Proliferation promoter and undifferentiated state maintenance agent for skin stem cells

Abstract

The objective is to discover a novel substance that has high proliferation-promoting activity for skin stem cells and to provide it as a proliferation-promoting agent for skin stem cells. [Solution] A skin stem cell proliferation promoter and undifferentiated state maintenance agent containing, as an active ingredient, an extract of soapwort or an extract of soapwort cultivated by irradiating with a combination of two types of artificial light with different wavelength ranges.

Description

Technical Field

[0001] The present invention relates to a promoter for the proliferation of skin stem cells and an agent for maintaining an undifferentiated state.

Background Art

[0002] The skin is roughly divided into three layers: the epidermis, the dermis, and the subcutaneous tissue. Among these, the outermost epidermis is mainly composed of epidermal keratinocytes. The epidermal tissue consists of four different cell layers: the basal layer, the spinous layer, the granular layer, and the stratum corneum, in order from the bottom layer. Epidermal keratinocytes divide in the basal layer, move upward, lose their ability to divide, differentiate, reach the stratum corneum, and differentiate into corneocytes. The stratum corneum is the final differentiation product of epidermal keratinocytes and plays an important role in the barrier function against various environmental factors (such as ultraviolet rays and dryness) from the outside, and then eventually peels off from the skin. This process of proliferation and differentiation of epidermal keratinocytes is called turnover.

[0003] Epidermal keratinocytes maintain epidermal homeostasis through this turnover process, where cells are periodically regenerated. The starting point of this turnover is epidermal stem cells located in the basal layer, and they are thought to play a crucial role in this process. Epidermal stem cells specifically express high levels of marker genes such as ITGA6, ITGB1, and CD271 as undifferentiated cells. Cells that detach from the skin's basement membrane migrate to the upper layers and mature into keratinocytes while expressing differentiation markers such as KRT10, FLG, and IVL. In recent years, it has been reported that the number of these epidermal stem cells decreases with age (Non-Patent Literature 1), and that INHBA / Activin-A is a factor that suppresses the proliferation of epidermal stem cells (Non-Patent Literature 2). On the other hand, dermal fibroblasts are essential cells for maintaining youthful skin free from wrinkles and sagging. Dermal stem cells, which give rise to these dermal fibroblasts, are located directly beneath the dermal papillary layer. They proliferate and differentiate as needed, constantly supplying new dermal fibroblasts to the dermis, resulting in continuous regeneration of dermal tissue (Non-Patent Literature 3). Since dermal components such as collagen and elastin are actively produced by dermal fibroblasts derived from dermal stem cells, dermal stem cells, like epidermal stem cells, play an important role in skin regeneration. However, it has been shown that their number decreases with age (Non-Patent Literature 1). Skin aging is caused by a decline in the regenerative capacity of these skin stem cells and a lengthening of cell turnover. Therefore, if we can target these skin stem cells, especially epidermal stem cells which are the starting point of cell turnover, and dermal stem cells which are the source of dermal fibroblasts that produce dermal components such as collagen and elastin, and promote the proliferation of epidermal and dermal cells, thereby suppressing the decrease in stem cells due to aging, it is thought that we can promote more fundamental and sustainable anti-aging and regeneration of the skin. Furthermore, it is believed that culturing epidermal stem cells and dermal stem cells in vitro will enable the efficient acquisition of cultured skin, which is expected to be extremely beneficial in transplant medicine for burns and wounds, as well as in cosmetic applications.However, while methods for promoting cell proliferation in epidermal keratinocytes and dermal fibroblasts have been reported (Patent Documents 1 and 2, etc.), methods for promoting proliferation specifically in epidermal stem cells and dermal stem cells, and their mechanisms, have not been generalized, and there is currently no prospect of practical application.

[0004] Soapwort (scientific name: Saponaria officinalis) is a perennial plant belonging to the genus Saponaria in the family Caryophyllaceae. Soapwort or its extract is known to have lipase inhibitory activity (Patent Document 3) and trypsin inhibitory activity (Patent Document 4), among others.

[0005] On the other hand, methods for enhancing the medicinal effects of plants through cultivation techniques are known, such as those that specifically increase the levels of functional substances like vitamins, polyphenols, and rutin within the plant. For example, Patent Document 5 discloses a method for improving the carotene content and vitamin A efficacy of soybean sprouts by irradiating them with light in the near-ultraviolet to blue wavelength range. Patent Document 6 discloses a cultivation method for increasing the content of functional substances like α-tocopherol and vitamin C in komatsuna (Japanese mustard spinach) by irradiating it with artificial ultraviolet light for 5 minutes a day. Patent Document 7 discloses a method for increasing the vitamin C and vitamin A content of komatsuna and lettuce by adjusting the intensity of blue, red, and far-red artificial light. However, as mentioned above, methods for enhancing the medicinal effects of plants by irradiating them with light are not applicable to all plants, as it is known that the responsiveness of photosynthesis differs depending on the plant species, and that the light required also differs depending on the plant species. Furthermore, the irradiation conditions for light vary considerably even within the same plant species, depending on the desired medicinal effect, and it is not easy to identify these irradiation conditions. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2019-214533 [Patent Document 2] Japanese Patent Application Publication No. 4-305528 [Patent Document 3] Japanese Patent Publication No. 2002-179586 [Patent Document 4] Japanese Patent Publication No. 2006-182732 [Patent Document 5] Japanese Patent Application Publication No. 11-103680 [Patent Document 6] Japanese Patent Publication No. 2004-305040 [Patent Document 7] Japanese Patent Application Publication No. 8-205677 [Non-patent literature]

[0007] [Non-Patent Document 1] Akamatsu H. et al., Journal of Dermatology. 2016;Vol.43,No.3,pp.311-313, Age-related decrease in CD271+ cells in human skin [Non-Patent Document 2] Kawagishi-Hotta M. et al.,J Dermatol Sci. 2022;106(3), pp.150-158, Increase in inhibin beta A / Activin-A expression in the human epidermis and the suppression of epidermal stem / progenitor cell proliferation with aging [Non-Patent Document 3] Hasebe Y. et al., J. Dermatol. Sci., 2016, Vol.89, pp.205-207 [Overview of the project] [Problems that the invention aims to solve]

[0008] In view of the circumstances described above, the present invention aims to find a new substance that has high proliferation-promoting activity for skin stem cells and to provide it as a proliferation-promoting agent for skin stem cells. [Means for solving the problem]

[0009] The inventors of this invention conducted intensive research to solve the above problems and, as a result, discovered that an extract of soapwort has an excellent proliferation-promoting effect and an undifferentiated state-maintaining effect on skin stem cells, thus completing the present invention.

[0010] In other words, the present invention encompasses the following inventions. (1) A skin stem cell proliferation promoter containing soapwort extract as an active ingredient. (2) A skin stem cell proliferation promoter containing an extract of soapwort cultivated by irradiating with a combination of two types of artificial light with different wavelength ranges as an active ingredient. (3) The skin stem cell proliferation promoter according to (2), wherein the two types of artificial light with different wavelength ranges are red light with a wavelength range of 570-730 nm and blue light with a wavelength range of 400-515 nm. (4) The skin stem cell proliferation promoter according to (3), wherein the ratio of the photosynthetic photon flux density (PPFD) of red light to blue light is 4:1 to 2:1. (5) A skin stem cell proliferation promoter according to any one of (1) to (4), wherein the skin stem cells are epidermal stem cells and / or dermal stem cells. (6) A skin stem cell maintenance agent containing soapwort extract as an active ingredient. (7) A skin stem cell undifferentiated state maintenance agent containing an extract of soapwort cultivated by irradiating with a combination of two types of artificial light with different wavelength ranges as an active ingredient. (8) The undifferentiated state maintenance agent for skin stem cells as described in (7), wherein the two types of artificial light with different wavelength ranges are red light with a wavelength range of 570-730 nm and blue light with a wavelength range of 400-515 nm. (9) The undifferentiated state maintenance agent for skin stem cells as described in (8), wherein the ratio of photosynthetic photon flux density (PPFD) of red light to blue light is 4:1 to 2:1. (10) An agent for maintaining the undifferentiated state of skin stem cells according to any one of (6) to (9), wherein the skin stem cells are epidermal stem cells and / or dermal stem cells. (11) An expression promoter for undifferentiated genes in skin stem cells, containing an extract of soapwort as an active ingredient. (12) An expression promoter for undifferentiated genes in skin stem cells, containing an extract of soapwort cultivated by irradiating with a combination of two types of artificial light having different wavelength ranges as an active ingredient. (13) The expression promoter for undifferentiated genes in skin stem cells according to (12), wherein the two types of artificial light having different wavelength ranges are red light with a wavelength range of 570 to 730 nm and blue light with a wavelength range of 400 to 515 nm. (14) The expression promoter for undifferentiated genes in skin stem cells according to (13), wherein the ratio of the photosynthetic photon flux density (PPFD) of the red light and the blue light is 4:1 to 2:1. (15) The expression promoter for undifferentiated genes in skin stem cells according to any one of (11) to (14), wherein the skin stem cells are epidermal stem cells and / or dermal stem cells. (16) A composition for promoting the proliferation of skin stem cells, containing a proliferation promoter for skin stem cells according to any one of (1) to (4). (17) A method for culturing skin stem cells, including the step of culturing skin stem cells in a medium containing an extract of soapwort. (18) A method for culturing skin stem cells, including the step of culturing skin stem cells in a medium containing an extract of soapwort cultivated by irradiating with a combination of two types of artificial light having different wavelength ranges. (19) The method for culturing skin stem cells according to (18), wherein the two types of artificial light having different wavelength ranges are red light with a wavelength range of 570 to 730 nm and blue light with a wavelength range of 400 to 515 nm. (20) The method for culturing skin stem cells according to (19), wherein the ratio of the photosynthetic photon flux density (PPFD) of the red light and the blue light is 4:1 to 2:1. (21) The method for culturing skin stem cells according to any one of (17) to (20), wherein the skin stem cells are epidermal stem cells and / or dermal stem cells.

Advantages of the Invention

[0011] The present invention provides a skin stem cell proliferation promoter and an undifferentiated state maintaining agent that can efficiently proliferate epidermal stem cells and dermal stem cells while maintaining their undifferentiated state. Therefore, the skin stem cell proliferation promoter and undifferentiated state maintaining agent of the present invention are effective in treating, improving, and preventing various skin symptoms caused by dryness, ultraviolet rays, aging, etc. (skin diseases such as atopic dermatitis and dry skin, decreased barrier function and turnover, age spots, wrinkles, sagging, decreased firmness and elasticity, etc.), and can make a significant contribution to the fields of regenerative medicine, regenerative beauty, and anti-aging. [Modes for carrying out the invention]

[0012] The present invention will be described in detail below. 1. Proliferation promoter and undifferentiated state maintenance agent for skin stem cells The skin stem cell proliferation promoter and undifferentiated state maintenance agent according to the present invention contains a soapwort extract as an active ingredient.

[0013] In this invention, "skin stem cells" are not particularly limited as long as they are stem cells present in the epidermis, dermis, or subcutaneous adipose tissue. In this invention, "epidermal stem cells" refer to cells capable of differentiating into epidermal keratinocytes, and "dermal stem cells" refer to cells capable of differentiating into dermal fibroblasts. Furthermore, "adipose stem cells" refer to cells capable of differentiating into adipocytes, chondrocytes, osteoblasts, etc. The origin of the skin stem cells is not limited, and the invention can be effective against skin stem cells from mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cattle, sheep, goats, and pigs.

[0014] The soapwort (scientific name: Saponaria officinalis) used in this invention is also known as soapwort and is a perennial plant belonging to the genus Saponaria in the family Caryophyllaceae. In this invention, the soapwort extract refers to an extract of a part of the plant such as its flowers, fruits, seeds, leaves, stems, roots, etc., or the whole plant (whole plant), or a mixture thereof. However, in this invention, the part used as the raw material for extraction is preferably the leaves. Furthermore, the plant may be used as is for extraction, or it may be processed by drying, crushing, or finely chopping.

[0015] Furthermore, in a preferred embodiment of the present invention, soapwort cultivated under a combination of two types of artificial light with different wavelength ranges is used as the extraction material. Soapwort can be cultivated using soil or hydroponics. When using hydroponics, the seeds can be used for hydroponic cultivation after they have rooted. Cultivation is preferably carried out in a facility where temperature, light, and carbon dioxide concentration are controlled. The cultivation temperature is 15 to 30°C, preferably 20 to 25°C. The cultivation period varies depending on the conditions under which the artificial light is irradiated, but it can generally be harvested in 20 to 50 days. It is also possible to cultivate for a longer period.

[0016] The light source for irradiating artificial light can be any light source capable of selectively irradiating a specific range of wavelengths, and examples include light-emitting diodes (LEDs) and laser diodes, but LEDs are preferred.

[0017] Preferably, the two types of artificial light with different wavelength ranges used to irradiate the soapwort are red light with a wavelength range of 570-730 nm and blue light with a wavelength range of 400-515 nm, and more preferably red light with a wavelength range of 630-680 nm and blue light with a wavelength range of 430-460 nm. It is most preferable to irradiate with these lights simultaneously. The wavelength at this time refers to the maximum wavelength (peak wavelength) of the irradiation spectrum. Any light source having such a peak wavelength can be used, whether it is a custom-made or commercially available one. Optical filters may also be used to selectively irradiate with the above wavelengths. In addition to the two types of artificial light described above, sunlight or fluorescent lamps can also be used as light sources.

[0018] The amount of light irradiated is expressed as photosynthetic photon flux density (PPFD). When two types of light-emitting devices are used for irradiation, it refers to the total amount of light emitted. This amount of light is 10-300 μmol·m³ after germination. -2 s -1 Preferably, 50-200 μmol·m -2 s -1This is even more preferable. If the light intensity is outside this range, growth disorders or poor growth may occur. It is preferable to irradiate from a position 10 to 50 cm above the soapwort. The irradiation time can be appropriately changed depending on the characteristics and purpose of the plant, but 6 hours or more per day is preferable, and 12 to 18 hours is more preferable.

[0019] The ratio of red light to blue light intensity refers to the ratio of their respective PPFDs, and can be selected according to the purpose, such as yield or effectiveness.

[0020] For the extraction of soapwort as described above, the plant can be used as is, or it may be processed by drying, crushing, or finely chopping.

[0021] In the present invention, the extraction material for obtaining the soapwort extract may be soapwort grown under natural light, or soapwort grown under a combination of two types of artificial light with different wavelength ranges. The method for extracting the extract from soapwort is not particularly limited and may be any of the following: a heat extraction method (e.g., 40-100°C), a room temperature extraction method (e.g., 15-25°C), or a low-temperature extraction method (e.g., 0-15°C). Examples of solvents used for extraction include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). Preferably, polar solvents such as water, lower alcohols, and liquid polyhydric alcohols are used, and more preferably, water, ethanol, 1,3-butylene glycol, and propylene glycol. These solvents may be used individually or in mixtures of two or more. Particularly preferred extraction solvents include water, a mixed polar solvent of water and ethanol, or a mixed polar solvent of water and 1,3-butylene glycol. Among these, it is preferable to contain 20 to 100% by weight of ethanol or 1,3-butylene glycol, and most preferably 50 to 100% by weight. In addition, a solvent whose pH has been adjusted by adding an acid or alkali to the above extraction solvent can also be used.

[0022] The dielectric constant of an extraction solvent correlates with its polarity, and similar dielectric constants can be used to extract equivalent components. For example, the 30% by weight ethanol extract of soapwort used in this invention has a similar component composition to extracts using 20-60% by weight liquid polyhydric alcohols, and is particularly equivalent to the component composition of 30-50% by weight 1,3-butylene glycol extracts.

[0023] The soapwort is subjected to solvent extraction using the solvent described above. There are no particular limitations on the amount of solvent used; it should be 5 times or more, preferably 10 times or more, relative to the dry weight of the soapwort plant, but it is preferable to use 100 times or less for convenience in operations such as filtration and concentration after extraction. The extraction temperature and time can be appropriately selected depending on the type of solvent used and the pressure during extraction.

[0024] The above extract may be used as is, but if necessary, it may be used after treatment such as concentration (concentration by vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization and deodorization with activated carbon, ethanol precipitation, etc., to the extent that the effects of the present invention are achieved. Furthermore, the extracted solution may be treated by concentration to dryness, spray drying, freeze-drying, etc., and used as a dried product.

[0025] The soapwort extract obtained in this manner has the effect of proliferating skin stem cells at the biological level (in vivo) or at the culture level (extra vivo). Therefore, the skin stem cell proliferation promoter of the present invention can be incorporated into and applied to pharmaceuticals, quasi-drugs, cosmetics, food and beverages, etc., as a drug to proliferate skin stem cells when administered to mammals including humans (monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cattle, sheep, goats, pigs, etc.).

[0026] Furthermore, because the soapwort extract has the effect of maintaining the undifferentiated state of skin stem cells and promoting the expression of undifferentiated genes in skin stem cells, it can also be used as an agent for maintaining the undifferentiated state of skin stem cells and as an agent for promoting the expression of undifferentiated genes in skin stem cells. Examples of undifferentiated genes in skin stem cells include CD271, PROCR, and NANOG. In this invention, "promotion of undifferentiated gene expression" means promoting the expression of the above undifferentiated genes at the biological or culture level. In addition, in this invention, "promotion of undifferentiated gene expression" means promoting the mRNA expression and protein expression of the above undifferentiated genes. Moreover, the soapwort extract can also be used as a culture medium additive for promoting the proliferation of skin stem cells and for producing skin stem cells, as well as as a research reagent and a medical reagent.

[0027] The skin stem cell proliferation promoter of the present invention is effective in treating, improving, and preventing diseases or conditions caused by the inability to form epidermal keratinocytes or dermal fibroblasts normally due to decreased or impaired proliferative capacity of epidermal stem cells or dermal stem cells, because the active ingredient, an extract of soapwort, has a proliferation-promoting effect on either or both epidermal stem cells or dermal stem cells. Diseases or conditions caused by the inability to form epidermal keratinocytes normally due to decreased or impaired proliferative capacity of epidermal stem cells include, for example, atopic dermatitis, psoriasis (accompanied by erythema, scaling, and desquamation), delayed healing of burns and wounds, rough skin, dry skin, sensitive skin, keratin thickening, melasma, age spots, dullness, and enlarged pores. Furthermore, diseases or conditions resulting from the inability to form dermal fibroblasts normally due to decreased or impaired proliferative capacity of dermal stem cells include, for example, wrinkles, sagging, nasolabial folds, marionette lines, loss of firmness and elasticity, lack of moisture and luster, roughness, dullness, photoelastic fibrosis, scleroderma, fibrosarcoma, xeroderma pigmentosum, cutaneous histiocytoma, linear cutaneous atrophy (striata), wounds, burns, pressure ulcers, scars, and birthmarks.

[0028] The amount of the soapwort extract in the skin stem cell proliferation promoter of the present invention is not particularly limited, but depending on the properties of the extract (extract, concentrate, or dried product), for example, 0.0001 to 10% by weight is preferred, and 0.001 to 1% by weight is more preferred. If the amount is less than 0.0001% by weight, the effect may not be sufficiently exhibited. If it exceeds 10% by weight, the enhancement of the effect is unlikely to be observed and it is uneconomical.

[0029] When administering the skin stem cell proliferation promoter of the present invention into a living organism, it can be administered as is. However, it can also be incorporated into various compositions such as cosmetics, quasi-drugs, pharmaceuticals, and food and beverages with appropriate additives, as long as the effects of the present invention are not impaired, and provided as a composition for promoting the proliferation of skin stem cells. The pharmaceuticals of the present invention also include drugs used in animals, i.e., veterinary drugs.

[0030] When the skin stem cell proliferation promoter of the present invention is incorporated into cosmetics or quasi-drugs, the dosage form may be any of the following: aqueous solution, solubilized, emulsified, powder, powder dispersion, oil-liquid, gel, ointment, aerosol, water-oil two-layer system, or water-oil-powder three-layer system. Furthermore, such cosmetics and quasi-drugs can be manufactured by selecting and appropriately incorporating various components, additives, bases, etc., commonly used in topical skin compositions, along with the skin stem cell proliferation promoter of the present invention, according to the methods known in the art. The form may be any of the following: liquid, emulsion, cream, gel, paste, spray, etc. Examples of ingredients in topical skin compositions include oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), and esters (isopropyl myristate, palmitic acid, etc.). Examples of ingredients include isopropyl tinate, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extracts, various surfactants, humectants, UV absorbers, antioxidants, stabilizers, preservatives, disinfectants, fragrances, etc.

[0031] Examples of cosmetics and quasi-drugs include lotions, emulsions, gels, serums, general creams, sunscreens, packs, masks, facial cleansers, cosmetic soaps, foundations, face powders, bath additives, body lotions, and body shampoos.

[0032] When incorporating the skin stem cell proliferation promoter of the present invention into a pharmaceutical product, it can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various formulations suitable for application to the affected area. Pharmacologically and pharmaceutically acceptable additives may include, depending on the dosage form and application, formulation bases or carriers, excipients, diluents, binders, lubricants, coatings, disintegrants or disintegration aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH adjusters, propellants, colorants, sweeteners, flavoring agents, fragrances, etc., which can be appropriately added to prepare various formulations that can be administered orally or parenterally systemically or topically by various known methods. When providing the pharmaceutical product of the present invention in any of the above forms, it can be manufactured by methods commonly used by those skilled in the art, such as those indicated in the individual articles of the General Provisions for Formulations of the Japanese Pharmacopoeia [2].

[0033] The form of the pharmaceutical product of the present invention is not particularly limited, but examples include oral preparations such as tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs; parenteral preparations such as injectable preparations (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drips, suppositories, ointments, lotions, eye drops, sprays, transdermal preparations, transmucosal preparations, and patches. It may also be provided as a dried product that is redissolved before use, and in the case of injectable preparations, it is provided in the form of unit dose ampoules or multi-dose containers.

[0034] Oral formulations may use, but are not limited to, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; disintegrants or disintegration aids such as carboxymethylcellulose, carboxymethylcellulose calcium, starch, or hydroxypropylcellulose; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc; coating agents such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol, or titanium dioxide; and bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat.

[0035] Parenteral formulations may use, but are not limited to, solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, alum solution, and vegetable oil; isotonic agents such as glucose, sodium chloride, and D-mannitol; and pH adjusters such as inorganic acids, organic acids, inorganic bases, or organic bases.

[0036] When the skin stem cell proliferation promoter of the present invention is used as a pharmaceutical for treating, improving, and preventing skin-related injuries and diseases, the most suitable form is a topical formulation, such as ointments, creams, gels, liquids, patches (poultices, plasters), foams, sprays, and aerosols. Ointments refer to homogeneous, semi-solid topical formulations and include oily ointments, emulsion ointments, and water-soluble ointments. Gels refer to topical formulations in which a water-insoluble water-holding compound is suspended in an aqueous solution. Liquids refer to liquid topical formulations and include lotions, suspensions, emulsions, liniments, and the like.

[0037] The pharmaceutical product of the present invention functions as a preventive agent to suppress the onset of the above-mentioned diseases and / or as a therapeutic agent to improve them to a normal state. When the skin stem cell proliferation promoter of the present invention is used as a pharmaceutical product for the treatment, improvement, and prevention of the aforementioned diseases, it can be administered orally or parenterally to mammals such as humans, mice, rats, rabbits, dogs, and cats in a wide range of dosages.

[0038] The dosage of the pharmaceutical product of the present invention can be appropriately determined according to the type of disease, the age, sex, weight, and severity of symptoms of the recipient. For example, when administered orally to an adult, the daily dose is 0.1 to 1000 mg, preferably 1 to 500 mg, and more preferably 5 to 300 mg, of soapwort extract.

[0039] The content of the skin stem cell proliferation promoter of the present invention in the cosmetics, quasi-drugs, and pharmaceuticals of the present invention is not particularly limited, but it is preferably 0.001 to 30% by weight, and more preferably 0.01 to 10% by weight, relative to the total weight of the formulation (composition). The above amounts are merely examples and should be set and adjusted as appropriate considering the type and form of the composition, the general amount used, efficacy and effects, etc. Furthermore, the method of adding the active ingredient in formulation may be added in advance or added during manufacturing, and the appropriate method should be selected considering workability.

[0040] Furthermore, the skin stem cell proliferation promoter of the present invention can also be incorporated into food and beverages. Providing it in the form of food and beverages makes it easy to ingest the active ingredients of the present invention on a daily or continuous basis. In the present invention, "food and beverages" is used to mean not only general food and beverages, but also foods other than pharmaceuticals that can be consumed for the purpose of maintaining or promoting health, such as health foods, functional foods, health functional foods, or foods for special dietary uses. Health foods include foods provided under names such as nutritional supplements, health supplements, and supplements. Health functional foods are defined by the Food Sanitation Act or the Food Promotion Act and include Foods for Specified Health Uses and Foods with Nutrient Function Claims, which can display specific health effects, the functions of nutritional components, and reduction of disease risk. The form of food and beverages may be any form suitable for consumption, such as solid, liquid, granular, granular, powder, capsule, cream, or paste.

[0041] Examples of food and beverage products include, but are not limited to, bread, noodles, confectionery, dairy products, processed seafood and livestock products, oils and fats and processed oils and fats products, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, dairy drinks, etc.), and concentrated liquids and powders for adjusting such beverages.

[0042] The food and beverages of the present invention may contain additives that are commonly used depending on their type. Any additive that is permissible from a food hygiene standpoint can be used, but examples include sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; acidulants such as citric acid, malic acid, and tartaric acid; excipients such as dextrin and starch; binders, diluents, flavorings, colorings, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, and antiseptics.

[0043] The amount of the soapwort extract used in the food and beverage of the present invention should be sufficient to exert a skin stem cell proliferation-promoting effect, but it should be set appropriately considering the general intake amount of the food and beverage, the form of the food and beverage, efficacy and effects, taste, palatability, and cost.

[0044] 2. Method for culturing skin stem cells The present invention also relates to a method for culturing skin stem cells, comprising the step of culturing skin stem cells in a medium containing an extract of soapwort.

[0045] In the culture method of the present invention, commercially available epidermal keratinocytes and fibroblasts, preferably primary cultured epidermal keratinocytes and fibroblasts, can be used as skin stem cells. Stem cells and progenitor cells can be isolated from these cells using FACS or the like, with the expression of a marker to evaluate the undifferentiated nature of stem cells as an indicator. Alternatively, immortalized cells that proliferate indefinitely while maintaining stem cell properties may be used as skin stem cells. The immortalization method is not limited to any method that immortalizes cultured cells without inducing cell death, but examples include introducing an immortalization gene into skin stem cells. Here, "immortalization gene" refers to a gene that immortalizes cells and gives them the ability to proliferate indefinitely. Examples include the telomerase reverse transcriptase (TERT) gene, genes that regulate telomerase expression or activity (e.g., Myc gene, Ras gene, etc.), and viral genes (SV40T, HPV E6-E7, EBV, etc.). The telomerase reverse transcriptase (TERT) gene is preferred, and the human telomerase reverse transcriptase (hTERT) gene is more preferred.

[0046] In the culture method of the present invention, the culture medium for culturing skin stem cells and the additives used simultaneously are not particularly limited, and any culture medium and additives commonly used for the proliferation of skin stem cells (epidermal stem cells, dermal stem cells, adipose stem cells) may be used.

[0047] Specifically, the culture medium used for culturing skin stem cells includes basic media containing components necessary for stem cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, and fatty acids), such as Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM), and Hank's balanced salt solution. Furthermore, to increase the rate of cell proliferation, the above culture medium may contain, as needed, growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, bovine serum albumin (BSA), fibronectin, progesterone, selenite, B27 supplement, N2 supplement, ITS supplement, etc., and may also contain antibiotics (penicillin, streptomycin, etc.). Each component of the culture medium should be sterilized by an appropriate method before use.

[0048] In addition to the above, it is preferable that the product contains serum (e.g., 10% FBS) at a concentration of 1-20%. However, since the composition of serum varies depending on the lot and its effectiveness can vary, it is preferable to use the product after performing a lot check.

[0049] Commercial culture media can be used to culture skin stem cells. Examples of commercially available media include Invitrogen's mesenchymal stem cell basal medium, Sanko Junyaku's mesenchymal stem cell basal medium, TOYOBO's MF medium, and Sigma's Hank's balanced salt solution.

[0050] In accordance with the skin stem cell proliferation promoter or culture method of the present invention described above, the above-mentioned soapwort extract can also be provided alone, separately from or mixed with a culture medium, as a reagent kit for promoting the proliferation of skin stem cells. The kit may include instructions for use, if necessary. Alternatively, the above-mentioned soapwort extract can be mixed with a culture medium and provided as a culture medium for promoting the proliferation of skin stem cells.

[0051] The culture vessel used for culturing skin stem cells is not particularly limited as long as it is capable of culturing stem cells, but examples include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. The culture vessel may be cell-non-adherent or cell-adherent, and can be appropriately selected according to the purpose. Cell-adherent culture vessels may be treated with cell-supporting substrates such as extracellular matrix to improve adhesion to cells. Examples of cell-supporting substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin.

[0052] The concentration of the above-mentioned soapwort extract added to the culture medium used for culturing skin stem cells can be appropriately determined in accordance with the content of the above-mentioned soapwort extract in the skin stem cell proliferation promoter according to the present invention, but for example, concentrations of 1 to 1000 μg / mL, preferably 10 to 400 μg / mL, are possible. In addition, the above-mentioned soapwort extract may be added to the culture medium periodically during the stem cell culture period.

[0053] The culture conditions for skin stem cells can follow the usual conditions used for stem cell culture, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30-40°C, preferably about 36-37°C. The CO2 gas concentration is, for example, about 1-10%, preferably about 2-5%. The culture medium is preferably changed once every 2-3 days, and more preferably daily. The culture conditions can also be adjusted as appropriate within a range in which the stem cells can survive and proliferate.

[0054] The promotion of skin stem cell proliferation can be evaluated, for example, by determining whether the number of cells in a skin stem cell proliferation promoter cultured in the presence of the skin stem cell proliferation promoter according to the present invention is significantly increased compared to stem cells cultured in the absence of the skin stem cell proliferation promoter according to the present invention. The number of cells can be measured using a commercially available cell counting kit, for example, by the MTT method or WST method. If the number of stem cells cultured for a predetermined time in the presence of the skin stem cell proliferation promoter according to the present invention is greater than the relative ratio (control) when cultured in the absence of the skin stem cell proliferation promoter according to the present invention, it can be determined that the proliferation of skin stem cells has been promoted.

[0055] The culture method of the present invention allows for the efficient proliferation of skin stem cells. Furthermore, skin stem cells produced by this culture method can generally be transplanted directly into wounds or areas where tissue regeneration is desired after being cultured in vitro. In other words, skin stem cells produced by the culture method of the present invention can be used as transplant material (cell transplant agent). [Examples]

[0056] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples.

[0057] [Example 1] Preparation of soapwort extract (1) Experimental materials and growth conditions Soapwort seeds were sown in moist vermiculite and germinated under fluorescent lighting at a temperature of 20-25°C, then grown into seedlings. Afterward, the seedlings were wrapped in sponges and grown in a hydroponic system set to a room temperature of 20-25°C. Red LEDs (peak wavelength 660nm) and blue LEDs (peak wavelength 450nm) were simultaneously irradiated from 30cm directly above the plants, resulting in a total photosynthetic photon flux density of 100 μmol·m³ from the combined red and blue LEDs. -2 s -1The plants were cultivated under the following conditions. The light intensity ratio was set to 4:1 to 2:1 for red LEDs and blue LEDs. The irradiation time was set to alternate between 16 consecutive hours of illumination and 8 consecutive hours of darkness, with a 24-hour cycle. The light intensity ratio was not changed during cultivation. As a comparative example, cultivation was also carried out under sunlight. In both cases, after 4 weeks of cultivation, the leaves were harvested and dried soapwort was obtained by hot-air drying at approximately 60°C.

[0058] (2) Example of production of soapwort extract Using the leaves of soapwort cultivated under sunlight obtained in (1), and the leaves of soapwort cultivated with varying light intensity ratios of red and blue LEDs, as extraction materials, an extract of soapwort was produced as follows.

[0059] (Manufacturing Example 1) Preparation of hot water extract of soapwort grown in sunlight 10 g of dried leaves of soapwort grown in sunlight were added to 200 mL of water and extracted at 95-100°C for 2 hours. The resulting extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 3.7 g of soapwort hot water extract.

[0060] (Manufacturing Example 2) Preparation of a 50% ethanol extract of soapwort grown in sunlight 10 g of dried leaves of soapwort grown in sunlight were immersed in 200 mL of a 50% ethanol aqueous solution at room temperature for 7 days to extract the contents. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 3.5 g of a 50% ethanol extract of soapwort.

[0061] (Manufacturing Example 3) Preparation of 100% ethanol extract of soapwort grown under sunlight 10 g of dried leaves of soapwort grown in sunlight were immersed in 200 mL of 100% ethanol at room temperature for 7 days to extract the contents. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 0.6 g of 100% ethanol extract of soapwort.

[0062] (Manufacturing Example 4) Preparation of a hot water extract of soapwort grown with a red LED:blue LED light intensity ratio of 2:1. 10g of dried soapwort leaves grown under a red LED:blue LED light intensity ratio of 2:1 was added to 200mL of water and extracted at 95-100°C for 2 hours. The resulting extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 4.0g of soapwort hot water extract.

[0063] (Manufacturing Example 5) Preparation of a 50% ethanol extract of soapwort using a red LED:blue LED light intensity ratio of 2:1. 10g of dried leaves of soapwort cultivated under a red LED:blue LED light intensity ratio of 2:1 was immersed in 200mL of 50% ethanol aqueous solution at room temperature for 7 days to extract the material. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 3.6g of a 50% ethanol extract of soapwort.

[0064] (Manufacturing Example 6) Preparation of 100% ethanol extract of soapwort cultivated with a red LED:blue LED light intensity ratio of 2:1 10g of dried leaves of soapwort cultivated under a red LED:blue LED light intensity ratio of 2:1 was immersed in 200mL of 100% ethanol at room temperature for 7 days to extract the material. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 0.4g of 100% ethanol extract of soapwort.

[0065] (Manufacturing Example 7) Preparation of a hot water extract of soapwort grown with a red LED:blue LED light intensity ratio of 3:1. 10g of dried soapwort leaves grown under a red LED:blue LED light intensity ratio of 3:1 was added to 200mL of water and extracted at 95-100°C for 2 hours. The resulting extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 4.0g of soapwort hot water extract.

[0066] (Manufacturing Example 8) Preparation of a 50% ethanol extract of soapwort using a red LED:blue LED light intensity ratio of 3:1. 10g of dried leaves of soapwort cultivated under a red LED:blue LED light intensity ratio of 3:1 was immersed in 200mL of 50% ethanol aqueous solution at room temperature for 7 days to extract the material. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 3.8g of a 50% ethanol extract of soapwort.

[0067] (Manufacturing Example 9) Preparation of 100% ethanol extract of soapwort cultivated with a red LED:blue LED light intensity ratio of 3:1 10g of dried leaves of soapwort cultivated under a red LED:blue LED light intensity ratio of 3:1 was immersed in 200mL of 100% ethanol at room temperature for 7 days to extract the solution. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 0.1g of 100% ethanol extract of soapwort.

[0068] (Manufacturing Example 10) Preparation of a hot water extract of soapwort grown with a red LED:blue LED light intensity ratio of 4:1. 10g of dried soapwort leaves grown under a red LED:blue LED light intensity ratio of 4:1 was added to 200mL of water and extracted at 95-100°C for 2 hours. The resulting extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 3.9g of soapwort hot water extract.

[0069] (Manufacturing Example 11) Preparation of a 50% ethanol extract of soapwort grown with a red LED:blue LED light intensity ratio of 4:1. 10g of dried leaves of soapwort cultivated under a red LED:blue LED light intensity ratio of 4:1 was immersed in 200mL of 50% ethanol aqueous solution at room temperature for 7 days to extract the material. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 3.6g of a 50% ethanol extract of soapwort.

[0070] (Manufacturing Example 12) Preparation of 100% ethanol extract of soapwort grown with a red LED:blue LED light intensity ratio of 4:1 10g of dried leaves of soapwort cultivated under a red LED:blue LED light intensity ratio of 4:1 was immersed in 200mL of 100% ethanol at room temperature for 7 days to extract the material. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 0.4g of 100% ethanol extract of soapwort.

[0071] [Example 2] Evaluation of the proliferation-promoting effect of each extract on skin stem cells For the Epidermal stem cells, commercially available normal human epidermal keratinocytes (Kurabo Industries Ltd.) maintained in HuMedia-KG2 medium (Kurabo Industries Ltd.) were used in the experiment. For the Dermal stem cells, commercially available normal human fibroblasts (Kurabo Industries Ltd.) maintained in DMEM medium containing 10% FBS (Nacalai Tesque Corporation) were used in the experiment. These cells were cultured in 10 cm dishes, harvested with trypsin-EDTA, and then immunostained with the stem cell marker anti-CD271 antibody (Origene). From the stained cells, the CD271-positive cell fraction was isolated using FACS Melody (Becton Dickinson, Inc.), and epidermal stem cells and dermal stem cells were obtained.

[0072] The collected epidermal stem cells and non-epidermal stem cells were each placed in 96-well plates (FALCON) in a 3x10⁶ arrangement. 4 Seeds were seeded at a density of 1 / well. The following day, each sample (extract from Preparation Examples 1-12) was added to the culture medium to a final concentration of 10 μg / mL, and the cultures were incubated for 48 hours.

[0073] The culture medium was replaced with a chromogenic reagent (CCK8: Dojindo Co., Ltd.), and the cell proliferation-promoting effect was evaluated by measuring the relative number of epidermal and dermal stem cells cultured under the sample-added conditions compared to the number of epidermal and dermal stem cells cultured under the sample-free conditions (control) (set as 100%). The results of these tests are shown in Table 1 below.

[0074] [Table 1]

[0075] As shown in Table 1, the soapwort extract exhibited excellent proliferation-promoting effects on skin stem cells (epidermal stem cells and / or dermal stem cells). Furthermore, the proliferation-promoting effect was even greater in the soapwort extract irradiated with red and blue LEDs, and the highest proliferation-promoting effect was observed in the soapwort extract with a red-to-blue LED light intensity ratio of 3:1. On the other hand, these extracts did not show any proliferation-promoting effect on non-epidermal stem cells and non-dermal stem cells, which are CD271-negative cell fractions.

[0076] [Example 3] Evaluation of the effect of each extract on maintaining the undifferentiated state of skin stem cells In Example 2, the epidermal stem cells and dermal stem cells were each placed in 12-well plates (FALCON) in a 1x10⁶ arrangement. 5 Seeds were sown at a rate of 1 / well. The following day, each sample (extract from Preparation Examples 1-12) was added to achieve a final concentration of 10 μg / mL, and the culture was incubated for 24 hours.

[0077] After culturing, the cells were washed twice with PBS(-), and RNA was extracted from the cells using Trizol Reagent (Invitrogen). The extracted RNA was reverse transcribed into cDNA using a 2-STEP Real-Time PCR Kit (Applied Biosystems). Real-time PCR (95°C: 15 seconds, 60°C: 30 seconds, 40 cycles) was then performed using an ABI7300 (Applied Biosystems) with the following primer set. In addition to the expression level of CD271, an undifferentiated marker used for sorting, the expression levels of PROCR, another epidermal stem cell marker, and NANOG, another dermal stem cell marker, were evaluated as indicators of undifferentiation. Other procedures were performed according to the prescribed method.

[0078] Primer set for CD271: 5'-CATCCTGGCTGCTGTGGTT-3'(Sequence ID 1) 5'-TGCAGCTGTTCCACCTCTTG-3'(Sequence ID 2) PROCR Primer Set: 5'-GTCTTCTTCGAAGTGGCTGTG-3'(Sequence ID 3) 5'-TTGTTTGGCTCCCTTTCGTG-3'(Sequence ID 4) Primer set for NANOG: 5'-CCTTCCTCCATGGATCTGCTT-3'(Sequence ID 5) 5'-AAGTGGGTTGTTTGCCTTTGG-3'(Sequence ID 6) Primer set for 18srRNA (internal standard): 5'-CCGAGCCGCCTGGATAC-3'(Sequence ID 7) 5'-CAGTTCCGAAAACCAACAAAATAGA-3'(Sequence ID 8)

[0079] The expression of CD271, PROCR, and NANOG (hereinafter referred to as "undifferentiated marker genes") was evaluated by calculating the relative expression level of undifferentiated marker genes (undifferentiated marker gene expression level / 18srRNA gene expression level), which was calculated as the ratio of the mRNA expression level of undifferentiated marker genes in cells without the added sample to the expression level of the internal standard 18s ribosomal RNA (18srRNA), with the value set to 100. In contrast, the relative expression level of undifferentiated marker genes in cells cultured with the added sample was calculated and evaluated. The results of these tests are shown in Table 2 below.

[0080] [Table 2]

[0081] As shown in Table 2, the soapwort extract showed excellent undifferentiated state-maintaining effects on skin stem cells (epidermal stem cells and / or dermal stem cells). Furthermore, the undifferentiated state-maintaining effect was even greater in soapwort extracts irradiated with red and blue LEDs, and among these, the highest undifferentiated state-maintaining effect was observed in the soapwort extract with a red-to-blue LED light intensity ratio of 3:1. On the other hand, these extracts did not show an undifferentiated state-maintaining effect on non-epidermal stem cells and non-dermal stem cells, which are CD271-negative cell fractions. [Industrial applicability]

[0082] The skin stem cell proliferation promoter of the present invention can promote proliferation of skin stem cells in vivo or in vitro while maintaining their undifferentiated state. Therefore, the present invention can be used in the field of manufacturing cosmetics and pharmaceuticals for treating, improving, and preventing skin diseases and conditions caused by dysfunction or failure of epidermal stem cells and dermal stem cells, as well as in the field of manufacturing transplant materials for regenerative medicine and regenerative beauty.

Claims

1. A skin stem cell proliferation promoter containing soapwort extract as an active ingredient.

2. A skin stem cell proliferation promoter containing an extract of soapwort cultivated under a combination of two types of artificial light with different wavelengths as its active ingredient.

3. The skin stem cell proliferation promoter according to claim 2, wherein the two types of artificial light with different wavelength ranges are red light with a wavelength range of 570 to 730 nm and blue light with a wavelength range of 400 to 515 nm.

4. The skin stem cell proliferation promoter according to claim 3, wherein the ratio of photosynthetic photon flux density (PPFD) of red light to blue light is 4:1 to 2:

1.

5. The skin stem cell proliferation promoter according to any one of claims 1 to 4, wherein the skin stem cells are epidermal stem cells and / or dermal stem cells.

6. A skin stem cell maintenance agent containing soapwort extract as an active ingredient.

7. This agent maintains the undifferentiated state of skin stem cells and contains an extract of soapwort cultivated under a combination of two types of artificial light with different wavelength ranges as its active ingredient.

8. The undifferentiated state maintenance agent for skin stem cells according to claim 7, wherein the two types of artificial light with different wavelength ranges are red light with a wavelength range of 570 to 730 nm and blue light with a wavelength range of 400 to 515 nm.

9. The undifferentiated state-maintaining agent for skin stem cells according to claim 8, wherein the ratio of photosynthetic photon flux density (PPFD) of red light to blue light is 4:1 to 2:

1.

10. The undifferentiated state-maintaining agent for skin stem cells according to any one of claims 6 to 9, wherein the skin stem cells are epidermal stem cells and / or dermal stem cells.

11. An agent that promotes the expression of undifferentiated genes in skin stem cells, containing soapwort extract as an active ingredient.

12. An agent that promotes the expression of undifferentiated genes in skin stem cells, containing as an active ingredient an extract of soapwort cultivated under irradiation with a combination of two types of artificial light with different wavelength ranges.

13. The expression promoter for undifferentiated genes in skin stem cells according to claim 12, wherein the two types of artificial light with different wavelength ranges are red light with a wavelength range of 570 to 730 nm and blue light with a wavelength range of 400 to 515 nm.

14. The expression promoter of undifferentiated genes in skin stem cells according to claim 13, wherein the ratio of photosynthetic photon flux density (PPFD) of red light to blue light is 4:1 to 2:

1.

15. The skin stem cell expression promoter in skin stem cells according to any one of claims 11 to 14, wherein the skin stem cells are epidermal stem cells and / or dermal stem cells.

16. A composition for promoting the proliferation of skin stem cells, comprising a skin stem cell proliferation promoter according to any one of claims 1 to 4.

17. A method for culturing skin stem cells, comprising the step of culturing skin stem cells in a culture medium containing an extract of soapwort.

18. A method for culturing skin stem cells, comprising the step of culturing skin stem cells in a culture medium containing an extract of soapwort grown by irradiating them with a combination of two types of artificial light with different wavelength ranges.

19. The method for culturing skin stem cells according to claim 18, wherein the two types of artificial light with different wavelength ranges are red light with a wavelength range of 570 to 730 nm and blue light with a wavelength range of 400 to 515 nm.

20. The method for culturing skin stem cells according to claim 19, wherein the ratio of photosynthetic photon flux density (PPFD) of red light to blue light is 4:1 to 2:

1.

21. The method for culturing skin stem cells according to any one of claims 17 to 20, wherein the skin stem cells are epidermal stem cells and / or dermal stem cells.

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