JP7214191B2 - Stem cell undifferentiated state maintenance agent and growth promoter - Google Patents

Description

TECHNICAL FIELD The present invention relates to an agent for maintaining an undifferentiated state of stem cells, an agent for promoting proliferation of stem cells, and a method for maintaining an undifferentiated state of stem cells and a method for promoting proliferation of stem cells.

In the tissues of vertebrates (particularly mammals), when cells/organs are damaged due to injury, disease, aging, or the like, the regeneration system works to recover the damaged cells/organs. Stem cells (tissue stem cells, somatic stem cells, adult stem cells) provided in the tissue play a major role in this action. Stem cells have pluripotency to differentiate into all kinds of cells and organs, and it is believed that this property leads to recovery by supplementing damaged parts of cells and organs. Expectations are high for regenerative medicine, which is a next-generation medical treatment that applies such stem cells.

The most advanced tissue for stem cell research in mammals is the bone marrow. Bone marrow contains the body's hematopoietic stem cells and has been shown to be the source of all blood cell regeneration. Furthermore, bone marrow is reported to contain stem cells capable of differentiating into organs and tissues (e.g., bone, cartilage, muscle, fat, etc.) in addition to hematopoietic stem cells (see Non-Patent Document 1).

Furthermore, in recent years, it has been clarified that stem cells exist in all organs and tissues other than bone marrow, such as skin, liver, pancreas, and fat, and are responsible for the regeneration and maintenance of homeostasis of each organ and tissue. I have come to understand (see Non-Patent Documents 2 to 5). In addition, stem cells present in each organ and tissue have excellent plasticity, and may be used to regenerate organs and tissues that have been unable to self-renew until now.

On the other hand, some of these stem cells are known to decrease with age, and research is being actively conducted on techniques to prevent the decrease of stem cells in order to maintain the homeostasis of each tissue ( Non-Patent Document 6). In recent years, in order to apply the ability (pluripotency) of stem cells to the regeneration of organs and tissues, in the fields of cell transplantation therapy and tissue engineering (regenerative medicine and regenerative beauty), stem cells are isolated from living tissues and then cultured. Techniques for proliferation are being developed (Non-Patent Documents 7 and 8).

In particular, when stem cells are cultured in vitro, it is extremely important to proliferate them while maintaining their pluripotency, which is the ability of stem cells, that is, maintaining their undifferentiated state. If the undifferentiated state of the stem cells cannot be maintained during this culture and the induction of differentiation proceeds, the ability (pluripotency) of the finally prepared stem cells will be lost, resulting in the desired effect ( Regeneration of organs and tissues, etc.) cannot be demonstrated.

Based on the above, when stem cells are used for cell transplantation therapy and tissue engineering (regenerative medicine and regenerative cosmetics) to regenerate organs and tissues, stem cells must be cultured while maintaining their undifferentiated state.

To date, there have been several reports on techniques for growing stem cells while maintaining their undifferentiated state, but they are still under development. For example, embryonic stem cells (ES cells) and hematopoietic stem cells can be maintained undifferentiated by co-culturing with supporting cells (stromal cells or feeder cells) (see Patent Document 1 and Non-Patent Documents 9-11). ). However, recently, cases of infection between heterologous animals by endogenous viruses derived from feeder cells have been reported (see Non-Patent Document 12), and culturing of stem cells using feeder cells has been proposed for medical purposes. not suitable for culturing

Another method is to maintain the undifferentiated state of stem cells by complex combinations of cytokines. For example, mouse ES cells are maintained undifferentiated by adding LIF (Leukemia Inhibitory Factor) to the medium (see Patent Document 2 and Non-Patent Document 13). Other embryonic stem cells that remain undifferentiated in the presence of early-acting cytokines thrombopoietin (TPO), interleukin-6 (IL-6), FLT-3 ligand, and stem cell factor (SCF); It has been reported in somatic stem cells and the like (see Patent Document 3 and Non-Patent Document 14).

Cytokines, however, are expensive, and have problems such as collection raw materials and storage stability, and are difficult to use easily. In addition, it was revealed that the effect of LIF is limited to a very specific cell lineage, and that the undifferentiated state cannot be maintained especially in primate ES cells and somatic stem cells by the addition of LIF alone. (See Non-Patent Document 10).

As described above, all of the currently reported methods for maintaining an undifferentiated state of stem cells require complicated operations and are less effective in maintaining an undifferentiated state. Therefore, in order to utilize stem cells in regenerative medicine, a technique for growing stem cells while maintaining an undifferentiated state has been desired. In other words, there has been a demand for a technology that can safely, simply, and efficiently proliferate stem cells while maintaining their undifferentiated state.

Japanese pepper (scientific name: Zanthoxylum piperitum) is a deciduous shrub belonging to the genus Zanthoxylum of the Rutaceae family, and its mature pericarp and immature fruit are used as a spice. In addition, the herbal medicine sansho (sansho) is a mature pericarp with as few seeds removed as possible. It is used in Chinese herbal medicine to improve stomach pain and bloating due to gastrointestinal function and enhance gastrointestinal function. So far, Japanese pepper has been known to have an effect of improving gray hair by promoting tyrosinase activity (Patent Document 4), promoting blood circulation (Patent Document 5), and the like. However, nothing has been known about the proliferation-promoting effect of Japanese pepper and the effect of maintaining an undifferentiated state of stem cells.

Japanese Patent Application Laid-Open No. 2004-24089 Japanese Patent Publication No. 2002-525042 Japanese Patent No. 3573354 JP-A-11-079951 JP 2008-69134 A

Pittenger M. F. et al., Science, 1999, Vol. 284, pp. 143-147 Goodell M. A. et al., Nat. Med. , 1997, Vol. 3, pp. 1337-1345 Zulewski H. et al., Diabetes, 2001, Vol. 50, pp. 521-533 Suzuki A. et al., Hepatology, 2000, Vol. 32, pp. 1230-1239 Zuk P. A. et al., Tissue Engineering, 2001, Vol. 7, pp. 211-228 Yasushi Hasegawa, Aesthetic Dermatology, 2013, Vol. 23, pp. 1-11 Hiroshi Mabuchi et al., Journal of the Japanese Society for Regenerative Medicine, 2007, Vol. 6, pp. 263-268 Kitagawa, et al., Japanese Society for Regenerative Medicine, 2008, Vol. 7, pp. 14-18 ThomsonJ. A. et al., Proc. Natl. Acad. Sci. USA, 1995, Vol. 92, pp. 7844-7848 ThomsonJ. A. et al., Science, 1998, Vol. 282, pp. 1145-1147 Reubinoff B.; E. et al., Nature Biotech. , 2000, Vol. 18, pp. 399-404 van der Laan L. J. et al., Nature, 2000, Vol. 407, pp. 90-94 Smith A. G. et al., Dev. Biol. , 1987, Vol. 121, pp. 1-9 Madlambayan G.M. J. et al., J. et al. Hematother. Stem Cell Res. , 2001, Vol. 10, pp. 481-492

In view of the circumstances described above, the present invention seeks to find a new substance capable of efficiently proliferating stem cells while maintaining their undifferentiated state, and to provide the substance as an undifferentiated state-maintaining agent or proliferation-promoting agent for stem cells. is the subject.

The inventors of the present invention conducted intensive research to solve the above problems, and as a result, found that the extract of Japanese pepper has an excellent undifferentiated state-maintaining effect and proliferation-promoting effect on stem cells, which was hitherto unknown. The discovery led to the completion of the present invention.

That is, the present invention includes the following.
(1) An agent for maintaining an undifferentiated state of stem cells containing an extract of Japanese pepper as an active ingredient.
(2) A proliferating agent for stem cells containing an extract of Japanese pepper as an active ingredient.
(3) The agent according to (1) or (2), wherein the stem cells are hair follicle stem cells.
(4) The agent according to any one of (1) to (3), wherein the Japanese pepper is grape Japanese pepper.
(5) A method for producing stem cells, which comprises the step of culturing in a medium containing an extract of Japanese pepper.
(6) A method for maintaining an undifferentiated state of stem cells, which comprises the step of culturing stem cells in a medium containing an extract of Japanese pepper.
(7) A method for promoting growth of stem cells, comprising the step of culturing stem cells in a medium containing an extract of Japanese pepper.
(8) The method according to any one of (5) to (7), wherein the stem cells are hair follicle stem cells.
(9) The method according to any one of (5) to (8), wherein the Japanese pepper is grape Japanese pepper.

According to the present invention, stem cells can be efficiently proliferated while maintaining an undifferentiated state. Therefore, the present invention can greatly contribute to the fields of regenerative medicine and regenerative beauty.

The present invention will be described in detail below.
An agent for maintaining an undifferentiated state or promoting proliferation of stem cells according to the present invention contains an extract of Japanese pepper as an active ingredient.

Japanese pepper (scientific name: Zanthoxylum piperitum) used in the present invention is a deciduous shrub belonging to the genus Zanthoxylum of the Rutaceae family, and grows naturally in Japan. It is also the original plant of the herbal medicine sansho (Japanese name: sansho). Examples of the Japanese pepper that can be used in the present invention include grape Japanese pepper belonging to the genus Zanthoxylum of the Rutaceae family (scientific name: Zanthoxylum piperitum (L.) DC f. inerme Makino), Zanthoxylum piperitum (L.) DC forma inerme (Makino) Makino), Zanthoxylum schinifolium, and Zanthoxylum piperitum (L.) DC forma brevispinosum Makino, among which Grape Zanthoxylum is preferred.

In the present invention, the extract of Japanese pepper refers to an extract of a part or the whole plant such as flowers, fruits, pericarp, stems, leaves, branches, roots and seeds of Japanese pepper, or a mixture thereof. However, the part used as the raw material for extraction in the present invention is preferably the pericarp or the seed. Peel (powder Japanese pepper) can be used as a condiment.

The extraction method for obtaining an extract from Japanese pepper is not particularly limited, and may be, for example, a heat extraction method, a normal temperature extraction method, or a cold temperature extraction method. Solvents used for extraction include, for example, water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol , propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran , propyl ether, etc.). Among these solvents, water, lower alcohols and liquid polyhydric alcohols are preferred, and water and ethanol are more preferred. These solvents may be used singly or in combination of two or more. For example, 30 to 70 v/v % ethanol aqueous solution may be used. Also, a solvent whose pH is adjusted by adding an acid or an alkali to the extraction solvent can be used.

The amount of the solvent to be used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more, relative to the pericarp (dry weight) of the Japanese pepper. For convenience of operation, it is preferably 100 times or less. The extraction temperature and time depend on the type of solvent used, but can be exemplified, for example, at 10 to 100° C., preferably 30 to 90° C., for 30 minutes to 24 hours, preferably 1 to 10 hours. More specifically, an extract of Japanese pepper can be obtained by adding water to the pericarp of Japanese pepper and performing hot water extraction at 95 to 100°C. Alternatively, a lower alcohol (e.g., ethanol, etc.) or a liquid polyhydric alcohol (e.g., propylene glycol, 1,3-butylene glycol, etc.) is added to the pericarp of Japanese pepper, and extraction is performed at room temperature (e.g., 5 to 35 ° C.). Thus, an extract of Japanese pepper can be obtained.

The extract may be used as an extracted solution, but if necessary, concentration (concentration by organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, activated carbon, etc., within a range that does not affect the effect. It may be used after being subjected to treatments such as decolorization, deodorization, and ethanol precipitation. Furthermore, the extracted solution may be subjected to a treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

The extract of Japanese pepper obtained in this way has the effect of efficiently proliferating stem cells while maintaining an undifferentiated state at the biological level or at the culture level. Can be used as an agent. Furthermore, the agent for maintaining an undifferentiated state of stem cells or the agent for promoting proliferation of stem cells according to the present invention is also used as a medium additive for stem cell culture and a research reagent for efficiently proliferating stem cells while maintaining an undifferentiated state. be able to.

By applying the agent for maintaining an undifferentiated state of stem cells or the agent for promoting proliferation of stem cells according to the present invention to stem cells of mammals including humans, the undifferentiated state of stem cells can be maintained and proliferation of stem cells can be promoted. . The stem cells to which the agent for maintaining an undifferentiated state of stem cells or the agent for promoting growth of stem cells according to the present invention is applied are not particularly limited as long as they meet the purpose of the present invention. For example, embryonic stem cells (ES cells); , skin (epidermis, dermis, subcutaneous tissue), fat, hair follicles, brain, nerve, liver, pancreas, kidney, muscle, and other tissues; stem cells artificially produced by gene transfer, etc. (induced pluripotent stem cells: iPS cells). The agent for maintaining an undifferentiated state of stem cells or the agent for promoting growth of stem cells according to the present invention preferably exerts a greater effect on hair follicle stem cells, maintains the undifferentiated state of the hair follicle stem cells in the bulge region of the hair follicle, and differentiates them. can be promoted while suppressing ES cells include, for example, ES cells established by culturing pre-implantation early embryos, ES cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei, and ES cells in which the genes on the chromosome of these ES cells have been modified using genetic engineering techniques. Such ES cells can be produced, for example, by a method known per se, and can be obtained from predetermined institutions or purchased commercially. Moreover, these stem cells may be primary cultured cells, subcultured cells or frozen cells.

Furthermore, the agent for maintaining an undifferentiated state of stem cells or the agent for promoting proliferation of stem cells according to the present invention can be applied to stem cells derived from all mammals, as long as they have the same characteristics regarding the direction of differentiation and the process of differentiation of stem cells. It is possible. For example, the agent for maintaining an undifferentiated state of stem cells or the agent for promoting proliferation of stem cells according to the present invention can be stem cells from mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, and pigs. can be effective against

The agent for maintaining an undifferentiated state of stem cells or the agent for promoting proliferation of stem cells according to the present invention may be applied to stem cells either in vitro or in vivo, and the effect can be exhibited in either case. Therefore, the agent for maintaining an undifferentiated state of stem cells or the agent for promoting growth of stem cells according to the present invention can be obtained by culturing stem cells in a stem cell culture medium containing an effective amount thereof, or by administering it to mammals including humans. , can maintain the undifferentiated state of stem cells and promote proliferation.

The agent for maintaining an undifferentiated state of stem cells or the agent for promoting growth of stem cells according to the present invention is suitable for use in skin, osteoblasts, cartilage, since the extract of Japanese pepper, which is an active ingredient, has an excellent effect of maintaining an undifferentiated state and promoting proliferation of stem cells. , muscles, nerves, fat, liver, etc., and is effective in treating, ameliorating, and preventing injury or damage to tissues or organs in vivo. In addition, since stem cells decrease or their function deteriorates with aging, the agent for maintaining an undifferentiated state of stem cells or the agent for promoting growth of stem cells according to the present invention is effective against the decrease or function deterioration of stem cells in the tissue or organ in the living body. Effective in treating, ameliorating and preventing associated diseases. Here, diseases related to tissue or organ damage or damage, reduction or functional decline of stem cells include, for example, skin-related wrinkles, sagging, spots, dullness, rough skin, thickened skin, open pores, and acne scars. , wounds, scars, keloids, etc., including scalp and hair damage such as thinning hair and hair loss. In addition, bone-related diseases such as osteoporosis and fractures (vertebral compression fractures, femoral neck fractures, etc.), cartilage-related diseases such as osteoarthritis, rheumatoid arthritis, and intervertebral disc herniation, and nerve-related diseases such as spinal cord injury and facial paralysis. , Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, age-related memory loss, etc. Blood-related diseases such as aplastic anemia, leukemia, etc. Cardiovascular-related diseases such as myocardial infarction, arteriosclerosis obliterans, etc. Dentistry Related diseases include periodontal disease and alveolar bone damage due to alveolar pyorrhea; ophthalmic diseases such as retinitis pigmentosa, age-related macular degeneration, and glaucoma; liver and pancreatic diseases such as hepatitis, cirrhosis, and diabetes. is not limited to

In addition, the extract of Japanese pepper has the effect of promoting proliferation of hair follicle stem cells and the effect of maintaining an undifferentiated state. That is, the Japanese pepper extract can inhibit the differentiation or depletion of hair follicle stem cells in the bulge region of the hair follicle, thereby preventing the reduction or depletion of hair follicle stem cells and promoting the proliferation of hair follicle stem cells. Therefore, it can be used as an active ingredient of an agent for preventing and/or improving alopecia. In the present invention, "prevention and/or amelioration of alopecia" includes prevention of occurrence of hair loss or thinning hair, improvement of the degree (number or range) of hair loss or thinning hair, reduction in progress speed of hair loss or thinning hair, and reduction of hair loss or thinning hair. It includes the suppression of the decrease in luster and elasticity of hair associated with hair loss. In addition, the effect of preventing and/or improving alopecia includes both a direct action mechanism on the hair and a percutaneous action mechanism on the head. In the present invention, alopecia refers to a symptom in which part or all of the hair falls out due to aging, disease, various stresses such as ultraviolet rays and overwork, and part or all of the scalp becomes transparent. . Alopecia includes, for example, male pattern baldness (AGA), diffuse alopecia (FAGA), alopecia areata, senile alopecia, seborrheic alopecia, alopecia pityriasis, postpartum (postpartum) hair loss. disease, mechanical (pressure or traction) alopecia, congenital alopecia, alopecia after burns or trauma, drug-induced alopecia caused by anticancer drugs, scarring alopecia (lichen planus pilaris, erythematous lupus, folliculitis bald, papillary dermatitis of the head, etc.), trichotillomania (trichotillomania), etc., but not limited to these.

The content of the extract of Japanese pepper in the agent for maintaining an undifferentiated state of stem cells or the agent for promoting growth of stem cells according to the present invention is not particularly limited, but depending on the properties of the extract (extract, concentrate, or dried product), for example, It can be appropriately set in the range of 0.00001 to 100% by weight, preferably 0.0001 to 10% by weight, more preferably 0.001 to 1% by weight.

The present invention also relates to a method for promoting stem cell proliferation while maintaining an undifferentiated state of stem cells by culturing stem cells in a medium containing an extract of Japanese pepper. In other words, the method according to the present invention is a method for producing stem cells, a method for maintaining an undifferentiated state of stem cells, or a method for promoting proliferation of stem cells, which comprises a step of culturing stem cells in a medium containing an extract of Japanese pepper. be able to.

In the method according to the present invention, stem cells may be cultured using media that are commonly used for maintaining and proliferating stem cells in an undifferentiated state. For example, a basal medium containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids) necessary for the survival and proliferation of stem cells, specifically Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI1640, Basal Medium Eagle (BME), Ham's F12, Ham's F12K (Kaighn's modified of Ham's F12), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM/F-12), Glasgow inimum Essential Medium (Glasgow MEM), Hank's balanced salt solution, MCDB153 medium and the like. In addition, the medium contains growth factors [epidermal growth factor (EGF), cholera toxin, basic fibroblast growth factor (bFGF), leukocyte migration inhibitory factor (LIF), stem cell factor (SCF), etc.], hormones (insulin , hydrocortisone, etc.) are preferably added. In addition, if necessary, the medium contains tumor necrosis factor (TNF), vitamins, interleukins, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement , antibiotics and the like.

In addition to the above components, the medium preferably contains bovine pituitary extract (BPE), serum or serum substitute at a content of 0.1 to 20% (v/v). Any known serum or serum substitute can be used. For example, serum includes fetal bovine serum (FBS, FCS) and the like, and serum substitutes include KSR and the like. However, since the components of serum differ depending on the lot, and the effect varies, it is preferable to use serum after lot check.

Commercially available media include Invitrogen's mesenchymal stem cell basal medium, Sanko Junyaku's mesenchymal stem cell basal medium, TOYOBO's MF medium, Sigma's Hank's balanced salt solution, etc. can be used. In addition, when the stem cells are hair follicle stem cells, commercially available epithelial cell culture media containing components necessary for culturing epithelial cells (e.g., insulin, hydrocortisone, epidermal cell growth factor, etc.) may be used. Keratinocyte-SFM medium (Thermo Fisher Scientific), Humedia-KG2 medium (KURABO), CnT-PR medium (CellnTec), MCDB153 medium (Sigma), serum-free medium for normal human keratinocytes (DS Pharma Biomedical company) and the like.

The incubator used for culturing stem cells is not particularly limited as long as it is capable of culturing stem cells. .

The incubator may be cell non-adhesive or adhesive, and is appropriately selected depending on the purpose. Cell-adhesive incubators may be treated with cell-supporting substrates such as extracellular matrices for the purpose of improving adhesion to cells. Cell-supporting matrices include, for example, collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.

The concentration of the Japanese pepper extract added to the medium used for stem cell culture is appropriately determined according to the content of the Japanese pepper extract in the agent for maintaining an undifferentiated state of stem cells or the agent for promoting proliferation of stem cells according to the present invention. However, the concentration is, for example, 10 to 10,000 μg/mL, preferably 100 to 5,000 μg/mL in terms of dried Japanese pepper. Also, these extracts may be periodically added to the medium during the culture period of the stem cells.

The conditions for culturing stem cells may follow the usual conditions used for culturing stem cells, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30-40°C, preferably 36-37°C. The CO ₂ gas concentration is, for example, about 1-10%, preferably about 2-5%. The culture medium is preferably exchanged once every 2 to 3 days, more preferably every day. The culture conditions can also be set by appropriately varying them within a range in which stem cells can survive and proliferate.

For maintaining an undifferentiated state of stem cells, for example, compared with stem cells cultured in the absence of the agent for maintaining an undifferentiated state of stem cells according to the present invention, the stem cells are cultured in the presence of the agent for maintaining an undifferentiated state of stem cells according to the present invention. Evaluation can be made by determining whether the expression level of the undifferentiated stem cell marker gene is significantly maintained at the mRNA level or protein level in the stem cells that have been cultured, at a level comparable to that at the start of culture. Examples of undifferentiated stem cell marker genes include Nanog gene (Cell Res. 2007 Jan; 17(1): 42-9. Review. Nanog and transcriptional networks in embryonic stem cell pluripotency. Pan G, Thomson JA.). . Examples of hair follicle stem cell undifferentiated marker genes include KRT15 (Keratin, type I cytoskeletal 15).

Methods for measuring the stem cell undifferentiated marker gene expression level include, at the mRNA level, methods such as RT-PCR, quantitative PCR, and Northern blotting using primers and probes specific to the stem cell undifferentiated marker gene. At the protein level, immunological methods such as ELISA, flow cytometry, Western blotting, etc. using antibodies specific to proteins encoded by undifferentiated stem cell marker genes can be used.

As a result of measuring the expression level, the expression level of the stem cell undifferentiated marker gene in the stem cells at the start of culture (100% undifferentiated state) and the stem cell after culturing for a predetermined time in the presence of the agent for maintaining the undifferentiated state of stem cells of the present invention When the relative ratio to the expression level of the undifferentiated stem cell marker gene is greater than the same relative ratio (control) when the stem cells are cultured in the absence of the agent for maintaining the undifferentiated state of the stem cells of the present invention, the undifferentiated state of stem cells is indicated. can be determined to be maintained.

In addition, stem cell proliferation promotion is, for example, compared to stem cells cultured in the absence of the stem cell proliferation-promoting agent of the present invention. It can be evaluated by whether or not the number of cells is significantly increased. The cell count can be measured, for example, by the MTT method, WST method, or the like, using a commercially available cell count measurement kit. As a result of the measurement, the relative ratio between the number of stem cells at the start of culture and the number of stem cells after culturing for a predetermined period of time in the presence of the agent for promoting proliferation of stem cells of the present invention was It can be determined that the proliferation of stem cells has been promoted when the ratio is greater than the same relative ratio (control) when cultured in the presence.

Stem cells prepared by the above method according to the present invention can be used as a transplant material (cell transplant agent), and can be performed in the same manner as conventional bone marrow transplantation or umbilical cord blood transplantation.

According to the agent for maintaining an undifferentiated state of stem cells or the agent for promoting proliferation of stem cells according to the present invention or the method according to the present invention, the extract of Japanese pepper is used alone, separately from the medium, or mixed with the medium to produce stem cells. It can also be provided as a reagent kit for maintaining an undifferentiated state or promoting proliferation. The kit can contain an instruction manual and the like as necessary. Alternatively, a Japanese pepper extract can be mixed with a medium and provided as a medium for maintaining an undifferentiated state or promoting proliferation of stem cells.

When the agent for maintaining an undifferentiated state of stem cells or the agent for promoting proliferation of stem cells according to the present invention is administered in vivo, it may be administered as it is, but it may be administered together with an appropriate additive to the extent that the effects of the present invention are not impaired. It can be provided by blending in various compositions such as cosmetics, quasi-drugs, pharmaceuticals, and food and drink. The drug of the present invention also includes drugs used for animals, that is, veterinary drugs.

When the agent for maintaining an undifferentiated state of stem cells or the agent for promoting proliferation of stem cells according to the present invention is incorporated in cosmetics or quasi-drugs, the dosage form may be an aqueous solution system, a solubilization system, an emulsification system, a powder system, a powder dispersion system, Any of an oil-based system, a gel system, an ointment system, an aerosol system, a water-oil two-layer system, or a water-oil-powder three-layer system may be used. In addition, for the cosmetics and quasi-drugs, various ingredients, additives, bases, etc. that are usually used in external skin compositions are selected according to the type, along with the agent for maintaining the undifferentiated state of stem cells or the agent for promoting proliferation. and can be appropriately blended and manufactured according to techniques known in the art. The form may be liquid, emulsion, cream, gel, paste, spray, or the like. Examples of components of the external composition for skin 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, isopropyl palmitate, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid) acids, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant/animal extracts, various surfactants agents, moisturizing agents, ultraviolet absorbers, antioxidants, stabilizers, preservatives, bactericides, fragrances and the like.

Types of cosmetics and quasi-drugs include lotions, milky lotions, gels, serums, general creams, sunscreen creams, packs, masks, facial cleansers, toilet soaps, foundations, powders, bath agents, body lotions, body shampoos, hair shampoos, hair conditioners, hair restorers, and the like.

When the agent for maintaining an undifferentiated state of stem cells or the agent for promoting growth of stem cells according to the present invention is incorporated into a pharmaceutical, the dosage form is suitable for mixing with pharmacologically and pharmaceutically acceptable additives and applying to the affected area. It can be formulated into various formulations of Pharmacologically and pharmaceutically acceptable additives include formulation bases and carriers, excipients, diluents, binders, lubricants, and coatings that are appropriately selected according to the dosage form and application. agent, disintegrant or disintegration aid, stabilizer, preservative, preservative, bulking agent, dispersant, wetting agent, buffer, solubilizer or dissolution aid, tonicity agent, pH adjuster, propellant , colorants, sweeteners, corrigents, flavoring agents, etc., may be added as appropriate, and various formulations that can be administered systemically or locally via oral or parenteral administration may be prepared by various known methods. When the drug of the present invention is provided in each of the forms described above, it can be produced by a method commonly used by those skilled in the art, for example, the method shown in the Japanese Pharmacopoeia General Rules for Formulations [2] Each article for formulations.

The form of the pharmaceutical of the present invention is not particularly limited, but examples include tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs. oral agents such as agents, injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drips, suppositories, ointments, lotions, eye drops, sprays, oral Examples include parenteral agents such as skin absorbers, transmucosal absorbers, and patches. It may also be a dry product to be reconstituted for use, and in the case of an injectable formulation, provided in unit dose ampules or multi-dose containers.

When the agent for maintaining an undifferentiated state of stem cells or the agent for promoting proliferation of stem cells according to the present invention is used as a pharmaceutical for treating, ameliorating, and preventing the aforementioned skin-related injuries and diseases, a suitable form is an external preparation, for example, Examples include ointments, creams, gels, liquids, patches (plasts, plasters), foams, sprays, sprays, and the like. An ointment refers to a homogeneous semi-solid preparation for external use, including oleaginous ointment, emulsion ointment, and water-soluble ointment. A gel is an external preparation in which a water-insoluble hydrate compound is suspended in an aqueous solution. Liquid preparations refer to liquid preparations for external use, including lotions, suspensions, emulsions, liniments and the like.

The medicament of the present invention functions as a prophylactic drug that suppresses the onset of the above diseases and/or as a therapeutic drug that improves normal conditions. Since the active ingredient of the pharmaceutical of the present invention is derived from natural products, it is extremely safe and has no side effects. It can be administered orally or parenterally to mammals such as , dogs and cats in a wide range of doses.

The content of the agent for maintaining an undifferentiated state of stem cells or the agent for promoting growth of stem cells in the cosmetics, pharmaceuticals, and quasi-drugs of the present invention is not particularly limited. It is preferably 0.001 to 30% by weight, more preferably 0.01 to 10% by weight, in terms of a substance. The above amounts are only examples, and may be appropriately set and adjusted in consideration of the type and form of the composition, the general usage amount, efficacy and effects, and the like. In addition, the method of adding the active ingredient in formulation may be added in advance or may be added during the production, and may be appropriately selected in consideration of workability.

In addition, the agent for maintaining an undifferentiated state of stem cells or the agent for promoting growth of stem cells according to the present invention can also be added to food and drink. In addition, in the present invention, food and drink means general food and drink, as well as foods other than pharmaceuticals that can be ingested for the purpose of maintaining or improving health, such as health foods, functional foods, health functional foods, or special purpose foods. Used in the sense of including food. Health foods include foods provided under the names of nutritional supplements, health supplements, supplements, and the like. Foods with health claims are defined by the Food Sanitation Act or the Health Promotion Act. This includes foods with function claims that can display the content notified to the Commissioner of the Consumer Affairs Agency regarding the functionality of the food. Foods for special dietary uses include foods for the sick, foods for the elderly, foods for infants, foods for pregnant women, etc., which are labeled as suitable for specific subjects or patients with specific diseases. When the stem cells are hair follicle stem cells, the agent can be suitably used in health foods for preventing or improving hair loss and thinning hair. Here, indications such as specific health effects and functions of nutritional ingredients attached to foods and drinks are indicated on product containers, packaging, instructions, and attached documents, product flyers and pamphlets, newspapers and magazines, etc. can be advertisements for products, etc.

The form of food and drink may be any form suitable for eating, such as solid, liquid, granule, grain, powder, capsule, cream, or paste. In particular, the shape of the above-mentioned health food and the like is, for example, tablet, round, capsule, powder, granule, fine grain, troche, liquid (including syrup, milk, and suspension). etc. are preferred.

Types of food and drink include bread, noodles, confectionery, dairy products, marine and livestock processed foods, oils and fats processed foods, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks , dairy beverages, etc.), and concentrated stock solutions and powders for preparation of such beverages, but are not limited thereto.

The food or drink of the present invention may contain additives that are commonly used depending on the type thereof. Any additive can be used as long as it is acceptable under the Food Sanitation Act. Examples include sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; citric acid and malic acid. , acidulants such as tartaric acid; excipients such as dextrin and starch; binders, diluents, flavoring agents, coloring agents, buffering agents, thickening agents, gelling agents, A turbidity agent, a preservative, etc. are mentioned.

When the food or drink of the present invention is a general food or drink, it can be produced by including a step of adding an extract of Japanese pepper in the normal manufacturing process of the food or drink. In the case of health foods, the manufacturing method for pharmaceuticals may be followed. For example, in the case of tablet supplements, additives such as excipients are added to the extract of Japanese pepper, mixed, and processed by a tableting machine. It can be manufactured by molding by applying pressure such as. In addition, other materials (for example, vitamins such as vitamin C, vitamin B ₂ and vitamin B ₆ , minerals such as calcium, dietary fiber, etc.) can be added as necessary.

The amount of the Japanese pepper extract in the food and drink of the present invention may be any amount that can exhibit the effect of maintaining the undifferentiated state and the effect of promoting proliferation of stem cells. It may be appropriately set in consideration of form, efficacy/effect, taste, palatability, cost, and the like.

EXAMPLES The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[Example 1] Production example of extract of Japanese pepper An extract of Japanese pepper was produced as follows.
(Production Example 1) Production of 50% Ethanol Extract of Pericarp of Grape Zanthoxylum 10 g of dried grape zanthoxylum pericarp was immersed in 200 mL of a 50% (v/v) ethanol aqueous solution at room temperature for 4 days. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 1.0 g of a 50% ethanol extract of the pericarp of grape zanthoxylum.

(Production Example 2) Production of Hot Water Extract of Pericarp of Grape Zanthoxylum 200 mL of water was added to 10 g of dried grape zanthoxylum pericarp and extracted at 95 to 100° C. for 2 hours. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.3 g of a hot water extract of grape zanthoxylum pericarp.

(Production Example 3) Production of Ethanol Extract of Pericarp of Grape Zanthoxylum 10 g of dried grape zanthoxylum pericarp was immersed in 200 mL of an ethanol aqueous solution at room temperature for 4 days. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 0.5 g of an ethanol extract of the pericarp of grape zanthoxylum.

(Production Example 4) Production of 50% Ethanol Extract of Grape Zanthoxylum Seeds 10 g of dried grape Zanthoxylum seed was immersed in 200 mL of a 50% (v/v) aqueous ethanol solution at room temperature for 4 days. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 1.0 g of a 50% ethanol extract of grape zanthoxylum seeds.

(Production Example 5) Production of 50% Ethanol Extract of Pericarp of Zanthoxylum japonicum 10 g of dried pericarp of Zanthoxylum japonicum was immersed in 200 mL of a 50% (v/v) aqueous ethanol solution at room temperature for 4 days. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 1.0 g of a 50% ethanol extract of the pericarp of Asakura Zanthoxylum.

(Production Example 6) Production of hot water extract of pericarp of Asakula Zanthoxylum 200 mL of water was added to 10 g of dried pericarp of Asakula Zangtho, and extracted at 95 to 100°C for 2 hours. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.3 g of a hot water extract of pericarp of Asakura Zanthoxylum.

(Production Example 7) Production of Ethanol Extract of Pericarp of Asakura Zanthoxylum 10 g of dried pericarp of Asakura Zanthoxylum was immersed in 200 mL of an ethanol aqueous solution at room temperature for 4 days. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 0.5 g of an ethanol extract of the pericarp of Asakura Zanthoxylum.

(Production Example 8) Production of 50% Ethanol Extract of Seeds of Zanthoxylum japonicum 10 g of dried Zanthoxylum japonicum seeds were immersed in 200 mL of a 50% (v/v) aqueous ethanol solution at room temperature for 4 days. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 1.0 g of a 50% ethanol extract of Asakurazansho seeds.

[Example 2] Proliferation-promoting effect and undifferentiated state-maintaining effect of Japanese pepper extract on hair follicle stem cells The proliferation-promoting effect and undifferentiated state-maintaining effect on stem cells using the Japanese pepper extract produced in Example 1 will be described below. An experimental example of the state maintenance effect and its results are shown.

(Experimental example 1) Evaluation of the proliferation-promoting effect of Japanese pepper extract on hair follicle stem cells (1) Cultivation of human hair follicle stem cells The tissue containing After washing with PBS(-), treatment with trypsin (manufactured by BD Biosciences) was performed. Cells were then isolated and collected using a cell strainer (manufactured by FALCON). The collected cells were seeded on a culture plate and maintained until confluent using KG2 medium (manufactured by KURABO). The confluent cells were collected and reseeded on the culture plate, and then engrafted and proliferating cells were used as hair follicle stem cells in the following tests.

(2) Evaluating the proliferation-promoting activity of Japanese pepper extract on hair follicle stem cells Applying Japanese pepper extract to hair follicle stem cells to determine how the Japanese pepper extract is involved in the proliferation of hair follicle stem cells. evaluated. 1×10 ⁴ hair follicle stem cells cultured in KG2 medium (manufactured by KURABO) were seeded in a 24-well culture plate, and the test substance (extract of Japanese pepper of Production Examples 1 to 8) was added to a final concentration of 1000 μg. /ml, and cultured for 3 days.

The cell proliferation rate of hair follicle stem cells after 3 days was detected by MTT assay. MTT assay was performed using a commercially available MTT assay kit (manufactured by Cosmo Bio) according to the attached protocol. The total number of cells when no test substance was added was taken as a control, and the control was taken as 100 (%), and the change (%) in the number of cells when the test substance was added was calculated to evaluate the effect of promoting stem cell growth. The results of these tests are shown in Table 1 below.

As shown in Table 1, all of the extracts of Japanese pepper (Production Examples 1 to 8) were found to have a remarkable growth-promoting effect on hair follicle stem cells. In addition, when embryonic stem cells (ES cells) were also tested in the same manner as the stem cells used in this experimental example, a remarkable stem cell proliferation-promoting effect was observed.

(Experimental Example 2) Evaluation of the effect of extract of Japanese pepper on hair follicle stem cells to maintain an undifferentiated state The extract of Japanese pepper was applied to hair follicle stem cells to determine how the extract of Japanese pepper affects the undifferentiated state of hair follicle stem cells. and how they are involved. 1×10 ⁴ hair follicle stem cells cultured in KG2 medium (manufactured by KURABO) were seeded in a 24-well culture plate, and the test substance (extract of Japanese pepper of Production Examples 1 to 8) was added to a final concentration of 1000 μg. 3 days after the addition of the extract of Japanese pepper, the hair follicle stem cells were collected. was analyzed as an index.

Gene expression analysis of KRT15 was performed as follows. The hair follicle stem cells after addition of the extract of Japanese pepper were washed twice with PBS(-), and then RNA was extracted from the cells with Trizol Reagent (manufactured by Invitrogen). After reverse transcription of the extracted RNA into cDNA using a 2-STEP real-time PCR kit (manufactured by Applied Biosystems), real-time PCR (95°C: 15 sec. , 60°C: 30 seconds, 40 cycles), and the gene expression level of KRT15 was measured. Other operations were carried out according to prescribed methods.

Primer set for KRT15 (hair follicle stem cell undifferentiated marker):
5'-GATGCTGCTTGACATAAAGACACG-3' (SEQ ID NO: 1)
5'- ACCTGTCCATCCACTGACTCTTC-3' (SEQ ID NO: 2)
Primer set for 18srRNA (internal standard):
5'-CCGAGCCGCCTGGATAC-3' (SEQ ID NO: 3)
5'-CAGTTCCGAAAACCAACAAAATAGA-3' (SEQ ID NO: 4)

The effect of maintaining the undifferentiated state of hair follicle stem cells was calculated as the relative expression level of KRT15 gene ( KRT15 gene expression level/18srRNA gene expression level) was defined as 100% undifferentiated state, and the relative expression level of KRT15 gene in hair follicle stem cells after 3 days of culture was calculated and evaluated. The results of these tests are shown in Table 2 below.

As shown in Table 2, all of the extracts of Japanese pepper (Production Examples 1 to 8) were found to have a remarkable effect of maintaining an undifferentiated state on hair follicle stem cells. In addition, when embryonic stem cells (ES cells) were also tested in the same manner as the stem cells used in this experimental example, a remarkable effect of maintaining an undifferentiated state of stem cells was observed.

As shown in each of the above experimental examples, the application of the extract of Japanese pepper to stem cells promotes the proliferation of stem cells while maintaining their undifferentiated state more easily and efficiently than conventional techniques. became possible.

As an application example of the present invention, application to regenerative medicine and regenerative beauty is expected. For example, by using the present invention, it is possible to easily and efficiently prepare undifferentiated stem cells for use in regenerative medicine and regenerative cosmetics. Furthermore, the extract of Japanese pepper according to the present invention can be directly injected into stem cells after transplantation of stem cells or existing in tissues, or applied by oral administration, application, patching, etc. , it is possible to proliferate while maintaining an undifferentiated state.
That is, the present invention can be used as a method for producing stem cells and/or an agent for maintaining an undifferentiated state or promoting growth of stem cells in regenerative medicine and regenerative cosmetics.

Claims (7)

An agent for maintaining an undifferentiated state of hair follicle stem cells, containing as an active ingredient a water or hot water extract, an ethanol aqueous solution extract, or an ethanol extract of pericarp or seeds of Japanese pepper. A hair follicle stem cell proliferation promoting agent containing, as an active ingredient , a water or hot water extract, an ethanol aqueous solution extract, or an ethanol extract of pericarp or seeds of Japanese pepper. The agent according to claim 1 or 2 , wherein the Japanese pepper is grape Japanese pepper. A method for producing hair follicle stem cells, comprising the step of culturing in a medium containing a water or hot water extract, an ethanol aqueous solution extract, or an ethanol extract of pericarp or seeds of Japanese pepper. A method for maintaining an undifferentiated state of hair follicle stem cells, comprising the step of culturing hair follicle stem cells in a medium containing a water or hot water extract, an ethanol aqueous solution extract, or an ethanol extract of pericarp or seed of Japanese pepper. A method for promoting proliferation of hair follicle stem cells, comprising the step of culturing hair follicle stem cells in a medium containing a water or hot water extract, an ethanol aqueous solution extract, or an ethanol extract of pericarp or seeds of Japanese pepper. The method according to any one of claims 4 to 6 , wherein the Japanese pepper is grape Japanese pepper.