JP6571494B2 - Stem cell undifferentiated state maintenance agent and growth promoter using Ainu Wakame - Google Patents

Description

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

  When tissue of vertebrates (especially mammals) is damaged due to injury or disease, or with aging, etc., the regeneration system works and tries to recover the damage of cells / organs. Stem cells (tissue stem cells, somatic stem cells) provided in the tissue play a major role in this action. Stem cells have the pluripotency to differentiate into all cells and organs, and this property is thought to lead to recovery by compensating for damaged parts of cells and organs. Expectations are gathered for regenerative medicine, which is the next generation of medicine using such stem cells.

  The most advanced tissue in stem cell research in mammals is the bone marrow. The bone marrow contains living hematopoietic stem cells, which have been shown to be the source of all blood cell regeneration. Furthermore, it has been reported that the bone marrow contains stem cells that can be differentiated into other organs / tissues (eg, 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 / tissues other than bone marrow, such as skin, liver, pancreas, fat, etc., and is responsible for regeneration and homeostasis of each organ / tissue. It has been understood (see Non-Patent Documents 2 to 5). In addition, stem cells present in each tissue are excellent in plasticity and may be used for regeneration of organs and tissues that have been impossible to self-replicate until now.

  On the other hand, some of these stem cells are known to decrease with aging, and research on techniques to prevent the decrease of stem cells is actively conducted to maintain the homeostasis of each tissue ( Non-patent document 6). In recent years, stem cells have been isolated from living tissues in the field of cell transplantation and tissue engineering (regenerative medicine and regenerative beauty) in order to apply the ability (pluripotency) of stem cells to regeneration of organs and tissues. Development of techniques for propagation is in progress (Non-Patent Documents 7 and 8).

  In particular, when culturing stem cells in vitro, it is extremely important to proliferate while maintaining the pluripotency that is the ability of stem cells, that is, maintaining an undifferentiated state. If the undifferentiated state of the stem cells cannot be maintained during this culture and differentiation induction has progressed, the ability (pluripotency) of the finally prepared stem cells has been lost, and the desired effect ( Can not reproduce organs and tissues.

  As described above, when stem cells are used for cell transplantation treatment or tissue engineering (regenerative medicine or regenerative beauty) and regeneration of organs or tissues is desired, the stem cells must be able to be cultured while maintaining an undifferentiated state.

  To date, there have been several reports on techniques for proliferating stem cells while maintaining an 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-culture with supporting cells (stromal cells or feeder cells) (see Patent Literature 1 and Non-Patent Literatures 9 to 11). ). However, recently, there have been reports of cases of infection between different animals caused by feeder cells-derived endogenous viruses (see Non-Patent Document 12), and stem cell culture using feeder cells is a stem cell for medical purposes. It is not suitable for culturing.

  As another method, there is a method of maintaining an undifferentiated state of a stem cell by complexly combining 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). In addition, maintaining embryonic stem cells 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 for somatic stem cells (see Patent Literature 3 and Non-Patent Literature 14).

  However, cytokines are expensive and have problems such as collection raw materials and storage stability, and easy use is difficult. In addition, the effect of LIF is limited to a very specific cell lineage. In particular, in primate ES cells and somatic stem cells, it has been clarified that the addition of LIF alone cannot maintain an undifferentiated state. (See Non-Patent Document 10).

  Any of the currently reported methods for maintaining the undifferentiated state of stem cells requires complicated operations and has a low effect of maintaining the undifferentiated state. Therefore, in order to use stem cells for regenerative medicine, a technique for proliferating stem cells while maintaining an undifferentiated state has been demanded. That is, there has been a demand for a technique that can proliferate stem cells while maintaining an undifferentiated state in a safe, simple and efficient manner.

JP 2004-24089 A JP-T-2002-525042 Japanese Patent No. 3573354

Pittenger M.M. F. Et al., Science, 1999, Vol. 284, pp. 143-147 Goodell M.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.M. A. Et al., Tissue Engineering, 2001, Vol. 7, pp. 211-228 Koji Hasegawa, Aesthetic Dermatology, 2013, Vol. 23, pp. 1-11 Hiroshi Mabuchi et al., Journal of Japanese Society for Regenerative Medicine, 2007, Vol. 6, pp. 263-268 All Kitagawa et al., Journal of Japanese Society for Regenerative Medicine, 2008, Vol. 7, pp. 14-18 Thomson J.M. A. Et al., Proc. Natl. Acad. Sci. USA, 1995, Vol. 92, pp. 7844-7848 Thomson J.M. A. Et al., Science, 1998, Vol. 282, pp. 1145-1147 Reubinoff B.E. E. Et al., Nature Biotech. , 2000, Vol. 18, pp. 399-404 van der Laan L.L. J. et al. 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 above-described circumstances, an object of the present invention is to provide a method for efficiently proliferating stem cells while maintaining an undifferentiated state.

  As a result of intensive studies to solve the above problems, the present inventors have found that a subcritical water treated product of Ainu sea turtle or an extract thereof has an excellent undifferentiated state maintenance effect and proliferation promoting effect on stem cells. The headline and the present invention were completed.

That is, the present invention includes the following.
(1) An undifferentiated state of a stem cell containing, as an active ingredient, a subcritical water treated product of Ainu seaweed treated with water in a subcritical state at a temperature of 100 to 374 ° C. and a pressure equal to or higher than a saturated vapor pressure or an extract thereof Maintenance agent.
(2) A stem cell growth promoter containing, as an active ingredient, a subcritical water treated product of Ainu sea turtle treated with water in a subcritical state at a pressure of 100 to 374 ° C. or higher and a saturated vapor pressure or an extract thereof.
(3) including a step of culturing stem cells in a medium containing a subcritical water treated product of Ainu seaweed or an extract thereof treated with water in a subcritical state at 100 to 374 ° C. and a pressure equal to or higher than a saturated vapor pressure A method for producing stem cells.
(4) including a step of culturing a stem cell in a medium containing a subcritical water treated product of Ainu seaweed or an extract thereof treated with water in a subcritical state having a pressure equal to or higher than a saturated vapor pressure at 100 to 374 ° C. A method for maintaining the undifferentiated state of stem cells.
(5) including a step of culturing a stem cell in a medium containing a subcritical water treated product of Ainu seaweed or an extract thereof treated with water in a subcritical state having a pressure equal to or higher than a saturated vapor pressure at 100 to 374 ° C. A method for promoting the proliferation of stem cells.
(6) Cosmetics, pharmaceuticals, or quasi drugs containing the agent according to claim 1 or 2.
(7) Food / beverage products containing the agent of Claim 1 or 2.

  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.

Hereinafter, the present invention will be described in detail.
The stem cell undifferentiated state maintenance agent or growth promoter according to the present invention contains a subcritical water treated product of Ainu seaweed or an extract thereof as an active ingredient.

  The Ainu wakame used in the present invention is an Ainu wakame (Alaria prellalonga Kjellman) that grows in eastern Hokkaido, etc., and the Ainu wakame main body may be used as it is, or treatment such as drying, crushing, shredding, etc. You can also use what you did.

  The subcritical water treatment used in the present invention is to bring water in a subcritical state under the conditions of a predetermined temperature and pressure and the object to be treated (in the present invention, Ainu Wakame). For example, when water is raised to a pressure of 22.12 MPa and a temperature of 374.15 ° C., it shows a state where it is neither liquid nor gas. This point is called the critical point of water, and hot water at a temperature and pressure lower than the critical point is called subcritical water. This subcritical water has an excellent component extraction action and hydrolysis action due to a decrease in dielectric constant and an improvement in ionic product. The water supplied to the subcritical water treatment can be used in a liquid state or a gas state. That is, water vapor may be supplied to the treatment tank for subcritical water treatment, water may be supplied, or both of them may be supplied. At the time of subcritical water treatment, as a desired reaction field, the reaction is more likely to proceed in the liquid state than in the gas. Therefore, it is preferable to use water in a liquid state forcibly in a sealed container. More specifically, Ainu sea turtle and water as a treatment agent are put in a pressure vessel such as metal or ceramics, sealed, and in a subcritical state of water (temperature: 100 ° C. or higher, pressure: saturated vapor pressure or higher). The treated product obtained by performing the contact between them for a certain time or longer can be a subcritical water treated product.

  The Ainu seaweed subcritical water treated product used in the present invention is a product of Ainu seaweed treated with water in a subcritical state, and includes a mixture of Ainu seaweed and an aqueous solution (such as an aqueous solution) or a dried product thereof. Moreover, the filtrate which filtered the said mixture, or its dried material is included.

  The subcritical water treatment temperature for Ainu sea turtle is preferably between 100 and 300 ° C., because the active ingredient contained in the subcritical water treated product of Ainu sea turtle or its extract is easily obtained, preferably 120 to More preferably, the temperature is between 200 ° C and more preferably between 130 and 150 ° C. When the temperature is less than 100 ° C., the amount of the active ingredient tends to decrease. Moreover, when the temperature of subcritical water treatment exceeds 300 degreeC, it will become easy to cause the excessive decomposition | disassembly of an active ingredient.

  The subcritical water treatment pressure of Ainu Wakame may be equal to or higher than the saturated vapor pressure at each temperature, more preferably between 0.1 and 3.0 MPa, more preferably between 0.26 and 1.56 MPa, Most preferably between 0.28 and 0.48 MPa. When the pressure is less than 0.1 MPa, the amount of the active ingredient tends to decrease. In addition, excessive pressurization in which the pressure of the subcritical water treatment greatly exceeds the saturated vapor pressure requires the addition of a separate pressurization device and improvement of the pressure resistance of the equipment, which deteriorates the economics of extraction.

  The subcritical water treatment time for Ainu Wakame is preferably 5 to 90 minutes, more preferably 10 to 30 minutes. If it is less than 5 minutes, the amount of the active ingredient tends to decrease. Moreover, when the time of subcritical water treatment exceeds 90 minutes, it becomes easy to cause the excessive decomposition | disassembly of an active ingredient.

That is, as subcritical water treatment conditions for Ainu Wakame, the temperature is preferably 100 to 300 ° C., the pressure is 0.1 to 3.0 MPa, and the time is preferably 5 to 90 minutes. By carrying out under these conditions, the active ingredient of the subcritical water treated product of Ainu Wakame can be improved.
Furthermore, as subcritical water treatment conditions for Ainu Wakame, it is most preferable that the temperature is 130 to 150 ° C., the pressure is 0.28 to 0.48 MPa, and the time is 10 to 30 minutes. By carrying out under these conditions, the active ingredient of the subcritical water treated product of Ainu Wakame can be easily improved.

  The extract of Ainu Wakame subcritical water treated product used in the present invention includes an extract extracted by adding a solvent to the subcritical water treated product or a dried product thereof.

  Examples of the solvent used for extraction include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, Propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, Propyl ether, etc.). Preferably, water, lower alcohol and liquid polyhydric alcohol are preferable, and water and ethanol are particularly preferable. These solvents may be used alone or in combination of two or more. For example, an ethanol aqueous solution of 30 to 70 v / v% can also be used. Moreover, the acid which adjusted the pH by adding an acid or an alkali to the said extraction solvent can also be used. Examples of the extraction method include continuous extraction, immersion extraction, supercritical extraction, and subcritical water extraction. As subcritical extraction conditions, subcritical water extraction can be further performed under the same or different conditions as the above subcritical water treatment.

  Ainu Wakame subcritical water treated product is subjected to solvent extraction using the above-mentioned solvent. The ratio of the processed subcritical water of Ainu Wakame to the solvent is, for example, 1 to 50% (w / w), preferably 5 to 25% (w / w) in terms of solid content. For example, an extract of Ainu Wakame subcritical water treatment product can be obtained by adding water to a dried product of Ainu Wakame subcritical water treatment product and performing hot water extraction at 95 to 100 ° C. Or lower alcohol (for example, ethanol etc.) or liquid polyhydric alcohol (for example, propylene glycol, 1,3-butylene glycol, etc.) is added to the dried product of Ainu Wakame subcritical water treated product, and room temperature (for example, 5-35). The extract of Ainu Wakame subcritical water treatment product can be obtained by performing the extraction at (° C.).

  In the Ainu Wakame subcritical water treatment product or the solvent extract thereof, the obtained solution itself or the solvent phase itself can be used as an extract of the Ainu Wakame subcritical water treatment product. Alternatively, if necessary, the obtained solution itself or the solvent phase is subjected to concentration (vacuum concentration, concentration by membrane concentration, etc.), dilution, filtration, drying, etc., decolorization with activated carbon, deodorization treatment, etc. The obtained product can be used as an extract of a processed Ainu Wakame subcritical water. In particular, it is preferable that the extracted solution is subjected to a treatment such as concentration to dryness, spray drying, freeze drying, and the like, and the obtained dried product is used as an extract of a processed Ainu Wakame subcritical water.

  The Ainu Wakame subcritical water treatment product or the extract thus obtained does not proceed with differentiation when stem cells proliferate, and efficiently proliferates stem cells while maintaining an undifferentiated state at the biological level or culture level. Therefore, it can be used as an agent for maintaining an undifferentiated state of stem cells or a growth promoter. Furthermore, the stem cell undifferentiated state maintenance agent or growth promoter of the present invention can also be used as an additive for cell culture and a research reagent for efficiently proliferating while maintaining the undifferentiated state of stem cells. .

  The compounding amount of the Ainu Wakame subcritical water treated product or extract thereof in the undifferentiated state maintaining agent or growth promoting agent for stem cells according to the present invention is not particularly limited. For example, the total amount of the drug is converted to a dry product. It is preferably 0.00001 to 10% by weight, and more preferably 0.0001 to 1% by weight. If it is less than 0.00001% by weight, the effect may not be sufficiently exhibited.

  By applying the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention to stem cells of mammals including humans, the undifferentiated state of stem cells can be maintained and the proliferation of stem cells can be promoted. . Stem cells to which the stem cell undifferentiated state maintaining agent or growth promoting agent according to the present invention is applied are not particularly limited as long as they meet the object of the present invention. For example, embryonic stem cells (ES cells); bone marrow, blood , Somatic stem cells present in the skin (epidermis, dermis, subcutaneous tissue), fat, hair follicle, brain, nerve, liver, pancreas, kidney, muscle and other tissues; stem cells artificially prepared by gene transfer etc. (Artificial pluripotent stem cells: iPS cells). Preferably, the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention is more effective for stem cells derived from bone marrow, blood, skin, or adipose tissue. Examples of ES cells include ES cells established by culturing early embryos before implantation, ES cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei, And ES cells obtained by modifying genes on the chromosomes of those ES cells using genetic engineering techniques. Such ES cells can be prepared, for example, by a method known per se, but can be obtained from a predetermined institution, and further commercially available products can be purchased. In addition, these stem cells may be primary cultured cells, subcultured cells, or frozen cells.

  Furthermore, the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention can be applied to stem cells derived from all mammals as long as it has equivalent characteristics with respect to the direction of differentiation of the stem cells and the process of differentiation. Is possible. For example, the stem cell undifferentiated state maintaining agent or the growth promoting agent according to the present invention is a stem cell of a mammal such as a human, monkey, mouse, rat, guinea pig, rabbit, cat, dog, horse, cow, sheep, goat or pig. Can be effective.

  The application of the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention to the stem cell may be in vitro or in vivo, and in any case, the action can be exerted. Therefore, the stem cell undifferentiated state maintaining agent or growth promoting agent according to the present invention is obtained by culturing stem cells in a medium for culturing stem cells to which an effective amount thereof is added, or by administering to a mammal including a human. , Can maintain the undifferentiated state of stem cells and promote proliferation.

  In the case where the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention is administered in vivo, it can be administered as it is, but it is a cosmetic product with appropriate additives within a range not impairing the effects of the present invention, It can be provided by blending in various compositions such as quasi-drugs, pharmaceuticals, and foods and drinks. The pharmaceutical of the present invention includes drugs used for animals, that is, veterinary medicine.

  Stem cell undifferentiated state maintenance agent or growth promoter according to the present invention is an active ingredient Ainu Wakame subcritical water treatment product or extract thereof has an excellent stem cell undifferentiated state maintenance action and growth promotion action, It is also effective in treating, improving, and preventing damage or damage to tissues or organs by acting on stem cells of tissues or organs in the body such as skin, osteoblast, cartilage, muscle, nerve, fat, liver, etc. . In addition, since stem cells decrease or decrease in function with aging or the like, the stem cell undifferentiated state maintaining agent or growth promoting agent according to the present invention reduces the stem cells or functions of organs or organs in the living body. It is effective in treating, ameliorating and preventing related diseases. Here, as diseases related to tissue or organ damage or damage, stem cell decrease or functional decline, for example, in the case of skin, wrinkles, tarmi, spots, dullness, rough skin, thickened skin, open pores, acne scars , Wounds, pressure ulcers, burns, scars, keloids, and the like, including scalp and hair damage such as thinning and hair loss. For bone-related, osteoporosis, fracture (vertebral compression fracture, femoral neck fracture, etc.), etc. For cartilage disease, osteoarthritis, rheumatoid arthritis, intervertebral disc herniation, etc., for nerve-related, spinal cord injury, facial paralysis , Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, memory decline with age, blood related, aplastic anemia, leukemia, etc., cardiovascular related, myocardial infarction, obstructive arteriosclerosis, etc. Related to periodontal disease, alveolar bone damage due to alveolar pyorrhea, ophthalmic related, retinitis pigmentosa, age-related macular degeneration, glaucoma, etc., liver / pancreas related include hepatitis, cirrhosis, diabetes, etc. It is not limited to.

  In the case where the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention is added to cosmetics or quasi drugs, the dosage form is an aqueous solution, a solubilization system, an emulsion system, a powder system, a powder dispersion system, Any of an oil liquid 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, the cosmetics and quasi-drugs are selected according to the type of various components, additives, bases, etc. that are usually used in the composition for external use of skin, together with an undifferentiated state maintenance agent or growth promoter for stem cells. And it mix | blends suitably and can manufacture according to a well-known method in this field | area. The form may be liquid, emulsion, cream, gel, paste, spray or the like. Examples of the ingredients 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 (myristin) Isopropyl acid, isopropyl palmitate, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc., organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone cal Acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and salts thereof, vitamins, plant / animal extracts, various interfaces Examples include activators, humectants, ultraviolet absorbers, antioxidants, stabilizers, preservatives, bactericides, and fragrances.

  As types of cosmetics and quasi-drugs, for example, lotion, milky lotion, gel, beauty essence, general cream, sunscreen cream, pack, mask, face wash, cosmetic soap, foundation, funny, bath preparation, body lotion, Examples include body shampoos, hair shampoos, hair conditioners, hair restorers and the like.

  When the stem cell undifferentiated state maintenance agent or growth promoter according to the present invention is added to a pharmaceutical, it is mixed with a pharmacologically and pharmaceutically acceptable additive and is suitable for application to an affected area. Can be formulated into various preparations. The pharmacologically and pharmaceutically acceptable additives include pharmaceutical bases and carriers, excipients, diluents, binders, lubricants, and coatings that are appropriately selected according to the dosage form and application. Agents, disintegrants or disintegration aids, stabilizers, preservatives, preservatives, extenders, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH adjusters, propellants A coloring agent, a sweetening agent, a corrigent, a fragrance, and the like may be added as appropriate, and various preparations that can be administered systemically or locally orally or parenterally by various known methods may be used. When the pharmaceutical product of the present invention is provided in each of the above-described forms, it can be produced by a method usually used by those skilled in the art, for example, the method shown in the General Formulation [2] Formulation of the Japanese Pharmacopoeia.

  The form of the pharmaceutical product of the present invention is not particularly limited. For example, tablets, sugar-coated tablets, capsules, troches, granules, powders, liquids, pills, emulsions, syrups, suspensions, elixirs Oral preparations such as pills, injections (eg, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, suppositories, ointments, lotions, eye drops, sprays, transdermal Non-oral agents such as skin absorbents, transmucosal absorbents, patches and the like can be mentioned. Moreover, it may be a dry product which is redissolved when used, and in the case of an injectable preparation, it is provided in the form of a unit dose ampoule or a multi-dose container.

  A form suitable for using the undifferentiated state maintenance agent or growth promoter for stem cells according to the present invention as a pharmaceutical product for treating, ameliorating, and preventing the skin-related damage or disease is an external preparation, for example, Examples include ointments, creams, gels, solutions, patches and the like. The ointment refers to a homogeneous semi-solid external preparation, and includes an oily ointment, an emulsion ointment, and a water-soluble ointment. The gel is an external preparation in which a water-insoluble component hydrate compound is suspended in an aqueous liquid. The liquid preparation refers to a liquid external preparation and includes lotions, suspensions, emulsions, liniments and the like.

  The pharmaceutical agent of the present invention functions as a prophylactic agent that suppresses the onset of the above diseases and / or a therapeutic agent that improves the normal state. Since the active ingredient of the pharmaceutical of the present invention is derived from natural products, it is very safe and has no side effects. Therefore, when used as a medicament for the treatment, amelioration, and prevention of the aforementioned diseases, humans, mice, rats, rabbits It can be administered orally or parenterally in a wide range of doses to mammals such as dogs and cats.

  The content of the stem cell undifferentiated state maintaining agent or growth promoter in the cosmetics, pharmaceuticals, and quasi drugs of the present invention is not particularly limited, but the Ainu Wakame subcritical water treated product with respect to the total weight of the preparation (composition). Or 0.001-30 weight% is preferable and 0.01-10 weight% is more preferable in conversion into the dry solid content of the extract. If it is 0.001% by weight or less, the effect is low, and even if it exceeds 30% by weight, the effect is hardly increased. The above amounts are merely 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. Moreover, about the addition method of the active ingredient in formulation, it may add previously, may be added in the middle of manufacture, and should just select suitably in consideration of workability | operativity.

  Moreover, the undifferentiated state maintenance agent or growth promoter of stem cells according to the present invention can also be added to food and drink. In addition, in the present invention, the food and drink is a general food and drink, a food that can be ingested for the purpose of maintaining and promoting health other than pharmaceuticals, for example, health food, functional food, health functional food, or special use Used to include food. The health food includes foods provided under the names of nutritional supplements, health supplements, supplements, and the like. Functional health foods are defined by the Food Sanitation Law or Health Promotion Law, and include specific health foods and nutritional functional foods that can display the effects of specific health, the function of nutritional components, the reduction of disease risk, and the like.

  The form of the food or drink may be any form suitable for edible use, for example, solid, liquid, granular, granular, powder, capsule, cream, or paste. In particular, the shape in the case of the above health foods, for example, tablets, rounds, capsules, powders, granules, fine granules, troches, liquids (including syrups, milks, suspensions) Etc. are preferred.

  The types of food and drink include tea drinks (green tea, oolong tea, tea, etc.), soft drinks (sports drinks, mineral water, near water, etc.), carbonated drinks, milk drinks, coffee drinks, fruit / vegetable juice drinks, nutrition Various drinks such as drinks and jelly drinks, powdered soups, confectionery (candy, gummi, gum, tablet confectionery, etc.), noodles, breads, dairy products, processed fishery products, processed foods, fats and oils and processed foods, etc. However, it is not limited to these.

  The food / beverage products of the present invention may be appropriately blended with additives usually used depending on the type. Any additive that is acceptable under the Food Sanitation Law can be used as the additive. For example, sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, stevia; citric acid, malic acid Acidulants such as tartaric acid; excipients such as dextrin and starch; binders, diluents, fragrances, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspensions Examples include turbidity agents and preservatives.

  When the food / beverage products of the present invention are general food / beverage products, the food / beverage products can be produced by including a step of adding a processed Ainuwakame subcritical water product or an extract thereof in a normal production process of the food / beverage product. In addition, in the case of health foods, it may be according to the above-mentioned pharmaceutical production method. For example, in the case of tablet-like supplements, additives such as excipients are added to the processed Ainu Wakame subcritical water or the extract thereof. It can be manufactured by mixing and molding by applying pressure with a tableting machine or the like. Moreover, other materials (for example, vitamins such as vitamin C, vitamin B2, and vitamin B6, minerals such as calcium, dietary fiber, etc.) can be added as necessary.

  The amount of Ainu Wakame subcritical water-treated product or extract thereof in the food or drink of the present invention may be any amount that can exhibit the effect of maintaining the undifferentiated state of stem cells and the effect of promoting proliferation, What is necessary is just to set suitably in consideration of the amount of intake, the form of food and drink, efficacy and effect, taste, palatability and cost.

  The present invention also relates to a method of accelerating stem cell proliferation while maintaining the undifferentiated state of stem cells by culturing stem cells in a medium containing a processed Ainu Wakame subcritical water or an extract thereof. In other words, the method according to the present invention includes a step of culturing a stem cell in a medium containing a processed Ainu seaweed subcritical water or an extract thereof, a method for producing a stem cell, a method for maintaining an undifferentiated state of a stem cell, or a stem cell It can be said that it is a method for promoting the growth of

  In the method according to the present invention, a culture medium generally used for maintaining and proliferating the undifferentiated state of stem cells may be used for culturing stem cells. For example, a basic medium containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids) necessary for stem cell survival and proliferation, specifically, Dulbecco's Modified Eagle Medium (D-MEM) ), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium (Nentient Mixture F-12 (D-MEM / F-M / G). Hanks' solution (Hank's balanced salt solution) etc. are mentioned. In addition, basic fibroblast growth factor (bFGF) and / or leukocyte migration inhibitory factor (LIF) may be added to the medium as growth factors. Further, if necessary, the medium may be epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27. -It may contain supplements, N2-supplements, ITS-supplements, antibiotics and the like.

  In addition to the above, it is preferable that serum is contained in the medium at a content of 1 to 20%. However, since serum has different components depending on the lot and there are variations in its effects, it is preferably used after lot check.

  Commercially available media include Mesenchymal stem cell basal medium manufactured by Invitrogen, Mesenchymal stem cell basal medium manufactured by Sanko Junyaku Co., Ltd., MF medium manufactured by TOYOBO, Hanks' solution manufactured by Sigma (Hank's balanced salt solution) ) Etc. can be used.

  The incubator used for stem cell culture is not particularly limited as long as stem cells can be cultured. Examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. .

  The incubator may be non-cell-adhesive or adhesive, and is appropriately selected according to the purpose. As the cell-adhesive incubator, for the purpose of improving the adhesion to cells, a cell-treated culture vessel treated with a cell support substrate using an extracellular matrix or the like may be used. Examples of the extracellular matrix include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.

  The concentration of the Ainu Wakame subcritical water treatment product or extract thereof added to the medium used for stem cell culture is the Ainu Wakame subcritical water treatment product in the above-described stem cell undifferentiated state maintenance agent or growth promoter, or Although it can determine suitably according to content of the extract, it is 10-100,000 microgram / mL in conversion to solid content, Preferably the density | concentration of 100-10000 microgram / mL is mentioned. Further, during the stem cell culture period, the Ainu Wakame subcritical water treated product or the extract thereof may be periodically added to the medium.

Stem cell culture conditions may be in accordance with normal conditions used for stem cell culture, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30 to 40 ° C, preferably 36 to 37 ° C. The CO ₂ gas concentration is, for example, about 1 to 10%, preferably about 2 to 5%. In addition, it is preferable to perform culture | cultivation replacement | exchange once every 2-3 days, and it is more preferable to carry out every day. The culture conditions can also be set by varying as appropriate within the range in which the stem cells can survive and proliferate.

  Stem cell undifferentiated state maintenance is performed, for example, in the presence of the stem cell undifferentiated state maintaining agent according to the present invention as compared to the stem cell cultured in the absence of the stem cell undifferentiated state maintaining agent according to the present invention. The stem cell undifferentiated marker gene expression level in the stem cells can be evaluated by whether or not it is maintained at the mRNA level or the protein level at the same level as the expression level at the start of culture. Examples of the stem cell undifferentiated marker gene include CD44 gene (Mol Biol Cell. 2002 Dec; 13 (12): 4279-95. Human adipose tissue of a source of multiple cells.) And the like.

  Examples of the method for measuring the level of expression of stem cell undifferentiated marker gene include, for example, RT-PCR using primers and probes specific to stem cell undifferentiated marker gene, quantitative PCR, Northern blotting and the like at the mRNA level. At the protein level, for example, immunological methods such as immunostaining using an antibody specific for a protein encoded by a stem cell undifferentiation marker gene, ELISA, flow cytometry, Western blotting and the like can be mentioned.

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

  In addition, the proliferation promotion of the stem cell is achieved by, for example, the stem cell cultured in the presence of the stem cell proliferation promoter according to the present invention, as compared with the stem cell cultured in the absence of the stem cell proliferation promoter according to the present invention. It can be evaluated by whether or not the number of cells is significantly increased. The number of cells can be measured using, for example, a commercially available cell number measurement kit by the MTT method, the WST method, or the like. 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 cultured for a predetermined time in the presence of the stem cell proliferation promoter of the present invention is the non-existence of the stem cell proliferation promoter of the present invention. It can be determined that the proliferation of stem cells could be promoted when the relative ratio (control) was greater when cultured in the presence.

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

  According to the above-described stem cell undifferentiated state maintenance agent or growth promoter or the method according to the present invention, the Ainu Wakame subcritical water treated product or the extract thereof is used alone or separately from the medium or the medium. And a reagent kit for maintaining the undifferentiated state of stem cells or promoting proliferation thereof. The kit can include an instruction manual or the like as necessary. Alternatively, the Ainu Wakame subcritical water treatment product or an extract thereof can be mixed with a medium and provided as a medium for maintaining the undifferentiated state of stem cells or promoting proliferation.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

[Example 1] Ainu Wakame subcritical water treated product and extract thereof (1) Ainu Wakame subcritical water treated product Production Example 1
35 g of Ainu Wakame was put into a subcritical water treatment can, and a subcritical water treatment was performed at a treatment temperature of 140 ° C., a treatment pressure of 0.37 MPa, and a treatment time of 10 minutes (subcritical water treatment condition # 1). After the completion of the subcritical water treatment, the treated product in the treatment can was freeze-dried to obtain 33.1 g of Ainu Wakame subcritical water treated product 1.

  Production Examples 2 to 5 (Ainu Wakame Subcritical Water Treatment Products 2 to 5) are production examples except that the treatment temperature, treatment pressure, and treatment time shown in the conditions list of the production examples shown in Table 1 are used. Same as 1. The recovered amounts of Production Examples 2 to 5 are shown in Table 1.

(2) Ainuwakame subcritical water extract
Production Example 6A To 10 g of the hot water extract Ainu Wakame subcritical water treated product 1 (Production Example 1), 200 mL of purified water was added and extracted at 95 to 100 ° C. for 2 hours. After filtration, the filtrate was concentrated and lyophilized to obtain a hot water extract.

Production Example 6B 50% ethanol extract 200 g of 50% ethanol aqueous solution was added to 10 g of Ainu Wakame subcritical water treated product 1 (Production Example 1), and extracted all day and night. After filtration, the filtrate was concentrated under reduced pressure and freeze-dried to obtain a 50% ethanol extract.

Production Example 6C Ethanol extract To 10 g of Ainu Wakame subcritical water treated product 1 (Production Example 1), 200 mL of ethanol was added and extracted all day and night. After filtration, the filtrate was concentrated under reduced pressure and freeze-dried to obtain an ethanol extract.

  Extracts (Production Examples 7A to 10C) of Ainu Wakame subcritical water treated products were produced in the same manner as in Production Examples 6A to 6C using Production Examples 2 to 5 (Table 2).

Production Example 11 Ainu Wakame Subcritical Water Treatment Product 6
35 g of Ainu Wakame was put into a subcritical water treatment can, and the subcritical water treatment was performed at a treatment temperature of 140 ° C., a treatment pressure of 0.37 MPa, and a treatment time of 10 minutes. After completion of the subcritical water treatment, the treated product in the treatment can was filtered, and the filtrate was concentrated and freeze-dried to obtain 11.5 g of Ainu Wakame subcritical water treated product 6.

Comparative Production Example 1 Ainu Wakame hot water extract 10 g of Ainu Wakame was added with 2 L of purified water and extracted at 95-100 ° C. for 2 hours. After filtration, the filtrate was concentrated and freeze-dried to obtain 1.5 g of a hot water extract of Ainu Wakame.

[Example 2] Evaluation of undifferentiated state maintaining effect and growth promoting effect on stem cells of extract of Ainu Wakame subcritical water treatment product Extract of Ainu Wakame subcritical water treatment product produced in Example 1 is used below. The experimental example of the undifferentiated state maintenance effect with respect to the stem cell and the proliferation promotion effect and the result are shown.

(Experimental example 1) Evaluation of an undifferentiated state maintenance effect with respect to a stem cell Dulbecco's Modified Eagle Medium culture solution (made by Sigma), fetal bovine serum (FBS, 1%, made by Sigma), ITS-X (100-fold dilution, 5 × 10 ⁴ human adult stem cells (manufactured by DS Pharma Biomedical) were seeded in a 24 well dish using a medium prepared by adding GIBCO) and Hydrocortison (0.4 μg / mL, manufactured by SIGMA), and each extraction. The product (Comparative Production Example 1, Production Example 6A, Production Example 7A) was added to the medium so that the final concentration was 625 μg / mL, and the culture was continued for 3 days. As a positive control, FGF-2 (10 ng / mL, manufactured by Pepro Teck) was used.

After 3 days of culture, the cells were washed twice with PBS (−), 4% paraformaldehyde was added, and the cells were fixed by incubation at room temperature for 10 minutes. After fixation, the cells were washed twice with PBS (−), and anti-CD44 antibody (diluted 200-fold, Santa Cruz Biotechnology) was added to PBS (−) containing 0.1% bovine serum albumin (BSA, manufactured by Wako Pure Chemical Industries, Ltd.). And a primary antibody solution added thereto was added and incubated at 37 ° C. for 1 hour. Next, the reaction was performed at 37 ° C. for 60 minutes with the secondary antibody Alexa488 (diluted 500 times, manufactured by Molecular Probes). The cells were washed twice with PBS (−) and observed using a fluorescence microscope (manufactured by Olympus), and images were taken with CD44 expression as green fluorescence. For the acquired images, the relative expression level of CD44 (fluorescence intensity per 100 μm ² ) was calculated using commercially available analysis software. The relative expression level of CD44 in cells cultured without adding the sample was set to 1, and the relative expression level of CD44 in cells cultured with the sample added was calculated and evaluated. The results are shown in Table 3.

  As shown in Table 3, a remarkable effect of maintaining the undifferentiated state of stem cells was observed in the extract of Ainu Wakame subcritical water treated (Production Examples 6A and 7A). From the above, it was clarified that the extract of the Ainu Wakame subcritical water treated product was excellent in maintaining the undifferentiated state of stem cells. In addition to the stem cells used in this experimental example, a similar test was performed on embryonic stem cells (ES cells), and a remarkable effect of maintaining the undifferentiated state of stem cells was observed. In addition, Ainu Wakame's subcritical water treated product 6 (Production Example 11) also showed an excellent effect of maintaining the undifferentiated state of stem cells.

(Experimental example 2) Evaluation of proliferation promoting effect on stem cells 3 × 10 ⁵ adult human stem cells (manufactured by Toyobo Co., Ltd.) cultured using human stem cell culture solution (manufactured by Toyobo Co., Ltd.) are seeded in a 6 cm dish, and each extract is extracted. (Production Example 6A, Production Example 7A and Comparative Production Example 1) were added so that the final concentration was 625 μg / mL, and the culture was continued for 3 days.

  After culturing for 3 days, the cells were washed three times with PBS (−) and then collected by a rubber policeman, and the number of each cell was counted.

  Increase / decrease (%) in the number of cells when each extract (Production Example 6A, Production Example 7A and Comparative Production Example 1) was added, with the total number of cells when no extract was added as a control and the control as 100 (%) ) Was calculated, and the stem cell proliferation promoting effect was evaluated. The test results are shown in Table 4 below.

  As shown in Table 4, the extract of Ainu Wakame subcritical water treated product (Production Examples 6A and 7A) showed a significant stem cell proliferation promoting effect. When the control value described above was 100%, the number of adult human stem cells at the start of culture was 27%. From the above, the extremely excellent stem cell proliferation promoting effect of the extract of Ainu Wakame subcritical water treatment was clarified. In addition to the stem cells used in this experimental example, a similar test was performed on embryonic stem cells (ES cells), and a remarkable effect of promoting stem cell proliferation was observed. In addition, Ainu Wakame's subcritical water treated product 6 (Production Example 11) also showed an excellent stem cell proliferation promoting effect.

  As described above, by applying the Ainu Wakame subcritical water treatment product or its extract to stem cells, the proliferation of stem cells can be achieved while maintaining an undifferentiated state more easily and efficiently than conventional techniques. It became possible to promote.

[Example 3] Formulation example of product A formulation example of a product containing an extract of Ainu Wakame subcritical water treated product is shown below.
(Formulation Example 1) Lotion Formulation Blending amount (% by weight)
1. Hot water extract of Ainu Wakame subcritical water treated product (Production Example 6A) 0.1
2. 1,3-butylene glycol 8.0
3. Glycerin 2.0
4). Xanthan gum 0.02
5. Citric acid 0.01
6). Sodium citrate 0.1
7). Ethanol 5.0
8). Methyl paraoxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40E.O.) 0.1
10. Fragrance 0.1
11. Purified water remaining

  Components 1 to 6 and 11 and components 7 to 10 are uniformly dissolved, then both are mixed and filtered to prepare a lotion.

(Formulation Example 2) Cream Formulation Formulation amount (% by weight)
1. Hot water extract of Ainu Wakame subcritical water treated product (Production Example 7A) 0.1
2. Squalane 5.5
3. Olive oil 3.0
4). Stearic acid 2.0
5. Beeswax 2.0
6). Octyldodecyl myristate 3.5
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Behenyl alcohol 1.5
9. Glycerol monostearate2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.2
12 Ethyl paraoxybenzoate 0.05
13.1,3-Butylene glycol 8.5
14 Purified water remaining

  Ingredients 2-9 are dissolved by heating and mixed, and kept at 70 ° C. to obtain an oil phase. Ingredients 1 and 11 to 14 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. Next, the aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. The component 10 is added at 45 ° C., and further cooled to 30 ° C. to obtain a product.

(Prescription Example 3) Emulsion
Formulation amount (% by weight)
1. Hot water extract of Ainu Wakame subcritical water treated product (Production Example 8A) 0.1
2. Squalane 5.0
3. Olive oil 5.0
4). Jojoba oil 5.0
5. Cetanol 1.5
6). Glycerol monostearate 2.0
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Polyoxyethylene sorbitan monooleate (20E.O.) 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12 Methyl paraoxybenzoate 0.2
13. Purified water remaining

  Ingredients 2 to 8 are dissolved by heating and mixed, and kept at 70 ° C. to obtain an oil phase. Ingredients 1 and 10-13 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. The component 9 is added at 45 ° C., and further cooled to 30 ° C. to obtain a product.

(Formulation example 4) Gel formulation Formulation amount (% by weight)
1. 50% ethanol extract of Ainu Wakame subcritical water treated product (Production Example 8B) 0.1
2. Ethanol 5.0
3. Methyl paraoxybenzoate 0.1
4). Polyoxyethylene hydrogenated castor oil (40E.O.) 0.1
5. Perfume appropriate amount 6.1,3-butylene glycol 5.0
7). Glycerin 5.0
8). Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. Purified water remaining

  Ingredients 2 to 5 and ingredients 1 and 6 to 11 are uniformly dissolved, and both are mixed to obtain a product.

(Formulation Example 5) Ointment Formulation Formulation amount (wt%)
1. 50% ethanol extract of Ainu Wakame subcritical water treated product (Production Example 9B) 2.0
2. Polyoxyethylene cetyl ether (30E.O.) 2.0
3. Glycerol monostearate 10.0
4). Liquid paraffin 5.0
5. Cetanol 6.0
6). Methyl paraoxybenzoate 0.1
7). Propylene glycol 10.0
8). Purified water remaining

  Ingredients 2 to 5 are dissolved by heating and mixed, and kept at 70 ° C. to obtain an oil phase. Ingredients 1 and 6 to 8 are dissolved by heating and mixed, and kept at 75 ° C. to obtain an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the product is cooled to 30 ° C. with stirring to obtain a product.

(Formulation example 6) Pack
Formulation amount (% by weight)
1. 50% ethanol extract of Ainu Wakame subcritical water treated product (Production Example 10B) 0.1
2. Polyvinyl alcohol 12.0
3. Ethanol 5.0
4.1,3-Butylene glycol 8.0
5. Methyl paraoxybenzoate 0.2
6). Polyoxyethylene hydrogenated castor oil (20 EO) 0.5
7). Citric acid 0.1
8). Sodium citrate 0.3
9. Perfume appropriate amount10. Purified water remaining

  Ingredients 1 to 10 are uniformly dissolved to obtain a product.

(Formulation Example 7) Tablet Formulation Formulation amount (% by weight)
1. Ethanol extract of Ainu Wakame subcritical water treated product (Production Example 6C) 1.0
2. Dried corn starch 25.0
3. Carboxymethylcellulose calcium 20.0
4). Microcrystalline cellulose 40.0
5. Polyvinylpyrrolidone 7.0
6). Talc 3.0

  Components 1-5 are mixed, then 10% water is added as a binder and dried after extrusion granulation. Ingredient 6 is added to the molded granules, mixed and compressed into tablets. One tablet is 0.52 g.

(Prescription Example 8) Beverage Formulation Blending amount (% by weight)
1. Ethanol extract of Ainu Wakame subcritical water treated product (Production Example 7C) 0.1
2. Stevia 0.05
3. Malic acid 5.0
4). Fragrance 0.1
5. Purified water remaining

  Components 1 to 4 are dissolved in a part of purified water of component 5 with stirring. The remaining purified water of Component 5 is then added and mixed, heated to 90 ° C. and filled into a 50 mL glass bottle.

  As an application example of the present invention, application to regenerative medicine and regenerative beauty is expected. For example, by utilizing the present invention, it becomes possible to easily and efficiently prepare undifferentiated stem cells used in regenerative medicine and regenerative beauty. Furthermore, the Ainu Wakame subcritical water-treated product or extract thereof according to the present invention is applied directly to the stem cells after transplantation of the stem cells or in the tissue, or applied by oral administration, application, pasting, etc. Thus, the stem cells can be grown while maintaining an undifferentiated state.

That is, the present invention can be used as a preparation method of stem cells and / or an undifferentiated state maintaining agent or growth promoting agent for regenerative medicine and regenerative beauty.

Claims (7)

  An agent for maintaining an undifferentiated state of a stem cell, which contains, as an active ingredient, a subcritical water treated product of Ainu sea turtle treated with water in a subcritical state at a temperature of 100 to 374 ° C. and a pressure equal to or higher than a saturated vapor pressure.   A stem cell growth promoter containing, as an active ingredient, a subcritical water treated product of Ainu sea turtle treated with water in a subcritical state at a temperature of 100 to 374 ° C. and a pressure equal to or higher than a saturated vapor pressure.   Culturing a stem cell in a medium containing a subcritical water treatment of Ainu seaweed or an extract thereof treated with water in a subcritical state at a temperature of 100 to 374 ° C. and a pressure equal to or higher than a saturated vapor pressure, A method for producing stem cells.   Culturing a stem cell in a medium containing a subcritical water treatment of Ainu seaweed or an extract thereof treated with water in a subcritical state at a temperature of 100 to 374 ° C. and a pressure equal to or higher than a saturated vapor pressure, A method for maintaining the undifferentiated state of stem cells.   Culturing a stem cell in a medium containing a subcritical water treatment of Ainu seaweed or an extract thereof treated with water in a subcritical state at a temperature of 100 to 374 ° C. and a pressure equal to or higher than a saturated vapor pressure, A method of promoting proliferation of stem cells.   Cosmetics, pharmaceuticals, or quasi drugs containing the agent according to claim 1 or 2. Food-drinks containing the agent of Claim 1 or 2.