JP7518513B2 - Pharmaceutical and cosmetic compositions - Google Patents

Description

The present invention relates to pharmaceutical compositions and cosmetic compositions.

Embryonic stem cells (ES cells) are stem cells established from early human or mouse embryos. ES cells have the pluripotency to differentiate into all cells present in the body. Currently, human ES cells can be used in cell transplantation therapy for many diseases, including Parkinson's disease, juvenile diabetes, and leukemia. However, there are obstacles to the transplantation of ES cells. In particular, the transplantation of ES cells can provoke an immune rejection response similar to the rejection that occurs following unsuccessful organ transplants. In addition, there is much criticism and opposition from an ethical standpoint to the use of ES cells, which are established by destroying human embryos.

Against this background, Professor Shinya Yamanaka of Kyoto University succeeded in establishing induced pluripotent stem cells (iPS cells) by introducing four genes: OCT3/4, KLF4, c-MYC, and SOX2 into somatic cells. For this, Professor Yamanaka was awarded the Nobel Prize in Physiology or Medicine in 2012 (see, for example, Patent Document 1). iPS cells are ideal pluripotent cells that are free from rejection reactions and ethical issues. Therefore, it is expected that iPS cells will be used in cell transplantation therapy. Meanwhile, there have been reports of reusing the medium used to culture iPS cells in pharmaceutical compositions (see, for example, Patent Document 2).

Patent No. 4183742 JP 2016-128396 A

However, the inventors' verification revealed that, since iPS cells differentiate in the culture method described in Patent Document 2, it is believed that the medium used to culture differentiated cells, rather than iPS cells, is actually being reused. One of the objects of the present invention is to provide a pharmaceutical composition and a cosmetic composition that effectively utilize the culture medium for iPS cells.

According to an aspect of the present invention, a pharmaceutical composition or a pharmaceutical composition raw material is provided, which comprises the supernatant of a culture medium used in reprogramming a somatic cell.

According to an aspect of the present invention, there is provided an agent for preventing and improving the formation of any of skin blemishes, wrinkles, and sagging, comprising the above-mentioned pharmaceutical composition or pharmaceutical composition raw material.

According to an aspect of the present invention, a cosmetic composition or a cosmetic composition ingredient is provided, which comprises the supernatant of a culture medium used in reprogramming somatic cells.

According to an aspect of the present invention, there is provided an agent for preventing and improving the formation of any of skin blemishes, wrinkles, and sagging, comprising the above-mentioned cosmetic composition or cosmetic composition ingredient.

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

According to an aspect of the present invention, a hair growth agent, a hair growth agent raw material, a hair restorer, or a hair restorer raw material is provided, which contains the supernatant of a culture medium used in reprogramming somatic cells.

According to an aspect of the present invention, there is provided a hair papilla cell activator or a hair papilla cell activator raw material, which comprises the supernatant of a culture medium used in reprogramming somatic cells.

According to an aspect of the present invention, a fibroblast growth factor family production promoter or a raw material for a fibroblast growth factor family production promoter is provided, which comprises the supernatant of a culture medium used in reprogramming somatic cells.

According to an aspect of the present invention, a vascular endothelial cell growth factor production promoter or a raw material for the vascular endothelial cell growth factor production promoter is provided, which contains the supernatant of a culture medium used in reprogramming somatic cells.

According to an aspect of the present invention, a wound treatment agent or a raw material for a wound treatment agent is provided, which comprises the supernatant of a culture medium used in reprogramming somatic cells.

According to an aspect of the present invention, an epidermal cell proliferation promoter or a raw material for an epidermal cell proliferation promoter is provided, which contains the supernatant of a culture medium used in reprogramming somatic cells.

According to an aspect of the present invention, a pharmaceutical composition or a pharmaceutical composition ingredient is provided, which comprises an extract of stem cells.

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

According to an aspect of the present invention, there is provided an agent for preventing and improving the formation of any of skin blemishes, wrinkles, and sagging, comprising the above-mentioned pharmaceutical composition or pharmaceutical composition raw material.

According to an aspect of the present invention, a cosmetic composition or a cosmetic composition ingredient is provided that contains an extract of stem cells.

In the above cosmetic composition or cosmetic composition ingredient, the stem cell extract may be in the form of a paste, or the stem cell extract may be freeze-dried.

According to an aspect of the present invention, there is provided an agent for preventing and improving the formation of any of blemishes, wrinkles, and sagging skin, comprising the above-mentioned cosmetic composition or cosmetic composition ingredient.

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

In the above-mentioned collagen production promoter or collagen production promoter raw material, the stem cell extract may be in the form of a paste, or the stem cell extract may be freeze-dried.

According to an aspect of the present invention, a hair growth agent, a hair growth agent raw material, a hair restorer, or a hair restorer raw material is provided, which contains an extract of stem cells.

In the above hair growth agent, hair growth agent raw material, hair restorer, or hair restorer raw material, the stem cell extract may be in the form of a paste, or the stem cell extract may be freeze-dried.

According to an aspect of the present invention, a hair papilla cell activator or a raw material for a hair papilla cell activator is provided, which contains an extract of stem cells.

In the above-mentioned dermal papilla cell activator or dermal papilla cell activator raw material, the stem cell extract may be in the form of a paste, or the stem cell extract may be freeze-dried.

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

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

According to an aspect of the present invention, a vascular endothelial cell growth factor production promoter or a raw material for a vascular endothelial cell growth factor production promoter is provided, which contains an extract of stem cells.

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

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

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

According to an aspect of the present invention, an epidermal cell proliferation promoter or a raw material for an epidermal cell proliferation promoter is provided, which contains an extract of stem cells.

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

According to an aspect of the present invention, there is provided a method for screening anti-UV substances, comprising: preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a patient with an aging disease or a patient with a skin disease; irradiating the skin cells with UV rays; culturing the UV-irradiated skin cells in each of a plurality of different solutions; and selecting a medium that causes less UV-induced damage to the skin cells or a solution that allows for rapid repair of UV-irradiated skin cells.

According to an aspect of the present invention, there is provided a method for screening an anti-desiccation substance, comprising: preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a patient with an aging disease or a patient with a skin disease; drying the skin cells; culturing the dried skin cells in each of a plurality of different solutions; and selecting a solution that provides a high survival rate for the skin cells.

According to an aspect of the present invention, there is provided a method for screening an anti-desiccation substance, comprising: preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a patient with an aging disease or a patient with a skin disease; drying the skin cells; culturing the dried skin cells in each of a plurality of different solutions; and selecting a solution that causes minimal damage to tight junctions in the skin cells.

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

According to an aspect of the present invention, there is provided a method for screening an antioxidant stress substance, comprising: preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a patient with an aging disease or a patient with a skin disease; applying oxidative stress to the skin cells; culturing the oxidatively stressed skin cells in each of a plurality of different solutions; and selecting a solution that provides a high survival rate for the skin cells.

According to an aspect of the present invention, there is provided a method for screening moisturizing-promoting substances, comprising: preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a patient with an aging disease or a patient with a skin disease; culturing the skin cells in each of a plurality of different solutions; and selecting from the plurality of different solutions a solution containing a large amount of natural moisturizing factors derived from the skin cells.

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

According to an aspect of the present invention, a method for testing UV resistance of skin is provided, which includes preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a subject, and testing the skin cells for damage caused by UV rays.

According to an aspect of the present invention, there is provided a method for testing the desiccation resistance of skin, comprising preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a subject, drying the skin cells, and testing the viability of the skin cells.

According to an aspect of the present invention, there is provided a method for testing the dryness resistance of skin, comprising preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a subject, drying the skin cells, and testing for damage to tight junctions in the skin cells.

In the above method, when examining damage to tight junctions in skin cells, at least one of occludin and claudin in the tight junctions may be analyzed.

According to an aspect of the present invention, a method for testing skin oxidative stress resistance is provided, which includes preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a subject, applying oxidative stress to the skin cells, and testing the viability of the skin cells.

According to an aspect of the present invention, there is provided a method for testing the moisturizing ability of skin, comprising preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a subject, culturing the skin cells, and testing the amount of natural moisturizing factors derived from the skin cells.

The present invention makes it possible to provide pharmaceutical and cosmetic compositions that effectively utilize iPS cell culture media.

1 is a graph showing the results of a test of hyaluronic acid production by fibroblasts in Example 2. 1 is a graph showing the results of a type I collagen production test using fibroblasts according to Example 4. 1 is a table showing the results of a type I collagen production test using fibroblasts according to Example 4. 1 is a graph showing the results of a proliferation test of hair follicle cells in Example 5. 1 is a table showing the results of a proliferation test of hair follicle cells according to Example 5. 1 is a graph showing the results of a test on VEGF production by hair papilla cells in Example 6. 1 is a table showing the results of a test on VEGF production by hair papilla cells according to Example 6. 1 is a graph showing the results of a test on FGF-7 production by hair papilla cells in Example 7. 1 is a table showing the results of a test of FGF-7 production by hair papilla cells according to Example 7. 13 is a photograph showing the results of a fibroblast migration test in Example 8. 13 is a photograph showing the results of a fibroblast migration test in Example 8. 13 is a graph showing the results of a UV irradiation test of skin fibroblasts according to Example 10. 1 is a graph showing the results of a dryness irritation test on skin fibroblasts in Example 11. 13 is a graph showing the results of an oxidative stress test of skin fibroblasts according to Example 12. 13 is a graph showing the results of an oxidative stress test of skin fibroblasts according to Example 12. 1 is a micrograph of cells according to a reference example. 1 is a micrograph of cells according to a reference example. 1 is a histogram showing the results of flow cytometry of cells according to a reference example.

The following describes in detail the embodiments of the present invention. Note that the embodiments shown below are merely examples of devices and methods for embodying the technical idea of this invention, and the technical idea of this invention is not limited to the combination of components described below. The technical idea of this invention can be modified in various ways within the scope of the claims.

First Embodiment
Each of the pharmaceutical composition, pharmaceutical composition raw material, cosmetic composition, and cosmetic composition raw material according to the first embodiment contains the supernatant of a culture medium used when somatic cells are reprogrammed into pluripotent stem cells such as iPS cells. Each of the pharmaceutical composition, pharmaceutical composition raw material, cosmetic composition, and cosmetic composition raw material according to the first embodiment may contain a lyophilized product of the supernatant of a culture medium used when somatic cells are reprogrammed into pluripotent stem cells such as iPS cells. Each of the pharmaceutical composition, pharmaceutical composition raw material, cosmetic composition, and cosmetic composition raw material according to the first embodiment may contain the supernatant of a culture medium used when somatic cells are reprogrammed into pluripotent stem cells such as iPS cells in a capsule such as a nanocapsule or an emulsion such as a nanoemulsion.

iPS cells are induced by introducing reprogramming factors such as OCT3/4, KLF4, c-MYC, and SOX2 into somatic cells, such as differentiated cells, such as blood cells and fibroblasts. Induction into iPS cells may be called reprogramming, reprogramming, transformation, transdifferentiation (transdifferentiation or lineage reprogramming), and cell fate reprogramming. Pluripotent stem cells may be induced while being three-dimensionally cultured, such as in suspension culture. During three-dimensional culture, a gel medium or a liquid medium may be used. For example, the positivity rate of any of TRA1-60, OCT3/4, SSEA3, SSEA4, TRA1-81, and NANOG is 30% or more, 50% or more, and preferably 80% or more. The gel medium does not have to contain feeder cells.

As a medium for induction culture, for example, human ES/iPS medium such as TeSR2 (STEMCELL Technologies) can be used. However, the medium is not limited to this, and various stem cell media can be used. For example, Primate ES Cell Medium, Reprostem, ReproFF, ReproFF2, ReproXF (Reprocell), mTeSR1, TeSRE8, ReproTeSR (STEMCELL Technologies), PluriS TEM (registered trademark) Human ES/iPS Medium (Merck), NutriStem (registered trademark) XF/FF Culture Medium for Human iPS and ES Cells, Pluriton reprogramming medium (Stemgent), PluriSTEM (registered trademark), Stemfit AK02N, Stemfit AK03 (Ajinomoto), ESC-Sure (registered trademark) serum and feeder free medium for hESC/iPS (Applied StemCell), L7 (registered trademark) hPSC Culture System (LONZA), and Primate ES Cell Medium (ReproCELL), etc. may also be used.

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

The gel medium may contain at least one polymer compound selected from the group consisting of gellan gum, hyaluronic acid, rhamsan gum, diutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, and salts thereof. The gel medium may also contain methylcellulose. By containing methylcellulose, aggregation of cells is further suppressed.

Alternatively, the gel medium may contain at least one temperature-sensitive gel selected from poly(glycerol monomethacrylate) (PGMA), poly(2-hydroxypropyl methacrylate) (PHPMA), Poly(N-isopropylacrylamide) (PNIPAM), amine terminated, carboxylic acid terminated, maleimide terminated, N-hydroxysuccinimide (NHS) ester terminated, triethoxysilane terminated, Poly(N-isopropylacrylamide-co-acrylamide), Poly(N-isopropylacrylamide-co-acrylic acid), Poly(N-isopropylacrylamide-co-butylacrylate), Poly(N-isopropylacrylamide-co-methacrylic acid), Poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-isopropylacrylamide.

Alternatively, each of the pharmaceutical composition, pharmaceutical composition raw material, cosmetic composition, and cosmetic composition raw material according to the first embodiment includes an extract of stem cells. The extract may be liquid. That is, the extract may be an extract. The stem cells include pluripotent stem cells such as iPS cells, and embryonic stem cells (ES cells). For example, the positivity rate of TRA1-60 or Oct3/4 in the stem cells is 30% or more, 50% or more, and preferably 80% or more. When the stem cells are cultured in an undifferentiated state, the medium contains b-FGF at 10 ng/ml or more, and preferably 40 ng/ml or more. Each of the pharmaceutical composition, pharmaceutical composition raw material, cosmetic composition, and cosmetic composition raw material may include a stem cell extract in the form of a stem cell paste. The stem cell paste is obtained by grinding iPS cells. The stem cell extract may be a lyophilized product. The stem cell extract may also be a powder. Alternatively, the stem cell extract may be a stem cell lysate.

The pharmaceutical composition according to the first embodiment may be a skin application composition. The pharmaceutical composition according to the first embodiment may be a skin disease treatment agent. Examples of diseases treatable with the skin disease treatment agent according to the first embodiment include acne vulgaris, psoriasis vulgaris, keloid, seborrheic dermatitis, contact dermatitis, atopic dermatitis, atopic dry dermatitis, dermatoporosis, actinic elastosis, solar keratosis, ptosis, alopecia areata, alopecia, eyelash hypotrichosis, melasma, senile pigmented spots, miliaria, freckles, delayed bilateral nevus of Ota, seborrheic keratosis, skin diseases due to progeria, and herpes simplex.

Examples of conditions that can be improved or eliminated by the cosmetic composition according to the first embodiment include age spots, freckles, wrinkles, sagging skin, creaking skin, loss of skin elasticity, dullness, sensitive skin, dry skin, and thinning hair. Effects of the cosmetic composition according to the first embodiment include conditioning the skin, conditioning the skin texture, keeping the skin healthy, preventing rough skin, tightening the skin, moisturizing the skin, replenishing and maintaining moisture and oil in the skin, maintaining the flexibility of the skin, protecting the skin, preventing dry skin, softening the skin, firming the skin, giving the skin a glossy finish, smoothing the skin, firming the skin, making age spots less noticeable, suppressing wrinkles, and brightening the skin. Furthermore, effects of the cosmetic composition according to the first embodiment on the scalp or hair include keeping the scalp healthy, promoting hair growth, preventing thinning hair, preventing itching, preventing hair loss, promoting hair growth, preventing hair loss after illness or after childbirth, and nourishing the hair.

The pharmaceutical composition according to the first embodiment may be a wound treatment agent, an epidermal cell proliferation promoter, an epidermal turnover promoter, a hair growth agent, a hair restorer, and an eyelash hypotrichosis treatment agent. The pharmaceutical composition and cosmetic composition according to the first embodiment may be a collagen production promoter, a hyaluronic acid production promoter, a hair growth agent, a fibroblast growth factor (FGF) family production promoter, and a vascular endothelial growth factor (VEGF) production promoter.

During hair growth, hair matrix cells in the hair root divide, and the cells that arise from these divide to form hair. Hair growth goes through a cycle called the hair cycle, which repeats the growth phase, regression phase, and resting phase. Dermal papilla cells affect the proliferation and differentiation of hair follicle epithelial stem cells through the production and release of growth factors, thereby controlling the hair cycle. It is said that the activation of dermal papilla cells and hair matrix cells contributes to the mechanism of hair growth. In addition, blood vessels are actively remodeled in hair follicles in response to the hair cycle, but if there is a problem with angiogenesis at this time, the supply of nutrients and oxygen required for hair formation becomes insufficient. It is said that insufficient blood flow from the hair follicle vascular network is involved in the pathology of male pattern baldness (AGA).

The following is known about the genes of hair papilla cells and hair growth and hair regeneration. That is, FGF-7, IGF-1, etc. are known as growth factors secreted by papilla cells to hair matrix cells. These factors have the effect of maintaining hair follicle growth. Vascular endothelial growth factor (VEGF) is secreted from hair papilla cells and is involved in the proliferation of hair follicle blood vessels, and also has the effect of autocrinely proliferating hair papilla cells, but the expression level decreases as the phase shifts from the anagen phase to the regressive phase. The expression of the VEGF gene is reduced in hair tissues of AGA (androgenetic alopecia). VEGFB binds competitively to VEGFR-1, which is the receptor on which VEGF acts. VEGFB has the activity of promoting the proliferation and permeability of vascular endothelial cells, but its effect on hair follicles is unknown.

The pharmaceutical composition and cosmetic composition according to the first embodiment act directly on the hair papilla, increasing the production of FGF-7, a hair growth promoting factor, and promoting hair growth, thereby lengthening the anagen phase of the hair cycle and helping thin, weak hair grow into thick, strong hair. It also increases vascular endothelial growth factor (VEGF), which is secreted by hair papilla cells and is involved in the proliferation of hair follicle blood vessels, and has the effect of autocrine proliferation of hair papilla cells. Therefore, the pharmaceutical composition and cosmetic composition according to the first embodiment can be used as an activator for hair papilla cells.

When the pharmaceutical composition and cosmetic composition according to the first embodiment are used as hair growth agents or hair regrowth agents, they may contain other active ingredients such as minoxidil, Swertia japonica, pantothenyl ethyl ether, tocopherol acetate, dipotassium glycyrrhizinate, and adenosine.

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

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

The pharmaceutical composition and cosmetic composition according to the first embodiment may contain pharma- ceutically acceptable carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, and physiological saline. Examples of excipients include lactose, starch, sorbitol, D-mannitol, and sucrose. Examples of disintegrants include carboxymethylcellulose and calcium carbonate. Examples of buffers include phosphates, citrates, and acetates. Examples of emulsifiers include gum arabic, sodium alginate, and tragacanth.

Examples of suspending agents include glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of soothing agents include benzyl alcohol, chlorobutanol, and sorbitol. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of preservatives include benzalkonium chloride, paraoxybenzoic acid, and chlorobutanol.

In addition, the pharmaceutical composition and cosmetic composition according to the first embodiment may contain water, alcohol, surfactants (cationic, anionic, nonionic, and amphoteric surfactants, etc.), moisturizers (glycerin, 1,3-butylene glycol, propylene glycol, propanediol, pentanediol, polyquaternium, amino acids, urea, pyrrolidone carboxylates, nucleic acids, monosaccharides, oligosaccharides, etc., and derivatives thereof, etc.), thickeners (polysaccharides, polyacrylates, carboxyvinyl polymers, polyvinylpyrrolidone, polyvinyl alcohol, chitin, chitosan, alginic acid, carrageenan, xanthan gum, methylcellulose, etc., and derivatives thereof, etc.), wax, petrolatum, hydrocarbon saturated fatty acids, unsaturated fatty acids, silicone oils, etc., and derivatives thereof, tri(caprylic/capric)glyceryl, and trioctyl esters. triglycerides such as glyceryl tanoate, ester oils such as isopropyl stearate, natural fats and oils (olive oil, camellia oil, avocado oil, almond oil, cacao butter, evening primrose oil, grape seed oil, macadamia nut oil, eucalyptus oil, rosehip oil, squalane, orange roughy oil, lanolin, ceramide, etc.), preservatives (oxybenzoic acid derivatives, dehydroacetate, photosensitizers, sorbic acid, phenoxyethanol, etc., and derivatives thereof, etc.), bactericides (sulfur, trichlorocarbanilide, salicylic acid, zinc pyrithione, hinokitiol, etc., and derivatives thereof, etc.), ultraviolet absorbers (para-aminobenzoic acid, methoxycinnamic acid, etc., and derivatives thereof, etc.), anti-inflammatory agents (allantoin, bisabolol, ε-aminocaproic acid, acetylphanesylcysteine, glycyrrhizic acid, etc., and and their derivatives), antioxidants (tocopherol, BHA, BHT, astaxanthin, and the like, and their derivatives), chelating agents (edetic acid, hydroxyethanediphosphonic acid, and the like, and their derivatives), animal and plant extracts (Angelica angelica, aloe, angelica tree, Chinese quince, chamomile, licorice, kiwi, cucumber, mulberry, white birch, angelica tree, garlic, peony, hops, horse chestnut, lavender, rosemary, eucalyptus, milk, various peptides, placenta, royal jelly, Euglena extract, hydrolyzed Euglena extract, Euglena oil, and the like, and the refined or fermented products of the components thereof), pH adjusters (inorganic acids, inorganic acid salts, organic acids, organic acid salts, and the like, and the derivatives thereof), vitamins (vitamin A, vitamin B, vitamin C, vitamin D, ubiquitin, and nicotinamide, and derivatives thereof), yeast, fermentation liquid of koji mold and lactic acid bacteria, Galactomyces culture liquid, whitening agents (tranexamic acid, cetyl tranexamate hydrochloride, 4-n-butylresorcinol, arbutin, kojic acid, ellagic acid, licorice flavonoids, niacinamide, and vitamin C derivatives), ceramide and ceramide derivatives, anti-wrinkle agents (retinol, retinal, and derivatives thereof, nicotinamide, oligopeptides, and derivatives thereof, natural and synthetic ingredients that inhibit neutrophil elastase, and MMP-1 and MMP-2), titanium oxide, talc, mica, silica, zinc oxide, iron oxide, silicon, and powders processed from these, etc., can be blended within a range that achieves the purpose of the pharmaceutical composition and cosmetic composition according to the first embodiment.

The ingredients that can be added to the pharmaceutical composition and cosmetic composition according to the first embodiment are not limited to those mentioned above, and any ingredients that can be used in pharmaceutical compositions and cosmetic compositions can be freely selected. When the pharmaceutical composition and cosmetic composition according to the first embodiment are used as poultices, in addition to the above ingredients, bases (kaolin, bentonite, etc.) and gelling agents (polyacrylates, polyvinyl alcohol, etc.) can be blended within the range that achieves the purpose. When the pharmaceutical composition and cosmetic composition according to the first embodiment are used as bath additives, sulfates, bicarbonates, borates, pigments, and moisturizers can be appropriately blended within the range that achieves the purpose, and the composition can be prepared as a powder type or liquid type.

The pharmaceutical composition and cosmetic composition according to the first embodiment can be produced by methods well known and commonly used in the art.

Second Embodiment
The screening method for anti-ultraviolet rays substances according to the second embodiment includes preparing skin cells induced to differentiate from iPS cells produced from somatic cells derived from an aging disease patient such as a patient with progeria or a patient with a skin disease, optionally irradiating the differentiated skin cells with ultraviolet rays, culturing the ultraviolet irradiated skin cells in each of a plurality of different solutions, and selecting a solution that causes less damage to the skin cells due to ultraviolet rays or a solution that quickly repairs the skin cells exposed to ultraviolet rays. Note that the patient is not limited to humans, and also includes non-human animals.

Examples of progeria include, but are not limited to, Werner syndrome, xeroderma pigmentosum, and Cockayne syndrome. Somatic cells derived from progeria patients can be, for example, fibroblasts,
Examples of the cells include, but are not limited to, blood cells, epithelial cells, somatic stem cells, keratinocytes, hair papilla cells, and dental pulp stem cells. iPS cells are induced by introducing reprogramming factors such as OCT3/4, KLF4, c-MYC, and SOX2 into somatic cells derived from a patient with progeria. When inducing differentiation of iPS cells into skin cells, for example, iPS cells are seeded in a low cell adhesion dish containing a culture medium that does not contain bFGF. Thereafter, the medium is replaced every two days, and embryoid bodies (EBs) formed after 8 days are seeded in the dish, the cells are allowed to adhere to the dish, and the cells are cultured in a 10% FBS medium. When the cells become confluent, the cells are detached from the dish with trypsin and subcultured. Subculture is repeated in the same manner for one month. Thereafter, it is confirmed that the cells have differentiated into fibroblasts using a fibroblast marker such as CD13. In addition, it may be possible to confirm whether or not undifferentiated iPS cells remain in the cells using an iPS cell marker such as TRA 1-60. The skin cells to be induced to differentiate are, for example, skin fibroblasts, but are not particularly limited thereto.

The differentiated skin cells are appropriately cultured and then irradiated with ultraviolet (UV) rays. The intensity, wavelength range, and irradiation time of the UV rays are appropriately set according to the application, method of use, dosage, etc. of the anti-UV substance to be screened. The UV-irradiated skin cells are cultured in each of a number of different solutions each containing a different substance to be screened. The solution may be a culture medium. The substance to be screened in the solution and the incubation time, etc. are appropriately set according to the application, method of use, dosage, etc. of the anti-UV substance to be screened.

Then, a solution that causes less damage to skin cells or a solution that causes quicker repair of skin cells is selected as a solution containing an anti-UV substance. For example, skin cells cultured in each of the multiple solutions are analyzed, and one or a group of solutions used to culture skin cells that cause less damage are selected, and one or a group of solutions used to culture skin cells that cause more damage are excluded. Alternatively, skin cells cultured in each of the multiple solutions are analyzed, and one or a group of solutions used to culture skin cells that repair quickly are selected, and one or a group of solutions used to culture skin cells that repair slowly or that do not repair are excluded.

According to the findings of the present inventors, skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases such as progeria and skin diseases, or patients with skin diseases, tend to be less resistant to UV irradiation than skin cells from healthy individuals. Therefore, it is possible to screen for effective anti-ultraviolet substances by using skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases such as progeria and skin diseases, or patients with skin diseases.

Third Embodiment
The UV resistance testing method for skin according to the third embodiment includes preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a subject, irradiating the skin cells with UV rays, and testing the skin cells for damage caused by the UV rays.

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

Fourth Embodiment
The screening method for anti-drying substances according to the fourth embodiment includes preparing skin cells that have been induced to differentiate from iPS cells produced from somatic cells derived from a patient with an aging disease or a patient with a skin disease, such as a patient with progeria or a patient with a skin disease, drying the skin cells, culturing the dried skin cells in each of a plurality of different solutions, and selecting a solution that provides a high survival rate for the skin cells.

As in the second embodiment, the skin cells induced to differentiate are appropriately cultured and then dried. When drying the skin cells, for example, the culture medium surrounding the skin cells is removed. The skin cells may be dried by applying an air current to them, or by using a desiccant or the like. The drying method and drying time of the skin cells are appropriately set according to the application, usage, dosage, etc. of the anti-drying substance to be screened. The dried skin cells are cultured in each of a plurality of different solutions each containing a different substance to be screened. The solution may be a culture medium. The substance to be screened and the culture time in the solution are appropriately set according to the application, usage, dosage, etc. of the anti-drying substance to be screened.

Then, the viability of the skin cells cultured in each of the multiple solutions is measured, and a solution having a high viability of the skin cells is selected as a solution containing an anti-drying substance. For example, the viability of the skin cells cultured in each of the multiple solutions is measured, and one or a group of solutions used to culture the skin cells having a high viability is selected, and one or a group of solutions used to culture the skin cells having a low viability is excluded.

In addition, instead of selecting a solution with a high survival rate of skin cells, a solution with less damage to the tight junctions of skin cells may be selected. In this case, at least one of occludin and claudins in tight junctions may be analyzed, and a solution with a high amount of at least one of occludin and claudins may be selected. In general, when skin cells dry out, the tight junctions are damaged, and the amounts of occludin and claudins decrease. Therefore, for example, the tight junctions of skin cells cultured in each of a plurality of solutions are analyzed, and one or a group of solutions used to culture skin cells with less damage to tight junctions is selected, and one or a group of solutions used to culture skin cells with greater damage to tight junctions is excluded.

According to the findings of the present inventors, skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases such as progeria and skin diseases, or patients with skin diseases, tend to be less resistant to dryness than skin cells from healthy individuals. Therefore, it is possible to screen for effective anti-drying substances by using skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases such as progeria and skin diseases, or patients with skin diseases.

Fifth Embodiment
The method for testing skin dryness resistance according to the fifth embodiment includes preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a subject, drying the skin cells, and testing the viability of the skin cells.

The subject may be a disease patient or a healthy individual. The somatic cells derived from the subject are obtained in advance from the subject, and the method does not have to include a step of obtaining the somatic cells from the subject. If the survival rate of the skin cells is high, it may be determined that the skin cells of the subject are resistant to desiccation. If the survival rate of the skin cells is low, it may be determined that the skin cells of the subject are not resistant to desiccation. According to the method, it is possible to test whether the skin cells of the subject are resistant to desiccation based on the test results of the survival rate of the skin cells.

Instead of testing the viability of skin cells, damage to tight junctions in skin cells may be tested. If the damage to tight junctions is small, it may be determined that the subject's skin cells have desiccation resistance, and if the damage to tight junctions is large, it may be determined that the subject's skin cells do not have desiccation resistance.

Sixth Embodiment
The screening method for anti-oxidative stress substances according to the sixth embodiment includes preparing skin cells induced to differentiate from iPS cells produced from somatic cells derived from a patient with an aging disease or a patient with a skin disease, such as a patient with progeria or a patient with a skin disease, subjecting the skin cells to oxidative stress, culturing the oxidatively stressed skin cells in each of a plurality of different solutions, and selecting a solution that provides a high survival rate for the skin cells.

The skin cells induced to differentiate as in the second embodiment are appropriately cultured and then subjected to oxidative stress. The method of applying oxidative stress to the skin cells is not particularly limited, but includes, for example, adding an oxidizing substance such as hydrogen peroxide to the culture medium in which the skin cells are cultured. The method of applying oxidative stress to the skin cells and the time for applying oxidative stress to the skin cells are appropriately set according to the application, method of use, dosage, etc. of the antioxidant stress substance to be screened. The skin cells that have been subjected to oxidative stress are cultured in each of a plurality of different solutions each containing a different substance to be screened, for example, after removing the substance that applies oxidative stress. The solution may be a culture medium. The substance to be screened in the solution and the culture time, etc. are appropriately set according to the application, method of use, dosage, etc. of the antioxidant stress substance to be screened.

Then, the viability of the skin cells cultured in each of the multiple solutions is measured, and a solution with a high viability of the skin cells is selected as a solution containing an antioxidant stress substance. For example, the viability of the skin cells cultured in each of the multiple solutions is measured, and one or a group of solutions used to culture the skin cells with a high viability is selected, and one or a group of solutions used to culture the skin cells with a low viability is excluded.

According to the findings of the present inventors, skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases such as progeria and skin diseases, or patients with skin diseases, tend to be less resistant to oxidative stress than skin cells from healthy individuals. Therefore, it is possible to screen for effective anti-oxidative stress substances by using skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases such as progeria and skin diseases, or patients with skin diseases.

Seventh Embodiment
The seventh embodiment of the method for testing skin oxidative stress resistance includes preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a subject, subjecting the skin cells to oxidative stress, and testing the viability of the skin cells.

The subject may be a disease patient or a healthy individual. The somatic cells derived from the subject are obtained in advance from the subject, and the method does not have to include a step of obtaining somatic cells from the subject. If the damage to the skin cells due to oxidative stress is large, it may be determined that the skin cells of the subject do not have oxidative stress resistance. If the damage to the skin cells due to oxidative stress is small, it may be determined that the skin cells of the subject have oxidative stress resistance. According to the method, it is possible to test whether the skin cells of the subject have oxidative stress resistance based on the test results of the damage to the skin cells due to oxidative stress.

Eighth embodiment
The screening method for moisturizing-promoting substances according to the eighth embodiment includes preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from patients with aging diseases or patients with skin diseases, such as patients with progeria and patients with skin diseases, culturing the skin cells in each of a plurality of different solutions, and selecting from the plurality of different solutions a solution having a high amount of natural moisturizing factors derived from the skin cells.

As in the second embodiment, the differentiated skin cells are cultured in a number of different solutions each containing a different substance to be screened. The substance to be screened in the solution and the culture time, etc. are appropriately set according to the application, method of use, dosage, etc. of the moisturizing promoting substance to be screened.

Then, the amount of natural moisturizing factor derived from skin cells is measured in each of the multiple solutions. Natural moisturizing factors include, but are not limited to, ceramide and filaggrin. A solution with a high amount of natural moisturizing factor is selected as a solution containing a moisturizing promoter. For example, the amount of natural moisturizing factor is measured in each of the multiple solutions, one or a group of solutions with a high amount of natural moisturizing factor is selected, and one or a group of solutions with a low amount of natural moisturizing factor is excluded.

According to the findings of the present inventors, the expression level of moisturizing promoters in skin cells tends to decrease as aging and skin diseases progress. Therefore, it is possible to screen for effective moisturizing promoters by using skin cells induced from iPS cells induced from somatic cells derived from patients with aging diseases such as premature aging and skin diseases, or patients with skin diseases.

Ninth embodiment
A method for testing the moisturizing ability of skin according to a ninth embodiment includes preparing skin cells induced to differentiate from pluripotent stem cells produced from somatic cells derived from a subject, culturing the skin cells, and testing the amount of natural moisturizing factors derived from the skin cells.

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

(Example 1: Preparation of medium for culturing blood cells while reprogramming them into iPS cells) Growth factors were added to serum-free and animal-derived component-free hematopoietic cell medium (StemspanACF, STEMCELL TECHNOLOGIES) to prepare hematopoietic cell medium. 2 x ¹⁰⁵ blood cells (peripheral blood mononuclear cells) were seeded in each well of a 12-well dish, and hematopoietic cell gel medium was dropped into each well to suspend the blood cells in the hematopoietic cell gel medium. The 12-well dish was then placed in a CO2 incubator at 37°C, and the cells were cultured in suspension.

Three days after the start of cell culture, hematopoietic cell gel medium was added to each well as needed. Six days after the start of cell culture, a Sendai virus vector kit for iPS cell production (CytoTune 2.0 Reprogramming Kit, registered trademark, Thermo Fisher) was added to the culture medium to introduce genes into each well so that the virus titer in each well was between 1 and 20, or an episomal plasmid (Thermo Fisher) was electroporated to introduce genes, and then gel medium was added to each well and the cells were cultured.

Gellan gum was added to the hES medium to a final concentration of 0.02%, to prepare a stem cell gel medium. Starting two days after infection, 2 mL of stem cell gel medium was added to each well once every two days. 14 days after infection, it was confirmed that iPS cell clusters had been induced, and the gel medium supernatant was collected. The collected gel medium supernatant was sterilized by filtering, and the gel medium supernatant that passed through the filter was used as the supernatant of the reprogramming medium according to Example 1.

(Reference Example 1: Preparation of medium for culturing iPS cells while maintaining them in an undifferentiated state)
Human iPS cells were cultured to maintain adhesion on a petri dish for adhesion culture coated with Matrigel (registered trademark, Corning) or Laminin 511 using mTeSR1 (registered trademark, STEMCELL Technologies) or StemFit (registered trademark, Ajinomoto). Human iPS cells were passaged every week. During passage, the cells were treated with ES cell dissociation solution (TrypLE Select (registered trademark, ThermoFisher).

The human iPS cells maintained and cultured as described above were detached from the petri dish for adhesion culture using an ES cell dissociation solution (TrypLE Select, registered trademark, ThermoFisher) and divided into single cells. Next, the human iPS cells were seeded in a stem cell medium gelled with the addition of gellan gum and 10 μmol/L of ROCK inhibitor (Selleck), and the human iPS cells were cultured in suspension for 14 days. When culturing in suspension for 14 days, the incubator was replenished with gelled stem cell medium once every two days.

Then, the gelled stem cell medium in which the human iPS cells were suspended was filtered through a mesh filter to remove cell clumps. The filtered gelled stem cell medium was then centrifuged at 1500 g for 5 minutes to precipitate the cells and gel, and the supernatant of the stem cell medium after centrifugation was collected again and centrifuged at 3000 rpm for 3 minutes. The supernatant of the stem cell medium after centrifugation was filtered through a 0.22 μm filter. The supernatant of the stem cell medium after filtration was used as the supernatant of the medium in which the iPS cells of Reference Example 1 were maintained and cultured.

In addition, it was confirmed that the iPS cells maintained in culture were positive for the undifferentiated markers NANOG, OCT3/4, and TRA 1-60.

(Example 2: Hyaluronic acid production test by fibroblasts)
DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin was prepared as growth medium A. Next, normal human fibroblasts derived from adults (KF-4109, Strain No. 01035, Kurabo) were suspended in growth medium A to a concentration of 5 x ¹⁰³ cells/0.1 mL/well, seeded in a 96-well plate, and cultured for 1 day in a _CO2 incubator (5% _CO2 , 37°C).

As test medium A, DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin was prepared. Next, the supernatant solutions of Example 1 and Reference Example 1 were mixed with test medium A to obtain supernatant-supplemented medium A of Example 1 and Reference Example 1 with a concentration of 10%. Growth medium A in some of the wells in which fibroblasts were cultured was replaced with supernatant-supplemented medium A of Example 1 and Reference Example 1, respectively. In addition, as a negative control, growth medium A in some of the wells was replaced with DMEM medium without added FBS, penicillin-streptomycin, or supernatant (supplement-free test medium A).

After replacing the medium, the fibroblasts were cultured for 3 days, the medium supernatant was collected, and the hyaluronic acid concentration in the medium supernatant was measured using DueSet Hyaluronan (Cat. No. DY3614, R&D Systems). The results are shown in Figure 1. It was confirmed that the fibroblasts cultured in the medium containing the supernatant of the reprogramming medium according to Example 1 produced more than twice as much hyaluronic acid as the fibroblasts cultured in the medium containing the supernatant of the medium for maintaining and culturing iPS cells according to Reference Example 1.

(Example 3: Preparation of iPS cell extract)
iPS cells were cultured on the Matrigel coat and the laminin coat using Teser1, Teser2, Stem Fit, Essential8, Teser-E8, and Nutri Stem, respectively. When the iPS cells reached 80% confluence, they were detached from the incubator using Triple Select, and the solution containing the detached iPS cells was centrifuged at 200 g for 5 minutes to collect the iPS cells in a 1.5 mL tube. The iPS cell mass was then ground with a pestle (Pestle in G-Tube, Thermo Fisher), and the iPS cell extract containing the paste of the ground iPS cells was flash frozen with liquid nitrogen. When using the iPS cell extract, the iPS cell extract was suspended in 5 mL of culture medium and incubated overnight at 4° C. The next day, the suspension was centrifuged at 1500 g for 10 minutes to remove cell debris from the solution, and the solution was then filtered through a filter, and the solution that passed through the filter was used as the iPS cell extract.

(Example 4: Type I collagen production test by fibroblasts)
As in Example 2, normal human fibroblasts derived from adults were cultured using growth medium A. Also, test medium A was prepared as in Example 2. Next, the extract of the iPS cells according to Example 3 and test medium A were mixed to obtain extract-added medium A containing the extract of the iPS cells according to Example 3 at a concentration of 50.0 v/v % or 100.0 v/v %. Growth medium A in some of the wells in which fibroblasts were cultured was replaced with extract-added medium A. Also, as a negative control, growth medium A in some of the wells was replaced with DMEM medium (additive-free test medium A) to which FBS, penicillin-streptomycin, and the extract of the iPS cells were not added.

After replacing the medium, the fibroblasts were cultured for 3 days, and the medium supernatant was collected and stored at -80°C. The medium supernatant was then thawed, and the concentration of type I collagen in the medium supernatant was measured using a human collagen type 1 ELISA kit (Cat. No. EC1-E105). The results are shown in Figures 2 and 3. It was confirmed that fibroblasts cultured in a medium containing an extract of iPS cells produced more collagen than fibroblasts cultured in a medium not containing an extract of iPS cells.

(Example 5: Proliferation test of hair papilla cells)
A dedicated medium for hair papilla cells (Cat. No. TMTPGM-250, TOYOBO) supplemented with dedicated additives (fetal bovine serum, insulin-transferrin-triiodothyronine mixture, bovine pituitary extract, cyproterone acetate) was prepared as growth medium B. Next, normal human hair papilla cells (Cat. No. CA60205a, Lot. No. 2868, TOYOBO) were suspended in growth medium B to a concentration of 1.2 x ¹⁰⁴ cells/0.3 mL/well, seeded on a type I collagen-coated 48-well plate, and cultured for 1 day in a _CO2 incubator (5% _CO2 , 37°C).

The extract of the iPS cells according to Example 3 was mixed with a dedicated medium for hair papilla cells to which no additives were added (additive-free test medium B) to obtain extract-added medium B containing an extract of the iPS cells according to Example 3 at a concentration of 50.0 v/v% or 100.0 v/v%. As a negative control, the growth medium B in some wells was replaced with a dedicated medium for hair papilla cells to which no additives or iPS cell extract were added (additive-free test medium B).

Hair papilla cells were cultured in the replaced medium for three days, and the number of viable cells was measured using the WST-8 method. The results are shown in Figures 4 and 5. It was confirmed that when additive-free test medium B containing the iPS cell extract of Example 3 was used, hair papilla cells proliferated more predominantly than when additive-free test medium B not containing the iPS cell extract was used. This suggests that the iPS cell extract has hair growth and hair regrowth effects, such as treating thinning hair, preventing hair loss, promoting hair growth, and promoting hair regrowth.

(Example 6: VEGF production test by hair papilla cells)
As in Example 5, normal human hair papilla cells were cultured for one day in growth medium B. Thereafter, the supernatant of the reprogramming medium according to Example 1 was added to the growth medium B in some of the wells at concentrations of 10.0 v/v% and 20.0 v/v%. In addition, the extract of the iPS cells according to Example 3 was added to the growth medium B in some of the wells at concentrations of 50.0 v/v% and 100.0 v/v%.

As a negative control, growth medium B in some wells was replaced with additive-free test medium B. In addition, as a reference control, growth medium B in some wells was replaced with adenosine-added medium in which 100 μmol/L of adenosine was added to a medium dedicated to dermal papilla cells, and minoxidil-added medium in which 30 μmol/L of minoxidil was added to a medium dedicated to dermal papilla cells. In addition, as a minoxidil vehicle control, growth medium B in some wells was replaced with DMSO-added medium in which 0.1% DMSO was added to a medium dedicated to dermal papilla cells.

After replacing the medium, the dermal papilla cells were cultured for 3 days, and the medium supernatant was collected and stored at -80°C. The medium supernatant was then thawed, and the vascular endothelial growth factor (VEGF) concentration in the medium supernatant was measured using a Human ELISA kit (Cat. No. ab100519, Abcam). The results are shown in Figures 6 and 7. It was shown that the supernatant of the medium in which iPS cells were induced and cultured, and the extract of iPS cells promote VEGF production in dermal papilla cells. This suggests that the supernatant of the medium in which iPS cells were induced and cultured, and the extract of iPS cells are effective for hair growth, hair regrowth, and hair thickening.

(Example 7: Test for FGF-7 production by hair papilla cells)
As in Example 5, normal human hair papilla cells were cultured for one day in growth medium B. Thereafter, the supernatant of the reprogramming medium according to Example 1 was added to the growth medium B in some of the wells at concentrations of 10.0 v/v% and 20.0 v/v%. In addition, the extract of the iPS cells according to Example 3 was added to the growth medium B in some of the wells at concentrations of 50.0 v/v% and 100.0 v/v%.

As a negative control, growth medium B in some wells was replaced with additive-free test medium B. In addition, as a reference control, growth medium B in some wells was replaced with adenosine-added medium in which 100 μmol/L of adenosine was added to a medium dedicated to dermal papilla cells, and minoxidil-added medium in which 30 μmol/L of minoxidil was added to a medium dedicated to dermal papilla cells. In addition, as a minoxidil vehicle control, growth medium B in some wells was replaced with DMSO-added medium in which 0.1% DMSO was added to a medium dedicated to dermal papilla cells.

After replacing the medium, the dermal papilla cells were cultured for 3 days, and the medium supernatant was collected and stored at -80°C. The medium supernatant was then thawed, and the fibroblast growth factor 7 (FGF-7) concentration in the medium supernatant was measured using an FGF-7 Human ELISA kit (Cat. No. ab100519, Abcam). The results are shown in Figures 8 and 9. It was shown that the supernatant of the medium in which iPS cells were induced and cultured, and the extract of iPS cells promote FGF-7 production by dermal papilla cells. This suggests that the supernatant of the medium in which iPS cells were induced and cultured, and the extract of iPS cells are effective for hair growth, hair regrowth, and hair thickening.

(Example 8: Fibroblast migration test)
Adult-derived normal human fibroblasts were suspended in 10% FBS medium at a concentration of 1 x ¹⁰⁵ to 2 x ¹⁰⁵ cells/well and seeded on a plate containing a kit for measuring migration ability (Radius Cell Migration Assay, registered trademark). Next, the human fibroblasts were treated with 10 μg/mL mitomycin C (Cat. No. 20898-21, Nacalai Tesque) for 2 hours to stop cell division of the human fibroblasts. The human fibroblasts were then cultured for 1 day in a _CO2 incubator (5% _CO2 , 37°C).

The medium in some of the plates was replaced with a medium in which an extract of iPS cells according to Example 3 was added to a 10% FBS medium to give a concentration of 10 v/v%. In addition, the medium in some of the plates was replaced with a medium in which the supernatant of the reprogramming medium according to Example 1 was added to a 10% FBS medium to give concentrations of 10.0 v/v% and 20.0 v/v%.

Growth medium B on some of the plates was replaced with epidermal cell medium containing no growth additives (additive-free test medium B) as a negative control.

Human fibroblasts were treated according to the protocol and a migration test was performed. During the wound healing process, human fibroblasts migrate toward the wound, causing it to shrink. In this example, a plate reader was used to analyze whether human fibroblasts migrated to areas that had been blocked with stoppers and to which human fibroblasts had not adhered prior to the migration test. Specifically, 23 hours after replacing the medium, epidermal cells were stained with Hechest and observed.

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

(Example 9: Preparation of cells derived from a patient with progeria)
Fibroblasts derived from a patient with Werner's syndrome (AG04110), a patient with xeroderma pigmentosum (GM16684 and GM16687), and a patient with Cockayne's syndrome (GM01098) were purchased from the Coriell Institute for Medical Research. These fibroblasts derived from patients with progeria were induced to become iPS cells. The iPS cells were then induced to differentiate into skin fibroblasts.

(Example 10: UV irradiation test of skin fibroblasts)
Normal human skin fibroblasts derived from an adult and skin fibroblasts induced from somatic cells of a progeria patient prepared in Example 9 were each suspended in test medium A to a concentration of 2 x ¹⁰⁵ cells/well, seeded into a 6-well plate, and cultured for 1 day in a _CO2 incubator (5% _CO2 , 37°C).

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

As a result, as shown in Figure 12, the cell death rate was increased in all skin fibroblasts irradiated with UV compared to cells not irradiated with UV. Furthermore, compared to normal skin fibroblasts, the mortality rate due to UV irradiation was higher in skin fibroblasts induced from somatic cells of patients with progeria. However, the survival rate of skin fibroblasts in a medium supplemented with the supernatant of the medium used for maintaining and culturing iPS cells was higher than that of skin fibroblasts in a medium not supplemented with the supernatant of the medium used for maintaining and culturing iPS cells. This suggests that skin fibroblasts induced from somatic cells of patients with progeria are sensitive to UV irradiation and are suitable for screening anti-UV substances.

(Example 11: Dryness Irritation Test on Skin Fibroblasts)
As in Example 10, normal human dermal fibroblasts derived from adults and dermal fibroblasts induced from somatic cells of a patient with progeria prepared in Example 9 were cultured for one day using test medium A. The next day, each well was dried for 40 seconds in the vent of a clean bench. Next, the test medium A in some of the wells was replaced with the supernatant-added medium A according to Reference Example 1. In addition, the supernatant solution according to Example 1 and the test medium A were mixed at a volume ratio of 10.00:90.00 to obtain the supernatant-added medium A according to Example 1 with a concentration of 10.00 v/v%. The test medium A in some of the wells was replaced with the supernatant-added medium A according to Example 1. The next day, all the cells were detached from the wells with trypsin, and the cells were stained with 7-aminoactinomycin D (7-AAD), and then the cell death rate was measured using a flow cytometer.

As a result, as shown in FIG. 13, the mortality rate due to dryness was higher in skin fibroblasts induced from somatic cells of patients with progeria compared to normal skin fibroblasts. However, skin fibroblasts in a medium supplemented with the supernatant of the medium used for maintaining and culturing iPS cells had a higher survival rate than skin fibroblasts in a medium not supplemented with the supernatant of the medium used for maintaining and culturing iPS cells. In addition, skin fibroblasts in a medium supplemented with the supernatant of the medium used for induced and culturing iPS cells from blood cells had a higher survival rate than skin fibroblasts in a medium supplemented with the supernatant of the medium used for maintaining and culturing iPS cells. This suggests that skin fibroblasts induced from somatic cells of patients with progeria are sensitive to dryness stimuli and are suitable for screening anti-drying stimuli substances.

(Example 12: Oxidative stress test of skin fibroblasts)
As in Example 10, normal human dermal fibroblasts derived from adults and dermal fibroblasts induced from somatic cells of a patient with progeria prepared in Example 9 were cultured for one day using test medium A. The next day, hydrogen peroxide was added to the test medium A in each well to a concentration of 0.03%. After 10 minutes, the medium in some of the wells was replaced with test medium A not containing hydrogen peroxide. In addition, the medium in some of the wells was replaced with the supernatant-added medium A according to Example 1 and Reference Example 1. Furthermore, the medium in some of the wells was replaced with the medium containing the extract of iPS cells according to Example 3. The next day, all the cells were detached from the wells with trypsin, and the cells were stained with 7-aminoactinomycin D (7-AAD), and then the cell death rate was measured using a flow cytometer.

As a result, as shown in Figures 14 and 15, the mortality rate due to dryness was higher in skin fibroblasts induced from somatic cells of patients with xeroderma pigmentosum compared to normal skin fibroblasts. Figure 15 shows the cell death rate of skin fibroblasts induced from somatic cells of patients with xeroderma pigmentosum. As shown in Figure 15, the survival rates of skin fibroblasts in the medium supplemented with the supernatant of the medium used for maintaining and culturing iPS cells, the medium supplemented with the supernatant of the medium used for inducing and culturing blood cells to iPS cells, and the medium supplemented with an extract of iPS cells were higher than those of skin fibroblasts in test medium A without the addition of any additives. This suggests that skin fibroblasts induced from somatic cells of patients with xeroderma pigmentosum are sensitive to oxidative stress and are suitable for screening antioxidant stress substances.

(Reference example)
Human iPS cells were cultured according to the example described in JP 2016-128396 A. That is, human iPS cells were cultured on feeder cells on a petri dish for adhesion culture using the same stem cell medium as in Example 1 to maintain adhesion. Human iPS cells were passaged every week. During passage, human iPS cells were treated with a detachment solution containing 0.25% trypsin, 0.1 mg/mL collagenase IV, 1 mmol/L CaCl ₂ , and 20% KSR.

Human iPS cells cultured as described above were detached from the petri dish for adhesion culture using ES cell dissociation solution (TrypLE Select, registered trademark, ThermoFisher). The detached human iPS cells were cultured in suspension for one week in non-gelled human iPS cells placed in a petri dish for non-adherent culture. As a result, embryoid bodies (EBs) were formed. The formed embryoid bodies were seeded on the petri dish for adhesion culture and outgrown for one week in DMEM containing 10% FBS and 1% Anti-Anti (registered trademark, antimycotic agent).

Next, the cells were detached from the petri dish for adhesion culture using a 0.05% trypsin-EDTA solution, and the cells that had split into single cells were seeded onto a new petri dish for adhesion culture. The cells were then cultured for one week using DMEM containing 10% FBS as the medium.

After confirming that the cells were 70% to 80% or more confluent, the cells were observed. A photograph of the cells cultured in this reference example is shown in FIG. 16(a). Usually, the morphology of undifferentiated iPS cells is as shown in the photograph in FIG. 16(b). Therefore, it was morphologically observed that the cells cultured in this reference example did not maintain an undifferentiated state. In addition, after culturing the cells according to this reference example for 21 days, the cells were treated with an anti-OCT3/4 antibody labeled with a fluorescent reagent and an anti-NANOG antibody labeled with a fluorescent reagent, and the results of observing the cells under a microscope are shown in FIG. 17. FIG. 17(a) shows a photograph of the cells observed without using excitation light. FIG. 17(b) shows a photograph of the cells observed using excitation light corresponding to the fluorescent reagent bound to the anti-OCT3/4 antibody. FIG. 17(c) shows a photograph of the cells observed using excitation light corresponding to the fluorescent reagent bound to the anti-NANOG antibody. No fluorescence was observed in Figures 17(b) and 17(c), confirming that the cells were OCT3/4-negative and NANOG-negative. Furthermore, when the cells were examined using a flow cytometer, it was confirmed that the cultured cells were negative for the undifferentiation markers NANOG, OCT3/4, and TRA 1-60. Therefore, it was confirmed that the cells did not maintain an undifferentiated state, but were differentiated.

Claims (2)

A wound treatment agent (excluding regenerating agents for UVA-damaged fibroblasts) containing the supernatant of the culture medium used in reprogramming somatic cells. A promoter of skin cell migration, including the supernatant of the culture medium used in reprogramming somatic cells (excluding the regenerator of UVA-damaged fibroblasts).