Skin stem cell proliferation promoter and skin regeneration promoter

Abstract

To find a new substance having a high proliferation promoting activity on skin stem cells and having a skin tissue regeneration promoting activity on both of an epidermal layer and a dermal layer and provide the found substance as a skin stem cell proliferation promoter or a skin regeneration promoter.SOLUTION: The present invention provides a skin stem cell proliferation promoter and a skin regeneration promoter each containing a compound represented by the general formula (A) as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a skin stem cell proliferation promoter and a skin regeneration promoter. [Background technology]

[0002] When cells or organs in the tissues of vertebrates (especially mammals) are damaged due to injury, disease, or aging, the regenerative system activates to repair the damaged cells or organs. Stem cells present in the tissue play a major role in this process. Stem cells have been found to exist in all organs and tissues, including bone marrow, liver, pancreas, skin, fat, and brain, and are known to be responsible for the regeneration and homeostasis of each organ or tissue.

[0003] In recent years, it has become clear that stem cells present in organs and tissues undergo aging (see Non-Patent Document 1). Specifically, stem cell aging refers to a decline in their proliferation and differentiation capabilities, which is thought to be the cause of a decline in the regenerative capacity of organs and tissues. For example, it has been reported that the proliferation capacity of neural stem cells present in the brain and hematopoietic stem cells that generate blood cells significantly declines with age (see Non-Patent Documents 2 and 3). It has also been reported that stem cells present in skin and subcutaneous fat tissue decrease in number and their differentiation capacity declines with age (see Non-Patent Documents 4 and 5). Therefore, technologies that improve the proliferation and differentiation capacity of stem cells present in various organs and tissues are thought to be extremely effective for anti-aging (anti-aging) applications, such as maintaining tissue homeostasis, repairing and regenerating damaged tissue, and preventing, treating, and improving various diseases. In particular, skin tissue has a complex three-dimensional structure and is located at the outermost layer of the human body, making it susceptible to damage from external injuries. Furthermore, because skin tissue is deeply related to a person's appearance and beauty, advances in regenerative technologies for this tissue are extremely important. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Beane OS et al., PLoS One, 2014, Vol. 9, No. 12, e115963 [Non-patent document 2] Molofsky AV et al., Nature, 2006, Vol. 443, No. 7110, pp. 448-452 [Non-patent document 3] Geiger H. et al., Nat. Rev. Immunol., 2013, Vol. 13, No. 5, pp. 376-389 [Non-patent document 4] Akamatsu H. et al., J. Dermatol., 2016, Vol. 43, pp. 311-313 [Non-Patent Document 5] Yamada T. et al., J. Dermatol. Sci., 2010, Vol. 58, pp. 36-42 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to discover a new substance that has high proliferation-promoting activity for skin stem cells and regeneration-promoting activity for skin tissues in both the epidermal and dermal layers, and to provide it as a skin stem cell proliferation promoter and a skin regeneration promoter. [Means for solving the problem]

[0006] As a result of intensive research to solve the above problems, the inventors discovered that certain compounds containing mesenchymal stem cells as a common structure have excellent effects of promoting the proliferation of skin stem cells and promoting skin regeneration, leading to the completion of the present invention.

[0007] That is, the present invention includes the following inventions. [1] The following general formula (A): [ka] (In the formula, R 1represents an unsubstituted alkyl group having 1 to 5 carbon atoms, or the following group (B-1) or (B-2): [ka] an alkyl group having 1 to 5 carbon atoms substituted with, or the following group (B-3): [ka] indicates, R 2 represents a hydrogen atom or one of the following groups (C-1), (C-2), or (C-3): [ka] A skin stem cell proliferation promoter comprising a compound represented by the formula (I): [2] The following general formula (A): [ka] (In the formula, R 1 represents an unsubstituted alkyl group having 1 to 5 carbon atoms, or the following group (B-1) or (B-2): [ka] an alkyl group having 1 to 5 carbon atoms substituted with, or the following group (B-3): [ka] indicates, R 2 represents a hydrogen atom or one of the following groups (C-1), (C-2), or (C-3): [ka] A skin regeneration promoter comprising a compound represented by the formula (I) as an active ingredient. [3] The agent according to [1] or [2], wherein the compound represented by the general formula (A) is a compound represented by any one of the following structural formulas (1) to (9): [ka]

[0008] [4] Skin stem cells are synthesized by the following general formula (A): [ka] (In the formula, R 1 represents an unsubstituted alkyl group having 1 to 5 carbon atoms, or the following group (B-1) or (B-2): [ka] an alkyl group having 1 to 5 carbon atoms substituted with, or the following group (B-3): [ka] indicates, R 2 represents a hydrogen atom or one of the following groups (C-1), (C-2), or (C-3): [ka] A culture method for growing skin stem cells, comprising a step of culturing in a medium containing a compound represented by the formula: [5] Skin stem cells are synthesized by the following general formula (A): [ka] (In the formula, R 1 represents an unsubstituted alkyl group having 1 to 5 carbon atoms, or the following group (B-1) or (B-2): [ka] an alkyl group having 1 to 5 carbon atoms substituted with, or the following group (B-3): [ka] indicates, R 2 represents a hydrogen atom or one of the following groups (C-1), (C-2), or (C-3): [ka] A method for producing three-dimensional cultured skin, comprising culturing the compound represented by the formula (I): [6] The method according to [4] or [5], wherein the compound represented by the general formula (A) is a compound represented by any one of the following structural formulas (1) to (9): [ka] [Effects of the Invention]

[0009] The present invention provides a skin stem cell proliferation promoter capable of efficiently proliferating epidermal stem cells and dermal stem cells, and a skin regeneration promoter capable of promoting the regeneration of the entire skin tissue, including the epidermal and dermal layers. Therefore, the skin stem cell proliferation promoter and skin regeneration promoter 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 cell turnover, age spots, wrinkles, sagging, and decreased firmness and elasticity, etc.), and can make a significant contribution to the fields of regenerative medicine, regenerative cosmetics, and anti-aging. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows stained images of epidermal tissue sections of three-dimensionally cultured skin produced by culturing skin stem cells in the absence of compound (control, DMSO) and in the presence of compound, as well as the thickness ratio (%) of the epidermal layer, and the appearance and diameter ratio (%) of the same three-dimensionally cultured skin.

[0011] The present invention will be described in detail below. 1. Skin stem cell proliferation promoter and skin regeneration promoter The skin stem cell proliferation promoter and skin regeneration promoter according to the present invention (hereinafter sometimes referred to as "the agent of the present invention") are compounds represented by the following general formula (A): [ka] (In the formula, R 1 represents an unsubstituted alkyl group having 1 to 5 carbon atoms, or the following group (B-1) or (B-2): [ka] an alkyl group having 1 to 5 carbon atoms substituted with, or the following group (B-3): [ka] indicates, R 2 represents a hydrogen atom or one of the following groups (C-1), (C-2), or (C-3): [ka] The compound represented by the formula (I) is contained as an active ingredient.

[0012] R 1 The "alkyl group having 1 to 5 carbon atoms" in the above means a linear saturated hydrocarbon group having 1 to 5 carbon atoms or a branched saturated hydrocarbon group having 3 to 5 carbon atoms, and examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, and a tert-pentyl group.

[0013] More specifically, examples of the compound represented by the above general formula (A) include compounds represented by the following structural formulas (1) to (9) (referred to as compounds 1 to 9, respectively).

[0014] [ka]

[0015] In the present invention, "skin stem cells" are not particularly limited as long as they are stem cells present in the epidermis, dermis, or subcutaneous tissue. In the present invention, "epidermal stem cells" refer to cells that can differentiate into epidermal keratinocytes, and "dermal stem cells" refer to cells that can differentiate into dermal fibroblasts. The origin of the skin stem cells is not limited, and the effect can be exerted on skin stem cells of mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, and pigs.

[0016] The agent of the present invention may use any one of the above compounds 1 to 9, which are specific examples of the compound represented by the above general formula (A) (hereinafter referred to as "compound A"), as an active ingredient, or may use a combination of two or more compounds.

[0017] Because compound A has the effect of proliferating skin stem cells and promoting skin regeneration both at the living body level (in vivo) and at the culture level (in vitro), the agent of the present invention can be administered to mammals, including humans (monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc.), as a drug for promoting skin stem cell proliferation and skin regeneration, and can be incorporated into or applied to pharmaceuticals, quasi-drugs, cosmetics, etc. The agent of the present invention can also promote the proliferation of skin stem cells and can be used as a culture medium additive, research reagent, or medical reagent for producing skin stem cells and cultured skin.

[0018] The agent of the present invention, containing Compound A as an active ingredient, has the effect of promoting the proliferation of either epidermal stem cells or dermal stem cells, or both, and is therefore effective in treating, ameliorating, and preventing diseases or pathologies caused by the failure of epidermal keratinocytes or dermal fibroblasts to form normally due to a decrease or insufficiency in the proliferation ability of epidermal stem cells or dermal stem cells. Examples of diseases or pathologies caused by the failure of epidermal keratinocytes to form normally due to a decrease or insufficiency in the proliferation ability of epidermal stem cells include atopic dermatitis, psoriasis (accompanied by erythema, scales, and desquamation), delayed healing of burns and wounds, rough skin, dry skin, sensitive skin, hyperkeratosis, blemishes, dullness, and enlarged pores. Furthermore, diseases or pathological conditions resulting from the failure of dermal fibroblasts to be formed normally due to a reduced or insufficient proliferation ability of dermal stem cells include, for example, wrinkles, sagging skin, nasolabial folds (nasolabial folds), marionette lines, loss of firmness and elasticity, lack of moisture and luster, stiffness, dullness, solar elastosis, scleroderma, fibrosarcoma, xeroderma pigmentosum, cutaneous histiocytoma, linear atrophy of the skin (straea), wounds, burns, pressure ulcers, scars, birthmarks, and melasma.

[0019] The amount of compound A in the agent of the present invention is not particularly limited, but is preferably 0.00001 to 10% by weight, more preferably 0.0001 to 1% by weight, based on the total amount of the agent. If the amount is less than 0.00001% by weight, the effect may not be fully exerted.

[0020] When the agent of the present invention is administered to a living body, it can be administered as is, but it can also be provided by being incorporated into various compositions such as cosmetics, quasi-drugs, pharmaceuticals, etc. together with appropriate additives within a range that does not impair the effects of the present invention. Note that the pharmaceuticals of the present invention also include drugs used for animals, i.e., veterinary drugs.

[0021] When the agent of the present invention is incorporated into cosmetics or quasi-drugs, the dosage form may be any of an aqueous solution, solubilized solution, emulsion, powder, powder dispersion, oil solution, gel, ointment, aerosol, water-oil two-layer system, or water-oil-powder three-layer system. Furthermore, the cosmetics or quasi-drugs can be produced according to techniques known in the art by appropriately blending various ingredients, additives, bases, etc. commonly used in topical skin compositions together with the agent of the present invention, selected according to their types. The form may be any of a liquid, emulsion, cream, gel, paste, spray, etc. Examples of ingredients included in the topical skin composition 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.), esters (isopropyl myristate, palmitic acid, hydroxypropyl methylcellulose ... These include: isopropyl myristate, cetyl octanoate, glycerin 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, moisturizers, UV absorbers, antioxidants, stabilizers, preservatives, disinfectants, fragrances, etc.

[0022] Examples of types of cosmetics and quasi-drugs include lotions, emulsions, gels, beauty serums, general creams, sunscreen creams, packs, masks, facial cleansers, cosmetic soaps, foundations, powders, bath additives, body lotions, body shampoos, hair shampoos, hair conditioners, and hair growth agents.

[0023] When the agent of the present invention is incorporated 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 include formulation bases, carriers, excipients, diluents, binders, lubricants, coating agents, disintegrants or disintegration aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonicity agents, pH adjusters, propellants, colorants, sweeteners, flavoring agents, and flavoring agents, and can be added as appropriate depending on the dosage form and intended use. Various formulations suitable for oral or parenteral systemic or local administration can be prepared by various known methods. When providing the pharmaceutical product of the present invention in the above-mentioned forms, it can be manufactured by methods commonly used by those skilled in the art, such as the methods set forth in the General Provisions for Preparations [2] of the Japanese Pharmacopoeia.

[0024] The form of the pharmaceutical of the present invention is not particularly limited, and examples thereof include oral preparations such as tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs, and parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections), drip infusions, suppositories, ointments, lotions, eye drops, sprays, transdermal absorption preparations, transmucosal absorption preparations, and patches. The pharmaceutical may also be in the form of a dried product that is reconstituted when used, and in the case of an injectable preparation, it is provided in the form of a unit-dose ampule or a multi-dose container.

[0025] For oral administration formulations, for example, 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 oxide; and bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat can be used, but are not limited to these.

[0026] Formulations for parenteral administration may contain, but are not limited to, solvents such as distilled water, saline, ethanol, glycerin, propylene glycol, macrogol, alum water, and vegetable oil; isotonicity agents such as glucose, sodium chloride, and D-mannitol; and pH adjusters such as inorganic acids, organic acids, inorganic bases, and organic bases.

[0027] When the agent of the present invention is used as a pharmaceutical for treating, improving, and preventing the above-mentioned skin-related damages and diseases, the form suitable for the agent is a topical preparation, such as an ointment, cream, gel, liquid, patch (poultice, plaster), foam, spray, or atomized preparation. Ointments refer to homogeneous semi-solid topical preparations, including oleaginous ointments, emulsion ointments, and water-soluble ointments. Gels refer to topical preparations in which a water-insoluble component, a hydrated compound, is suspended in an aqueous liquid. Solutions refer to liquid topical preparations, including lotions, suspensions, emulsions, liniments, and the like.

[0028] The pharmaceutical of the present invention functions as a preventive drug that suppresses the onset of the above-mentioned diseases and / or as a therapeutic drug that improves the condition to a normal state. When the agent of the present invention is used as a pharmaceutical for treating, improving, and preventing the above-mentioned 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.

[0029] The dosage of the pharmaceutical of the present invention can be appropriately determined depending on the type of disease, the age, sex, weight, severity of symptoms, etc. For example, when orally administered to an adult, the daily dosage is 0.1 to 1000 mg, preferably 1 to 500 mg, more preferably 5 to 300 mg of the compound.

[0030] The content of the agent of the present invention in the cosmetics, quasi-drugs, and pharmaceuticals of the present invention is not particularly limited, but the content of the compound is preferably 0.001 to 30 wt. % and more preferably 0.01 to 10 wt. % relative to the total weight of the formulation (composition). The above amounts are merely examples and may be set or adjusted as appropriate taking into account the type and form of the composition, the typical amount used, efficacy, and other factors. Furthermore, the method of adding the active ingredient in the formulation may be either added in advance or during production, and may be selected appropriately taking into account workability.

[0031] 2. Skin stem cell culture method The present invention also relates to a method for culturing skin stem cells, which comprises the step of culturing skin stem cells in a medium containing compound A.

[0032] In the culture method of the present invention, the medium for culturing skin stem cells and the additives used at the same time are not particularly limited, and any medium and additives commonly used for the proliferation of skin stem cells (epidermal stem cells or dermal stem cells) may be used.

[0033] Specifically, the medium used to culture skin stem cells is a basal medium containing components necessary for the survival and proliferation of stem cells (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), or Hank's Balanced Salt Solution. Furthermore, to increase the cell proliferation rate, the above-mentioned medium may contain, as necessary, 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., as well as antibiotics (penicillin, streptomycin, etc.). Each component of the medium should be sterilized by an appropriate method before use.

[0034] In addition to the above, it is preferable that serum (e.g., 10% FBS) is contained at a content of 1 to 20%. However, since serum contains different components depending on the lot and its effects vary, it is preferable to check the lot before use.

[0035] Commercially available media can be used to culture skin stem cells, including 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.

[0036] In accordance with the skin stem cell proliferation promoter of the present invention or the culture method of the present invention, the above-mentioned compound A can be provided as a reagent kit for promoting the proliferation of skin stem cells, either alone, separately from a culture medium, or mixed with a culture medium. The kit can include an instruction manual, etc., as needed. Alternatively, the above-mentioned compound A can be mixed with a culture medium and provided as a culture medium for promoting the proliferation of skin stem cells.

[0037] The culture vessel used for culturing skin stem cells is not particularly limited as long as it is capable of culturing stem cells, and examples include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, roller bottles, etc. The culture vessel may be either non-cell-adhesive or cell-adhesive and is selected appropriately depending on the purpose. Cell-adhesive culture vessels may be treated with a cell support substrate such as an extracellular matrix to improve cell adhesion. Examples of cell support substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin.

[0038] The concentration of compound A added to the medium used for culturing skin stem cells can be determined appropriately based on the content of compound A in the skin stem cell proliferation promoter of the present invention, and may be, for example, 1 to 1000 μg / mL, preferably 10 to 400 μg / mL. Compound A may also be added to the medium periodically during the stem cell culture period.

[0039] The culture conditions for skin stem cells may be the same as those typically used for stem cell culture, and no special controls are necessary. 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 medium is preferably changed every 2-3 days, and more preferably every day. The culture conditions can be adjusted as needed within a range that allows stem cells to survive and proliferate.

[0040] Promotion of skin stem cell proliferation can be evaluated, for example, by determining whether the number of stem cells cultured in the presence of a skin stem cell proliferation promoter of the present invention is significantly increased compared to stem cells cultured in the absence of the skin stem cell proliferation promoter of the present invention. Cell number can be measured, for example, by the MTT method or WST method using a commercially available cell count measurement kit. If the measurement results show that the relative ratio of the number of skin stem cells at the start of culture to the number of stem cells after a predetermined period of culture in the presence of a skin stem cell proliferation promoter of the present invention is greater than the same relative ratio when cultured in the absence of the skin stem cell proliferation promoter of the present invention (control), it can be determined that skin stem cell proliferation has been promoted.

[0041] The culture method of the present invention allows for efficient proliferation of skin stem cells, and the skin stem cells produced by the culture can generally be cultured in vitro and then transplanted into a wound or a site where tissue regeneration is desired, for example by direct injection. In other words, the skin stem cells produced by the culture method of the present invention can be used as a transplant material (cell transplant agent).

[0042] 3. Method for producing three-dimensional cultured skin The present invention also relates to a method for producing three-dimensionally cultured skin, comprising culturing skin stem cells in a medium containing Compound A. Skin stem cells can be derived from skin cells present in the epidermis, dermis, and subcutaneous tissue that make up the skin, specifically stem cells present in epidermal keratinocytes and dermal fibroblasts. The origin of the skin cells is not particularly limited as long as they are mammalian, and examples include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses, with humans being preferred. Furthermore, the skin cells used may be primary culture cells, but it is preferable to use immortalized cells to eliminate lot-to-lot differences.

[0043] Immortalization can be achieved by introducing an immortalization gene into cultured skin cells. Here, the term "immortalization gene" refers to a gene that immortalizes cells and confer the ability to proliferate indefinitely. It is not particularly limited as long as it immortalizes cultured epithelial skin cells, such as epidermal keratinocytes, without inducing cell death. The immortalization gene is an exogenous gene, meaning an immortalization gene newly introduced from outside the cell. Furthermore, the immortalization gene may be derived from a non-human source or may be an immortalization gene modified to be expressible in target cells. Examples of immortalization genes used in the present invention 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.

[0044] In addition to the immortalization gene, it is preferable to further introduce a gene encoding a cell cycle transition promoting factor. Examples of cell cycle transition promoting factors (positive regulators of the cell cycle) include: Examples include cyclin-dependent kinases (CDKs), which are involved in the progression from the G1 to S phase of the cell cycle, and their binding partners, cyclins (CCNs). The cyclin-dependent kinases (CDKs) and cyclins (CCNs) may be either one or both. Examples of cyclin-dependent kinases (CDKs) used in the present invention include CDK1, CDK2, CDK3, CDK4, CDK6, and CDK7. Among these, CDK4 and CDK6 are preferred, with CDK4 being more preferred. Furthermore, any cyclin (CCN) may be used as long as it can bind to and activate the above-mentioned CDKs, including, for example, D-type cyclins (CCND1, CCND2, and CCND3). Information on the nucleotide sequences of the CDK genes and / or CCN genes used in the present invention is available from the NCBI database. Furthermore, the CDK genes and / or CCN genes are preferably derived from mammals, and more preferably from humans.

[0045] The method for introducing the immortalizing gene or the gene encoding a cell cycle transition-promoting factor into skin cells is not limited as long as it is a method commonly used for gene introduction, and examples thereof include methods using viral vectors, lipofection, calcium phosphate coprecipitation, electroporation, etc., with methods using viral vectors being preferred. Examples of viral vectors include lentiviral vectors, retroviral vectors, adeno-associated viral (AAV) vectors, and adenoviral vectors.

[0046] Next, single cells are produced from the population of immortalized skin cells by single-cell cloning of the desired immortalized skin stem cells. Single cells can be produced by selecting and isolating immortalized stem cells with high differentiation potential, using the expression level of differentiation markers as an indicator when immortalized skin cells are differentiated with a differentiation-inducing factor. When producing immortalized epidermal stem cells, differentiation markers such as filaggrin (FLG) and involucrin (IVL) can be used, and when producing immortalized dermal stem cells, differentiation markers such as type I collagen (COL1A1) can be used.

[0047] Next, the prepared immortalized epidermal stem cells are subjected to three-dimensional culture, which consists of a proliferation culture step and a differentiation induction step, and can be performed according to methods known in the art for preparing conventional three-dimensional cultured skin, except that compound A is added to the medium in the differentiation induction step.

[0048] First, prior to the proliferation and culture step, the immortalized dermal stem cells obtained above are added to a culture insert and cultured to form a dermal layer of dermal fibroblasts. In this case, collagen gel, collagen sponge, etc. can also be used as a support for the immortalized dermal stem cells. In the proliferation and culture step, the immortalized epidermal stem cells obtained above (hereinafter simply referred to as "epidermal stem cells") are cultured on the dermal layer until confluent using a combination of immersion culture, air-liquid culture, etc. Specifically, the epidermal stem cells are dispersed in a cell proliferation medium, and this cell dispersion is seeded into the insert with the dermal layer formed. The exterior of the culture insert is also filled with the same cell proliferation medium, and the epidermal stem cells are cultured while immersed in the cell proliferation medium. A liquid-permeable membrane maintains communication between the interior and exterior of the culture insert, allowing the medium to pass through. The number of epidermal stem cells added to the upper dermal layer is not particularly limited, but is usually 15 x 10 4 ~120×10 4 cells / cm 2 is preferred, and 30 × 10 4 ~90×10 4 cells / cm 2 is more preferred.

[0049] The liquid-permeable membrane of the culture insert is not particularly limited as long as it can serve as a support on which seeded epidermal stem cells can adhere or be fixed and on which the epidermal stem cells can proliferate, and examples thereof include membranes of polycarbonate, polyethylene terephthalate, polystyrene, etc. Furthermore, the membrane may be coated with an extracellular matrix such as collagen, laminin, fibronectin, or a substance that aids cell adhesion such as poly-L-lysine.

[0050] The proliferation culture is carried out for, for example, 1 to 6 days, preferably 2 to 4 days. During this time, the medium may be replaced as appropriate. Whether the epidermal stem cells proliferated in the culture insert are in a confluent state can be confirmed using a cell staining reagent such as the CnT-ST-100 stain kit (manufactured by CELLnTEC).

[0051] Next, in the differentiation induction step, the media inside and outside the culture insert are changed from the cell growth medium to The cell differentiation medium is changed to one containing compound A, and the epidermal stem cells are immersed and cultured in this medium for approximately 6 to 48 hours. After that, all medium inside and outside the culture insert is removed with an aspirator, and cell differentiation medium is added to the outside of the insert. The epidermal stem cells inside the insert are exposed to air (atmospheric air) and cultured for 5 to 12 days to induce differentiation into epidermal keratinocytes.

[0052] The cell growth medium is not particularly limited as long as it is a basal medium suitable for the growth and subculture of epidermal keratinocytes, but a serum-free, low-calcium basal medium is preferred, and commercially available media such as MCDB153 medium (Sigma), HuMedia-KG2 (Kurabo), serum-free medium for normal human epidermal keratinocytes (DS Pharma Biomedical), and Keratinocyte-SFM (Life Technologies) may be used. The medium may contain growth factors such as basic fibroblast growth factor (bFGF), leukocyte migration inhibitory factor (LIF), and stem cell factor (SCF). To increase the growth rate, the medium may contain epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, bovine serum albumin (BSA), L-glutamine, fibronectin, progesterone, selenite, B27 supplement, N2 supplement, or ITS supplement, as needed. Antibiotics may also be added as needed. The calcium concentration of the cell growth medium is preferably about 0.03 to 0.15 mM.

[0053] The cell differentiation medium is not particularly limited as long as it is a basal medium suitable for inducing differentiation into epidermal keratinocytes, and commercially available media such as CnT-Prime 3D Barrier Culture Medium (manufactured by CELLnTEC) may be used. The calcium concentration of the cell differentiation medium is preferably about 1.2 to 1.5 mM.

[0054] The culture temperature for proliferation and differentiation induction varies depending on the origin of the cells, but for example, in the case of cells of human origin, it is preferably 30 to 40° C., more preferably 36 to 38° C. Furthermore, the CO gas concentration is preferably, for example, about 1 to 10%, more preferably about 2 to 5%.

[0055] The process of culturing the above-mentioned epidermal stem cells to produce stratified epidermal keratinocytes may be carried out using a commercially available kit for producing cultured epidermis, such as the Keratinocyte Three-Dimensional Culture Starter Kit (manufactured by Funakoshi Co., Ltd.), using the culture medium and culture inserts packaged in the kit and following the instructions attached to the kit. [Example]

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

[0057] [Example 1] Preparation of immortalized epidermal stem cells and dermal fibroblasts and evaluation of stemness (1) Establishment of immortalized epidermal stem cells and immortalized dermal stem cells We immortalized primary human keratinocytes cultured in a medium supplemented with the supplements provided with Humedia KG2 (Kurabo) and primary human fibroblasts cultured in DMEM medium (Nacalai Tesque) containing 10% FBS. Primary human keratinocytes and primary human fibroblasts were immortalized by transfecting them with three genes: telomerase reverse transcriptase (TERT), CDK4 (cyclin-dependent kinase 4), and cyclin D1. The immortalization genes were introduced into cultured skin cells by transfection using vectors containing the genes encoding each immortalization gene. Single cells were isolated from the cell population immortalized under the above conditions by single-cell cloning. For the obtained immortalized keratinocytes, the expression level of differentiation markers (FLG, IVL) when differentiated with 3 mM calcium was used as an indicator, and for the immortalized fibroblasts, the expression level of the collagen gene (COL1A1) when differentiated with TGFβ was used as an indicator to select single clone lines of immortalized epidermal stem cell models and immortalized dermal stem cell models with high differentiation potential.

[0058] (2) Evaluation of the effect of promoting stem cell proliferation The immortalized epidermal stem cell model and immortalized dermal stem cell model prepared in (1) were placed in a 96-well plate (FALCON) at 3 × 10 cells each. 4 Cells were seeded at 100 cells / well. The following day, cell engraftment was confirmed, and medium containing the test substances (compounds 1-9) shown in Table 1 below dissolved at a final concentration of 1 μM was added, followed by culturing for 48 hours in a CO2 incubator. After culturing, the cell proliferation rate was analyzed using a Cell Count Normalization Kit (DOJINDO). Wells containing only the solvent (DMSO) without the test substance were used as controls, and the increase / decrease (%) in cell count upon addition of the test substance was calculated relative to the control, setting the control at 100% to evaluate the effect of promoting stem cell proliferation. The test results are shown in Table 1.

[0059] [Table 1] TIFF0007754483000025.tif68161

[0060] As shown in Table 1, all of compounds 1 to 9 were found to have an excellent proliferation-promoting effect on skin stem cells (epidermal stem cells and / or dermal stem cells).

[0061] [Example 2] Preparation of three-dimensional cultured skin (1) Preparation of three-dimensional cultured skin The immortalized dermal stem cell model prepared in Example 1 was placed in Cellmatrix Type 1-A (manufactured by Nitta Gelatin Co., Ltd.) at 1 × 10 5 The cells were suspended at a concentration of 1000 cells / mL and poured into a cell culture insert (Greiner) to prepare an artificial dermis.

[0062] Next, a growth medium: CnT-Prime Epithelial Culture Medium (manufactured by CELLnTEC) was added to the outside of the culture insert, and then 1 × 10 immortalized epidermal stem cell models prepared in Example 1 were added to the top of the culture insert. 6The cells were then incubated in a CO2 incubator for 48 hours. After incubation, the medium inside and outside the culture insert was replaced with differentiation medium: CnT-Prime 3D Barrier Culture Medium (CELLnTEC), and the cells were incubated in a CO2 incubator for 24 hours. The medium inside the culture insert was then removed and the cells were exposed to air. The outside of the culture insert was replaced with medium containing the test substances (compounds 7, 8, and 9) at a final concentration of 1 μM, and the cells were cultured for 8 days, with the medium replaced every two days, to produce 3D cultured skin.

[0063] (2) Evaluation of epidermal tissue regeneration effect The three-dimensional cultured skin prepared in (1) was immersed in 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Industries, Ltd.) and fixed for 24 hours. After fixation, paraffin blocks were prepared using a tissue embedding machine, and thin sections were prepared using a microtome and stained using a hematoxylin-eosin staining kit (ScyTeK). Images of the stained sections were photographed under a microscope, and the thickness of the epidermal layer of the three-dimensional cultured skin was measured using image analysis software. The thickness of the epidermal layer prepared using medium containing only DMSO was used as the control (100%), and the relative value (%) of the thickness of the epidermal layer prepared using medium containing the test substance was calculated.

[0064] (3) Evaluation of dermal tissue regeneration effect The appearance of the three-dimensional cultured skin prepared in (1) was photographed using a stereomicroscope, and the diameter of the top surface of the three-dimensional cultured skin was measured using image analysis software. The diameter of the three-dimensional cultured skin prepared using medium containing only DMSO was used as the control (100%), and the relative value (%) of the diameter of the three-dimensional cultured skin prepared using medium containing the test substance was calculated. Note that a smaller diameter of the three-dimensional cultured skin indicates a higher rate of dermal layer regeneration.

[0065] The test results (stained images of epidermal tissue sections, thickness of the epidermal layer, appearance and diameter of the three-dimensional cultured skin) are shown in Figure 1.

[0066] As shown in Figure 1, the thickness of the epidermal layer of the 3D cultured skin prepared using a medium containing compounds 7, 8, and 9 was significantly increased compared to the control. In addition, the diameter of the 3D cultured skin was smaller than that of the control, indicating that regeneration of the dermal layer was promoted. [Industrial Applicability]

[0067] The skin stem cell proliferation promoter and skin regeneration promoter of the present invention can promote the proliferation of skin stem cells and promote the regeneration of skin tissue in vivo or ex vivo. Therefore, the present invention can be used in the fields of producing cosmetics and pharmaceuticals for treating, ameliorating, and preventing skin diseases and pathologies caused by impaired or insufficient function of epidermal stem cells or dermal stem cells, and in the fields of producing transplant materials for regenerative medicine and regenerative beauty.

Claims

1. An agent for promoting proliferation of epidermal stem cells and / or dermal stem cells, comprising as an active ingredient a compound represented by any one of the following structural formulas (1), (3), (5) to (8): 【Chemical 1】

2. A skin regeneration promoter containing one or more compounds represented by any one of the following structural formulas (1), (3), (5) to (8) as active ingredients. 【Chemistry 2】

3. A culture method for proliferating epidermal stem cells and / or dermal stem cells, comprising the step of culturing epidermal stem cells and / or dermal stem cells in a medium containing a compound represented by any one of the following structural formulas (1), (3), (5) to (8): 【Chemistry 3】

4. A method for producing three-dimensional cultured skin, comprising a step of culturing skin stem cells in a medium containing one or more compounds represented by any of the following structural formulas (1), (3), (5) to (8): 【Chemistry 4】

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