohol, chlorobutanol, and toluenemethyl ester. Examples of preservatives include chlorobenzoic acid, p-hydroxybenzoic acid, and chlorobutanol.

Additionally, within the scope of achieving the objectives of the pharmaceutical composition and cosmetic composition of the first embodiment, the following components may be incorporated into the pharmaceutical composition and cosmetic composition of the first embodiment, said components including: water, ethanol, surfactants (cationic, anionic, nonionic, and amphoteric surfactants, etc.), humectants (glycerin, 1,3-butanediol, 1,2-propanediol, 1,3-propanediol, pentanediol, polyquaternium salts, amino acids, urea, pyrrolidone carboxylates, nucleic acids, monosaccharides, and oligosaccharides, etc., and their derivatives, etc.), thickeners (polysaccharides, polyacrylates, carboxyvinyl polymers, polyvinylpyrrolidone, polyvinyl alcohol, chitin, chitosan, alginate, carrageenan, xanthan gum, and methylcellulose, etc., and their derivatives, etc.), waxes, petrolatum, hydrocarbons, etc. Saturated fatty acids, unsaturated fatty acids, and silicone oils, as well as their derivatives; triglycerides such as caprylic/capric triglycerides and caprylic acid triglycerides; ester oils such as isopropyl stearate; natural oils (olive oil, camellia oil, avocado oil, almond oil, cocoa butter, evening primrose oil, grapeseed oil, macadamia nut oil, eucalyptus oil, rosehip oil, squalane, neroli oil, lanolin, and ceramides, etc.); preservatives (hydroxybenzoic acid derivatives, dehydroacetates, photosensitizers, sorbic acid, and phenoxyethanol, as well as their derivatives, etc.); bactericides (sulfur, trichloroaniline, salicylic acid, zinc pyrithione, and juniper alcohol, as well as their derivatives, etc.); ultraviolet absorbers (para-aminobenzoic acid and methoxycinnamic acid, as well as their derivatives, etc.); anti-inflammatory drugs (allantoin, bisabolol, etc.). ε-aminohexanoic acid, acetylbenzoylcysteine, and glycyrrhizic acid, and their derivatives, etc.), antioxidants (tocopherol, BHA, BHT, and astaxanthin, and their derivatives, etc.), chelating agents (ethylenediaminetetraacetic acid and hydroxyethanediphosphonic acid, and their derivatives, etc.), plant and animal extracts (herbs such as *Hippophae rhamnoides*, aloe vera, *Citrus aurantium*, *Scutellaria baicalensis*, *Phellodendron chinense*, seaweed, papaya, chamomile, licorice, kiwifruit, 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, and their constituent components, purified products, or fermented products, etc.), pH adjusters (inorganic acids, inorganic acid salts, organic acids and organic acid salts, and their derivatives, etc.), vitamins. Vitamins (such as vitamins A, B, C, and D, ubiquinone, and nicotinamide, and their derivatives), fermentation broths of yeast, Aspergillus, and lactic acid bacteria, galactase culture media, whitening agents (tranexamic acid, cetyl tranexamic acid hydrochloride, 4-n-butylresorcinol, arbutin, kojic acid, ellagic acid, glycyrrhizin flavonoids, nicotinamide, and vitamin C derivatives), ceramide/ceramide derivatives, anti-wrinkle agents (retinol and retinaldehyde, and their derivatives, nicotinamide and oligopeptides, and their derivatives, neutrophil elastase inhibitors, and certain natural and synthetic components with MMP-1 and MMP-2 inhibitory effects), titanium dioxide, talc, mica, silica, zinc oxide, iron oxide, silicon, and powders obtained by processing them.

It should be noted that the ingredients that can be added to the pharmaceutical composition and cosmetic composition of the first embodiment are not limited to those described above; any ingredient that can be used in the pharmaceutical composition and cosmetic composition can be freely selected. When using the pharmaceutical composition and cosmetic composition of the first embodiment as a wet dressing, in addition to the above-mentioned ingredients, a matrix (such as kaolin and bentonite) and a gelling agent (such as polyacrylate and polyvinyl alcohol) can be added within the scope of achieving the purpose. When using the pharmaceutical composition and cosmetic composition of the first embodiment as a shower gel, sulfates, bicarbonates, borates, pigments, and moisturizers can be appropriately added within the scope of achieving the purpose and formulated into a powder or liquid form.

The pharmaceutical and cosmetic compositions of the first embodiment can be manufactured using conventional methods well known in the art.

(Second Implementation)

The screening method for anti-ultraviolet substances in the second embodiment includes: preparing skin cells derived from iPS cell differentiation, wherein the iPS cells are produced from somatic cells derived from patients with aging diseases such as progeria or skin diseases; irradiating the randomly differentiated skin cells with ultraviolet light; culturing the ultraviolet-irradiated skin cells in each of several different solutions; and selecting solutions that result in less damage to the skin cells caused by ultraviolet light or solutions that allow for faster repair of skin cells exposed to ultraviolet light. It should be noted that patients are not limited to humans, but also include non-human animals.

While Werner syndrome, xeroderma pigmentosum, and Cockayne syndrome are cited as examples of progeria, there are no specific limitations. Somatic cells derived from progeria patients can include, for example, fibroblasts, blood cells, epithelial cells, adult stem cells, keratinocytes, dermal papilla cells, and dental pulp stem cells, but there are no specific limitations. iPS cells are induced by introducing initialization factors such as OCT3/4, KLF4, c-MYC, and SOX2 into somatic cells derived from progeria patients. When differentiating skin cells from iPS cells, for example, iPS cells are seeded in low-adhesion culture dishes containing culture medium without bFGF. Then, the culture medium is changed every 2 days, and embryoid bodies (EBs) formed after 8 days are seeded into the culture dishes to allow cell adhesion, and the cells are cultured in 10% FBS medium. Even when the cells reach confluence, they are detached from the culture dishes using trypsin and passaged. This passage culture is repeated for one month. Then, fibroblast markers such as CD13 are used to confirm that the cells have differentiated into fibroblasts. Alternatively, iPS cell markers such as TRA 1-60 can be used to confirm whether undifferentiated iPS cells remain in the cells. Although differentiated skin cells can be induced, such as skin fibroblasts, there are no particular limitations.

Differentiation-induced skin cells are irradiated with ultraviolet (UV) light after appropriate culture. The intensity, wavelength range, and irradiation time of the UV light are appropriately set according to the intended use, method of application, and dosage of the selected UV-resistant substance. The UV-irradiated skin cells are cultured in multiple different solutions, each containing a different substance selected as the target substance. The solutions may be culture media. The target substance in the solutions and the culture time are appropriately set according to the intended use, method of application, and dosage of the selected UV-resistant substance.

Then, solutions with minimal skin cell damage or rapid skin cell repair are selected as the solutions containing UV-protective substances. For example, skin cells cultured in each of multiple solutions are analyzed, and one or more solutions used for culturing skin cells with minimal damage are selected, while one or more solutions used for culturing skin cells with significant damage are excluded. Alternatively, skin cells cultured in each of multiple solutions are analyzed, and one or more solutions used for culturing skin cells that repair rapidly are selected, while one or more solutions used for culturing skin cells that repair slowly or fail to repair are excluded.

According to the present invention, skin cells induced by iPS cells derived from somatic cells of patients with aging diseases such as progeria and skin diseases, compared to skin cells of healthy individuals, tend to be less resistant to UV radiation. Therefore, by using skin cells induced by iPS cells derived from somatic cells of patients with aging diseases such as progeria and skin diseases, effective UV-resistant substances can be screened.

(Third implementation method)

The method for testing the skin's ultraviolet tolerance according to the third embodiment includes: preparing skin cells derived from pluripotent stem cells differentiated and induced from somatic cells derived from the subject; irradiating the skin cells with ultraviolet light; and examining the skin cells for damage caused by ultraviolet light.

The subject can be a patient with a disease or a healthy individual. Somatic cells derived from the subject are obtained beforehand; this method may not require the step of obtaining somatic cells from the subject. If the skin cell damage caused by ultraviolet radiation is significant, the subject's skin cells can be determined to lack ultraviolet tolerance. If the skin cell damage caused by ultraviolet radiation is minor, the subject's skin cells can be determined to possess ultraviolet tolerance. According to this method, it is possible to examine whether a subject's skin cells possess ultraviolet tolerance based on the results of examinations of skin cell damage caused by ultraviolet radiation.

(Fourth Implementation)

The screening method for anti-drying substances according to the fourth embodiment includes: preparing skin cells induced from the differentiation of iPS cells, which are produced from somatic cells derived from patients with aging diseases such as progeria and skin diseases; drying the skin cells; culturing the dried skin cells in each of several different solutions; and selecting the solution with high survival rate of the skin cells.

Skin cells, induced to differentiate in the same manner as in the second embodiment, are dried after appropriate culture. During drying, the culture medium surrounding the skin cells is removed. Alternatively, airflow can be applied to the skin cells, or a desiccant can be used to dry them. The drying method and time for the skin cells are appropriately set according to the intended use, dosage, and amount of the selected anti-drying substance. The dried skin cells are cultured in multiple different solutions, each containing a different substance to be screened. The solutions can be culture media. The selected substances in the solutions and the culture time are appropriately set according to the intended use, dosage, and amount of the selected anti-drying substance.

Then, the viability of skin cells cultured in each of the multiple solutions is detected, and the solution with high skin cell viability is selected as the solution containing the anti-drying substance. For example, the viability of skin cells cultured in each of the multiple solutions is detected, and one or a group of solutions used for culturing skin cells with high viability are selected, while one or a group of solutions used for culturing skin cells with low viability are excluded.

It should be noted that solutions with minimal damage to the tight junctions of skin cells can be chosen instead of solutions with high skin cell viability. In this case, at least one of the closing proteins and dentin in the tight junctions can be analyzed, and the solution with a higher amount of either protein can be selected. Generally, if skin cells are dry, tight junctions are damaged, and the amounts of closing and dentin decrease. Therefore, for example, by analyzing the tight junctions of skin cells cultured in each of several solutions, one or more solutions used for culturing skin cells with minimal damage to tight junctions can be selected, while one or more solutions used for culturing skin cells with significant damage to tight junctions can be excluded.

According to the present invention, skin cells induced by iPS cells derived from somatic cells of patients with aging diseases such as progeria and skin diseases, compared to skin cells of healthy individuals, tend to be less resistant to dryness. Therefore, by using skin cells induced by iPS cells derived from somatic cells of patients with aging diseases such as progeria and skin diseases, effective anti-dryness substances can be screened.

(Fifth implementation method)

The fifth embodiment of the method for testing the dryness tolerance of skin includes: preparing skin cells derived from pluripotent stem cells differentiated and induced from somatic cells derived from a subject; drying the skin cells; and checking the survival rate of the skin cells.

The subject can be a patient with a disease or a healthy individual. Somatic cells derived from the subject are obtained beforehand from the subject; alternatively, the method may not include the step of obtaining somatic cells from the subject. If the skin cell survival rate is high, the subject's skin cells can be considered to have dryness tolerance. If the skin cell survival rate is low, the subject's skin cells can be considered to lack dryness tolerance. According to this method, it is possible to examine whether a subject's skin cells have dryness tolerance based on the results of skin cell survival rate testing.

It should be noted that examining damage to the tight junctions of skin cells can be used instead of examining skin cell viability. Small damage to tight junctions indicates that the subject's skin cells are tolerant to dryness, while large damage indicates that the subject's skin cells are not tolerant to dryness.

(Sixth Implementation Method)

The method for screening antioxidant stress substances according to the sixth embodiment includes: preparing skin cells induced from the differentiation of iPS cells, which are produced from somatic cells derived from patients with aging diseases such as progeria and skin diseases; subjecting the skin cells to oxidative stress; culturing the oxidatively stressed skin cells in each of a plurality of different solutions; and selecting solutions with high survival rates of skin cells.

Skin cells differentiated and induced in the same manner as in the second embodiment are subjected to oxidative stress after appropriate culture. While the method of administering oxidative stress to the skin cells is not particularly limited, it includes, for example, adding an oxidizing substance such as hydrogen peroxide to the culture medium in which the skin cells are cultured. The method and duration of administering oxidative stress to the skin cells are appropriately set according to the purpose, method of administration, and dosage of the selected antioxidant. After the oxidatively stressed skin cells are removed, for example, from the oxidative stress-inducing substance, they are cultured in each of several different solutions containing different substances that are the targets for screening. The solutions may be culture media. The target substances in the solutions and the culture time are appropriately set according to the purpose, method of administration, and dosage of the selected antioxidant.

Then, the viability of skin cells cultured in each of the multiple solutions was tested, and the solution with the high skin cell viability was selected as the solution containing the antioxidant stress agent. For example, the viability of skin cells cultured in each of the multiple solutions was tested, and one or a group of solutions used for culturing skin cells with high viability were selected, while one or a group of solutions used for culturing skin cells with low viability were excluded.

According to the present invention, skin cells induced by iPS cells derived from somatic cells of patients with aging diseases such as progeria and skin diseases, compared to skin cells of healthy individuals, tend to be less resistant to oxidative stress. Therefore, by using skin cells induced by iPS cells derived from somatic cells of patients with aging diseases such as progeria and skin diseases, effective antioxidants can be screened.

(Seventh Implementation)

The method for testing the oxidative stress tolerance of skin according to the seventh embodiment includes: preparing skin cells derived from pluripotent stem cells differentiated and induced from somatic cells derived from the subject; administering oxidative stress to the skin cells; and examining the survival rate of the skin cells.

The subject can be a patient with a disease or a healthy individual. Somatic cells derived from the subject are obtained beforehand from the subject; this method may not require the step of obtaining somatic cells from the subject. If the damage to skin cells due to oxidative stress is significant, the subject's skin cells can be considered to lack oxidative stress tolerance. If the damage to skin cells due to oxidative stress is minor, the subject's skin cells can be considered to have oxidative stress tolerance. According to this method, the oxidative stress tolerance of a subject's skin cells can be determined based on the examination results of skin cell damage caused by oxidative stress.

(Eighth Implementation)

The method for screening moisturizing promoting substances according to the eighth embodiment includes: preparing skin cells differentiated and induced from pluripotent stem cells, which are derived from somatic cells of patients with aging diseases such as progeria and skin diseases; culturing the skin cells in each of a plurality of different solutions; and selecting the solution from the plurality of different solutions that contains the largest amount of natural moisturizing factors derived from the skin cells.

Skin cells induced by differentiation in the same manner as in the second embodiment were cultured in each of several different solutions, each containing a different substance to be screened. The screening substance in the solution and the culture time were appropriately set according to the purpose, usage, and dosage of the moisturizing promoting substance being screened.

Next, the amount of natural moisturizing factors derived from skin cells in each of the multiple solutions is measured. While there are no specific limitations on natural moisturizing factors, ceramides and filaggrins are examples. Solutions with higher amounts of natural moisturizing factors are selected as those containing moisturizing-promoting substances. For example, by measuring the amount of natural moisturizing factors in each of the multiple solutions, one or a group of solutions with higher amounts of natural moisturizing factors is selected, and one or a group of solutions with lower amounts of natural moisturizing factors is excluded.

According to the present invention, the expression levels of moisturizing-promoting substances in skin cells tend to decrease as aging and/or skin diseases progress. Therefore, by using skin cells induced from iPS cells derived from somatic cells of patients with aging diseases such as progeria and skin diseases, it is possible to screen for effective moisturizing-promoting substances.

(Ninth Implementation)

The method for examining the skin's moisturizing ability according to the ninth embodiment includes: preparing skin cells derived from pluripotent stem cells differentiated and induced from somatic cells derived from the subject; culturing the skin cells; and examining the amount of natural moisturizing factors derived from the skin cells.

The subject can be a patient with a disease or a healthy individual. Somatic cells derived from the subject are obtained beforehand from the subject; this method may not require the step of obtaining somatic cells from the subject. If the amount of natural moisturizing factor derived from the skin cells is high, the subject's skin cells can be considered to have high moisturizing capacity. If the amount of natural moisturizing factor derived from the skin cells is low, the subject's skin cells can be considered to have low moisturizing capacity. According to this method, the moisturizing capacity of a subject's skin cells can be examined based on the amount of natural moisturizing factor derived from the skin cells.

Example

(Example 1: Preparation of a culture medium for simultaneously reprogramming blood cells into iPS cells)

Hematopoietic cell culture medium was prepared by adding growth factors to serum-free, animal-derived hematopoietic cell culture medium (Stemspan ACF, Stem Cell Technology Ltd.). 2 × ^10⁵ blood cells (peripheral blood mononuclear cells) were seeded into each well of a 12-well culture dish, and hematopoietic cell gel culture medium was added to each well to suspend the blood cells in the gel. The 12-well culture dishes were then placed in a _CO₂ incubator at 37°C for suspension culture of the cells.

Three days after the start of cell culture, add appropriate amounts of hematopoietic cell gel medium to each well. Six days after the start of cell culture, add the Sendai Virus Vector Kit for iPS Cell Production (CytoTune 2.0 Reprogramming Kit, registered trademark, Thermo Fisher Scientific) to the culture medium in each well to introduce the gene, either by setting the viral titer to 1-20, or by electroporating the episome plasmid (Thermo Fisher Scientific) to introduce the gene. Then add gel medium to each well and culture the cells.

A stem cell gel medium was prepared by adding gellan gel to the hES medium to achieve a final concentration of 0.02%. Starting two days after infection, 2 mL of stem cell gel medium was added to each well every two days. After 14 days of infection, iPS cell clusters were confirmed, and the gel medium supernatant was collected. The collected gel medium supernatant was filtered and sterilized, and the supernatant passing through the filter was used as the supernatant for the reprogramming medium in Example 1.

(Refer to Example 1: Preparation of a culture medium for culturing iPS cells while maintaining their undifferentiated state)

Human iPS cells were cultured for adhesion maintenance using mTeSR1 (registered trademark, Stem Cell Technology Co., Ltd.) 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, cells were treated with ES cell dissociation medium (TrypLE Select, registered trademark, Thermo Fisher Scientific Co., Ltd.).

Human iPS cells, maintained and cultured as described above, were detached from the adhesion culture dish and separated into single cells using ES cell dissociation medium (TrypLE Select, registered trademark, Thermo Fisher Scientific). The human iPS cells were then seeded into stem cell culture medium gelled with gellan gel and 10 μmol/L of the ROCK inhibitor (Selleck), and cultured in suspension for 14 days. During the 14-day suspension culture, the gelled stem cell culture medium was replenished to the culture vessel every two days.

Next, the gelled stem cell culture medium containing suspended human iPS cells was filtered through a sieve filter to remove cell clumps. Then, the filtered gelled stem cell culture medium was centrifuged at 1500g for 5 minutes to precipitate the cells and gel. The supernatant of the centrifuged stem cell culture medium was recovered and centrifuged again at 3000 rpm for 3 minutes. The supernatant of the centrifuged stem cell culture medium was then filtered through a 0.22μm filter. This filtered supernatant of the stem cell culture medium was used as the supernatant of the culture medium after maintaining iPS cell culture in Reference Example 1.

In addition, iPS cells obtained from maintenance culture were confirmed to be positive for NANOG, OCT3/4 and TRA1-60, which are markers of undifferentiated cells.

(Example 2: Hyaluronic acid production assay based on fibroblasts)

DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin was prepared as proliferation medium A. Then, normal human fibroblasts derived from adults (KF-4109, Strain No. 01035, Kurashiki Spinning Co., Ltd., Japan) were suspended in proliferation medium A at a concentration of 5× ^10³ cells/0.1 mL/well and seeded into 96-well plates, and cultured in a _CO₂ incubator (5% _CO₂ , 37℃) for 1 day.

DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin was prepared as test medium A. Then, the supernatants from Example 1 and Reference Example 1 were mixed with test medium A to obtain 10% supernatant-added medium A for Example 1 and Reference Example 1. The proliferation medium A in a portion of the wells containing fibroblasts was replaced with supernatant-added medium A from Example 1 and Reference Example 1, respectively. Additionally, a portion of the wells containing proliferation medium A was replaced with DMEM medium without FBS, penicillin-streptomycin, and supernatant (without test medium A) as a negative control.

After changing the culture medium, fibroblasts were cultured for 3 days. The supernatant of the culture medium was recovered, and the hyaluronic acid concentration in the supernatant was measured using a DueSet Hyaluronan kit (DueSet Hyaluronic Acid Assay Kit) (Cat. No.: DY3614, R&D Systems). The results are shown in Figure 1. It was confirmed that fibroblasts cultured in a medium supplemented with the supernatant of the reprogrammed culture medium of Example 1 produced several times more hyaluronic acid compared to fibroblasts cultured in a medium supplemented with the supernatant of the maintenance culture medium for iPS cells in Reference Example 1.

(Example 3: Preparation of iPS cell extract)

iPS cells were cultured on various matrix gel and laminin coatings using Teser1, Teser2, StemFit, Essential8, Teser-E8, and Nutri Stem. When the iPS cells reached 80% confluence, they were detached from the culture vessel using Tryple Select (cell dissociation medium). The solution containing the detached iPS cells was centrifuged at 200g for 5 minutes, and the iPS cells were collected in 1.5mL test tubes. The cell clusters were then ground using a mortar and pestle (Thermo Fisher Scientific). The iPS cell extract containing the ground iPS cell paste was flash-frozen with liquid nitrogen. When using the iPS cell extract, it was resuspended in 5mL of culture medium and incubated overnight at 4°C. The following day, the suspension was centrifuged at 1500g for 10 minutes to remove cell debris from the solution. The solution was then filtered, and the solution that passed through the filter was used as the extract for iPS cells.

(Example 4: Assay on type I collagen production based on fibroblasts)

Normal human fibroblasts derived from adults were cultured using proliferation medium A, as in Example 2. Additionally, test medium A was prepared in the same manner as in Example 2. Then, the iPS cell extract from Example 3 was mixed with test medium A to obtain extract-added medium A containing 50.0 v/v% or 100.0 v/v% of the iPS cell extract from Example 3. The proliferation medium A in a portion of the wells containing the fibroblasts was replaced with extract-added medium A. Furthermore, the proliferation medium A in a portion of the wells was replaced with DMEM medium (without test medium A) without FBS, penicillin-streptomycin, and the iPS cell extract as a negative control.

After changing the culture medium, fibroblasts were cultured for 3 days, and the supernatant was recovered and stored at -80°C. The supernatant was then thawed, and the concentration of type I collagen in the supernatant was detected using a human type I collagen enzyme-linked immunosorbent assay (ELISA) kit (model: EC1-E105). The results are shown in Figures 2 and 3. It was confirmed that fibroblasts cultured with iPS cell extract produced significantly more collagen compared to those cultured with medium without iPS cell extract.

(Example 5: Proliferative assay of dermal papilla cells)

Prepare a dermal papilla cell-specific culture medium (model: TMTPGM-250, TOYOBO) pre-added with special additives (fetal bovine serum, insulin-transferrin-triiodothyronine mixture, bovine pituitary extract, and cyproteron acetate) as proliferation medium B. Next, normal human dermal papilla cells (model: CA60205a, Lot No.: 2868, TOYOBO) were suspended in proliferation medium B at a concentration of 1.2 × ^10⁴ cells/0.3 mL/well and seeded into type I collagen-coated 48-well plates. The cells were then cultured for 1 day in a _CO₂ incubator (5% _CO₂ , 37°C).

The iPS cell extract from Example 3 was mixed with dermal papilla cell-specific culture medium (additive-free test medium B) without additives to obtain extract-added medium B containing 50.0 v/v% or 100.0 v/v% of the iPS cell extract from Example 3. A portion of the wells of proliferation medium B were replaced with dermal papilla cell-specific culture medium (additive-free test medium B) without additives and iPS cell extract as a negative control.

Hair papilla cells were cultured in the replaced culture medium for 3 days, and viable cell counts were determined using the WST-8 assay. The results are shown in Figures 4 and 5. It was confirmed that hair papilla cells proliferated better when using additive-free test medium B containing the extract from Example 3, compared to using additive-free test medium B without iPS cell extract. Therefore, this suggests that the iPS cell extract has therapeutic effects on thinning hair, prevention of hair loss, promotion of hair growth, and other hair regrowth-promoting effects.

(Example 6: VEGF production assay based on dermal papilla cells)

Normal human dermal papilla cells were cultured in proliferation medium B for 1 day, similar to Example 5. Then, the supernatant of the reprogrammed medium from Example 1 was added to a portion of the wells of proliferation medium B at concentrations of 10.0 v/v% and 20.0 v/v%. Additionally, the extract of iPS cells from Example 3 was added to a portion of the wells of proliferation medium B at concentrations of 50.0 v/v% and 100.0 v/v%.

A portion of the wells containing proliferation medium B were replaced with unadulterated test medium B as a negative control. Additionally, a portion of the wells containing proliferation medium B were replaced with adenosine-added medium (dermal papilla cell-specific medium with 100 μmol/L adenosine) and minoxidil-added medium (dermal papilla cell-specific medium with 30 μmol/L minoxidil) as reference controls. Furthermore, a portion of the wells containing proliferation medium B were replaced with DMSO-added medium (dermal papilla cell-specific medium with 0.1% DMSO) as a minoxidil excipient control (vehicle control).

After changing the culture medium, dermal papilla cells were cultured for 3 days. The supernatant was then collected and stored at -80°C. The supernatant was then thawed, and the concentration of vascular endothelial growth factor (VEGF) in the supernatant was detected using a human ELISA kit (model: ab100519, Abcam). The results are shown in Figures 6 and 7. The results show that the supernatant of the culture medium inducing iPS cell culture and the extract of iPS cells promote VEGF production by dermal papilla cells. This suggests that the supernatant of the culture medium inducing iPS cell culture and the extract of iPS cells are effective for hair growth, regrowth, and enhancement.

(Example 7: FGF-7 production assay based on dermal papilla cells)

Normal human dermal papilla cells were cultured in proliferation medium B for 1 day, similar to Example 5. Then, the supernatant of the reprogrammed medium from Example 1 was added to a portion of the wells of proliferation medium B at concentrations of 10.0 v/v% and 20.0 v/v%. Additionally, the extract of iPS cells from Example 3 was added to a portion of the wells of proliferation medium B at concentrations of 50.0 v/v% and 100.0 v/v%.

A portion of the wells containing proliferation medium B were replaced with unadulterated test medium B as a negative control. Additionally, a portion of the wells containing proliferation medium B were replaced with adenosine-added medium (dermal papilla cell-specific medium with 100 μmol/L adenosine) and minoxidil-added medium (dermal papilla cell-specific medium with 30 μmol/L minoxidil) as reference controls. Furthermore, a portion of the wells containing proliferation medium B were replaced with DMSO-added medium (dermal papilla cell-specific medium with 0.1% DMSO) as a minoxidil excipient control.

After changing the culture medium, dermal papilla cells were cultured for 3 days. The supernatant was then collected and stored at -80°C. The supernatant was then thawed, and the concentration of fibroblast growth factor 7 (FGF-7) in the supernatant was detected using an FGF-7 Human ELISA kit (model: ab100519, Abogen Biosciences). The results are shown in Figures 8 and 9. The results indicate that the supernatant of the culture medium inducing iPS cell culture and the extract of iPS cells promote FGF-7 production in dermal papilla cells. This suggests that the supernatant of the culture medium inducing iPS cell culture and the extract of iPS cells are effective for hair growth, regrowth, and enhancement.

(Example 8: Fibroblast migration assay)

Normal human fibroblasts derived from adults were suspended in 10% FBS medium at a concentration of 1× ^10⁵ to 2× ^10⁵ cells/well and seeded into plates using a migration assay kit (Radius Cell Migration Assay, registered trademark). Next, the human fibroblasts were treated with 10 μg/mL mitomycin C (model: 20898-21, Nacalai Tesque) for 2 hours to stop cell division. The human fibroblasts were then cultured in a _CO₂ incubator (5% _CO₂ , 37°C) for 1 day.

A portion of the culture medium in the plate was replaced with a culture medium obtained by adding the iPS cell extract from Example 3 to 10% FBS medium at a concentration of 10 v/v%. Additionally, a portion of the culture medium in the plate was replaced with a culture medium obtained by adding the supernatant of the reprogrammed culture medium described in Example 1 to 10% FBS medium at concentrations of 10.0 v/v and 20.0 v/v.

Replace a portion of the proliferation medium B on the plates with epidermal cell culture medium (without added experimental medium B) used as a negative control without the addition of proliferation additives.

Human fibroblasts were treated according to the experimental protocol to perform a migration assay. During wound healing, human fibroblasts migrate towards the wound as it contracts. In this embodiment, a microplate reader was used to analyze whether human fibroblasts migrated to the portion of the wound that was previously blocked by a stopper and had not yet adhered to them. Specifically, 23 hours after changing the culture medium, epidermal cells were stained with Hechest and observed.

The results are shown in Figures 10 and 11. It was confirmed that the extract of iPS cells and the supernatant of the culture medium inducing iPS cells promoted the migration ability of human fibroblasts. Therefore, it is shown that the extract of iPS cells and the supernatant of the culture medium inducing iPS cells are effective for wound healing.

(Example 9: Preparation of cells from progeria patients)

Fibroblasts derived from Werner syndrome patients (AG04110), xeroderma pigmentosum patients (GM16684 and GM16687), and Kockaine syndrome patients (GM01098) were purchased from the Coriell Institute for Medical Research. These fibroblasts from progeria patients were induced into iPS cells. The iPS cells were then further differentiated into skin fibroblasts.

(Example 10: UV irradiation experiment on skin fibroblasts)

Normal human skin fibroblasts derived from adults and skin fibroblasts induced from somatic cells of progeria patients prepared in Example 9 were suspended in test medium A at a concentration of 2 × ^10⁵ cells/well and seeded into 6-well plates and cultured for 1 day in a _CO₂ incubator (5% _CO₂ , 37°C).

The next day, the skin fibroblasts in each well were irradiated with 302 nm ultraviolet light for 15 minutes using a UV irradiation machine. Next, the supernatant solution from Reference Example 1 and test medium A were mixed at a volume ratio of 10.00:90.00 to obtain a 10.00 v/v% supernatant supplemented with medium A from Reference Example 1. A portion of the test medium A in each well was replaced with supernatant supplemented with medium A from Reference Example 1. The next day, all cells were detached from the wells using trypsin, stained with 7-aminoactinomycin D (7-AAD), and the cell death rate was determined by flow cytometry.

As a result, as shown in Figure 12, for any type of skin fibroblast, the cell death rate of UV-irradiated cells was higher than that of cells not exposed to UV radiation. Furthermore, skin fibroblasts induced from somatic cells of progeria patients had a higher mortality rate due to UV radiation compared to normal skin fibroblasts. However, skin fibroblasts in culture media containing the supernatant of the maintenance culture medium for iPS cells had a higher survival rate than those in media without the supernatant. Therefore, this suggests that skin fibroblasts induced from somatic cells of progeria patients are sensitive to UV radiation and are suitable for screening for anti-UV substances.

(Example 11: Dryness Stimulation Test of Skin Fibroblasts)

Similar to 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, each well was dried for 40 seconds in a ventilated area of a laminar flow hood. Next, a portion of the test medium A in the wells was replaced with the supernatant supplemented with medium A from Reference Example 1. Alternatively, the supernatant solution from Example 1 was mixed with test medium A at a volume ratio of 10.00:90.00 to obtain a 10.00 v/v% supernatant supplemented with medium A from Example 1. A portion of the test medium A in the wells was replaced with the supernatant supplemented with medium A from Example 1. On the second day, all cells were peeled from the wells with trypsin, stained with 7-aminoactinomycin D (7-AAD), and the cell death rate was detected by flow cytometry.

As a result, as shown in Figure 13, skin fibroblasts induced from somatic cells of progeria patients had a higher mortality rate due to dryness stimulation compared to normal skin fibroblasts. However, skin fibroblasts in culture media containing the supernatant of the culture medium used to maintain iPS cells had a higher survival rate than those in media without the supernatant. Furthermore, skin fibroblasts in culture media containing the supernatant of the culture medium used to induce iPS cells from blood cells had a higher survival rate than those in media containing the supernatant of the culture medium used to maintain iPS cells. Therefore, this suggests that skin fibroblasts induced from somatic cells of progeria patients are sensitive to dryness stimulation and are suitable for screening anti-dryness substances.

(Example 12: Oxidative stress test of skin fibroblasts)

Similar to 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 test medium A at a concentration of 0.03% in each well. After 10 minutes, a portion of the medium in the wells was poured back into test medium A without hydrogen peroxide. Additionally, a portion of the medium in the wells was replaced with the supernatant from Example 1 and Reference Example 1, respectively, and then supplemented with medium A. Furthermore, a portion of the medium in the wells was replaced with medium containing the extract of iPS cells from Example 3. On the second day, all cells were peeled from the wells with trypsin, stained with 7-aminoactinomycin D (7-AAD), and the cell death rate was detected by flow cytometry.

The results, as shown in Figures 14 and 15, indicate that skin fibroblasts induced from somatic cells of patients with xeroderma pigmentosum exhibit a higher mortality rate due to dryness stimulation compared to normal skin fibroblasts. Figure 15 shows the cell death rate of skin fibroblasts induced from somatic cells of patients with xeroderma pigmentosum. As shown in Figure 15, skin fibroblasts in culture media containing the supernatant of the culture medium used to maintain iPS cell culture, the supernatant of the culture medium used to induce blood cells into iPS cells, and the extract of iPS cells have higher survival rates compared to skin fibroblasts in unadulterated test medium A. Therefore, this suggests that skin fibroblasts induced from somatic cells of patients with xeroderma pigmentosum are sensitive to oxidative stress and are suitable for screening for antioxidants.

(Example for reference)

Human iPS cells were cultured according to the examples described in Japanese Patent Application Publication No. 2016-128396. Specifically, human iPS cells were cultured on feeder cells in an adhesion culture dish using the same stem cell culture medium as in Example 1. The human iPS cells were passaged weekly. During passage, the human iPS cells were treated with a stripping solution containing 0.25% trypsin, 0.1 mg/mL collagenase IV, ₁ mmol/L CaCl₂, and 20% KSR.

Human iPS cells cultured as described above were detached from adhesion culture dishes using ES cell dissociation medium (TrypLE Select, registered trademark, Thermo Fisher Scientific). The detached human iPS cells were then suspended and cultured in non-adhesion culture dishes containing ungelled human iPS cells for one week. This resulted in the formation of embryoids (EBs). The resulting embryoids were then seeded onto adhesion culture dishes and allowed to grow outgrowth for one week in DMEM containing 10% FBS and 1% anthraquinone (registered trademark, antifungal agent).

Next, cells were detached from the adhesion culture dish using a 0.05% trypsin-EDTA solution, and the cells, now divided into single cells, were seeded into new adhesion culture dishes. The cells were then cultured for one week in DMEM containing 10% FBS.

After confirming that the cells had reached a confluence of 70%–80% or more, the cells were observed. A photograph of the cells cultured in this reference example is shown in Figure 16(a). Typically, undifferentiated iPS cells have the morphology shown in Figure 16(b). Therefore, morphologically, the cells cultured in this reference example did not maintain an undifferentiated state. Furthermore, after culturing the cells of this reference example for 21 days, the cells were treated with fluorescently labeled anti-OCT3/4 antibody and fluorescently labeled anti-NANOG antibody, and the cells were observed under a microscope. The results are shown in Figure 17. Figure 17(a) shows a photograph of the cells observed without excitation light. Figure 17(b) shows a photograph of the cells observed using excitation light corresponding to the fluorescent reagent bound to the anti-OCT3/4 antibody. Figure 17(c) shows a photograph of the cells observed using excitation light corresponding to the fluorescent reagent bound to the anti-NANOG antibody. No fluorescence was observed in Figures 17(b) and 17(c), confirming that the cells were OCT3/4 negative and NANOG negative. Furthermore, flow cytometry examination confirmed that the cultured cells were negative for NANOG, OCT3/4, and TRA 1-60, markers of undifferentiated cells. Therefore, it was confirmed that the cells did not remain undifferentiated but had differentiated.

Claims (20)

1. An agent for preventing and improving the formation of any one of the following skin conditions: spots, wrinkles, and sagging, characterized in that it comprises an extract of pluripotent stem cells. 2. A collagen production promoter, characterized in that it comprises an extract of pluripotent stem cells. 3. A hair growth agent or hair regrowth agent, characterized in that it comprises an extract of pluripotent stem cells. 4. An activator for dermal papilla cells, characterized in that it comprises an extract of pluripotent stem cells. 5. A fibroblast growth factor family production promoter, characterized in that it comprises an extract of pluripotent stem cells. 6. A vascular endothelial cell proliferation factor production promoter, characterized in that it comprises an extract of pluripotent stem cells. 7. A wound treatment agent, characterized in that it comprises an extract of pluripotent stem cells. 8. An epidermal cell proliferation promoter, characterized in that it comprises an extract of pluripotent stem cells. 9. A secretion activator of IGF-1, FGF-7 or VEGF in dermal papilla cells, characterized in that it comprises the supernatant of the culture medium used when reprogramming somatic cells. 10. A hair follicle growth maintenance agent, characterized in that it comprises the supernatant of the culture medium used when reprogramming somatic cells. 11. A dermal papilla cell proliferation agent, characterized in that it comprises the supernatant of the culture medium used when reprogramming somatic cells. 12. A hyaluronic acid production promoter, characterized in that it comprises the supernatant of the culture medium used in reprogramming somatic cells. 13. A secretion activator of IGF-1, FGF-7 or VEGF in dermal papilla cells, characterized in that it comprises an extract of pluripotent stem cells. 14. A hair follicle growth maintenance agent, characterized in that it comprises an extract of pluripotent stem cells. 15. A dermal papilla cell proliferation agent, characterized in that it comprises an extract of pluripotent stem cells. 16. A hyaluronic acid production promoter, characterized in that it comprises an extract of pluripotent stem cells. 17. Use of the supernatant of a culture medium inducing pluripotent stem cells in the preparation of a pharmaceutical composition for preventing and improving any one of spots, wrinkles and sagging skin. 18. Use of the supernatant of a culture medium inducing pluripotent stem cells in the preparation of a cosmetic composition for preventing and improving any one of blemishes, wrinkles and sagging skin. 19. Use of the supernatant of a culture medium inducing pluripotent stem cells in the preparation of a drug for hair growth, hair development and hair maturation. 20. Use of the supernatant of a culture medium in which pluripotent stem cells have been induced to grow in the preparation of a wound treatment drug.