JP2011211957A - Undifferentiation-maintaining agent for stem cell and growth-promoting agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an undifferentiation-maintaining and growth-promoting agent for stem cells which exhibits a stem cell-specific growth-promoting effect while maintaining an undifferentiated state of stem cells of mammalian animals, a stem cell having a high survival rate to tissues at transplanting, and a technology for preparing the same.SOLUTION: Specific growth of stem cells is promoted while maintaining an undifferentiated state of stem cells by using an extract from fungus gall of Zizania latifolia for embryonic stem cells of mammalian animals, somatic stem cells in body tissues such as bone marrow, blood, fat and skin tissues, or artificially prepared stem cells. Further stem cells prepared in a culture solution containing the extracts have an improved effect on a survival rate to body tissues at the transplanting. Therefore, this technology can contribute greatly to the field of tissue regeneration and is expected to be applied to medical, pharmaceutical, quasi drug, cosmetic and health fields.

Description

  The present invention relates to an undifferentiated maintenance and / or growth promoter specific for stem cells, characterized by containing an extract of Makomotake as an active ingredient. In addition, the present invention relates to a method for preparing stem cells having a very high engraftment rate at the time of transplantation and / or stem cells prepared by using a culture solution containing an extract of Makomotake.

  In the case of vertebrate, particularly mammalian tissue, when cell or organ damage occurs due to injury or disease, or aging, the regeneration system works to try to recover the damage of the cell or organ. Stem cells provided in the tissue play a major role in this effect. Stem cells have pluripotency that differentiates into all types of cells, and this property is thought to lead to recovery by regenerating the cells and tissues in the damaged part. 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. 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 in the bone marrow, there are stem cells that can be differentiated into other organs (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 such as liver, pancreas, fat, etc. in addition to bone marrow, and it has been found that they are responsible for regeneration and homeostasis of each organ / tissue. (See Non-Patent Documents 2 to 5). Such stem cells present in each tissue are excellent in plasticity and may be used for fundamental regeneration of organs and tissues that have been impossible to self-replicate until now. In recent years, technologies such as embryonic stem cells (ES cells) and stem cells artificially prepared by gene transfer (artificial pluripotent stem cells: iPS cells) have also advanced, and various stem cells can be used for regenerative medicine. It is expected to be possible (see Patent Documents 1 and 2).

Pittenger M.M. F. , Et al. , Science, 1999, 284, 143-147. Goodell M.M. F. , Et al. , Nat. Med. , 1997, 3, 1337-1345. Zulewski H. , Et al. , Diabetes, 2001, 50, 521-533 Suzuki A. , Et al. , Hepatology, 2000, 32, 1230-1239 Zuk P.M. A. , Et al. , Tissue Engineering, 2001, 7, 211-228. Japanese Patent No. 4183742 Japanese Patent No. 4411363

  Under such circumstances, proliferation techniques and cultures for preparing stem cells used for treatment in order to make effective use of the ability (pluripotency) of these stem cells in the field of cell transplantation treatment and tissue engineering (regenerative medicine and regenerative beauty). Development of technology and the like is underway (see Non-Patent Documents 6 to 7).

Hiroshi Mabuchi, Journal of Japanese Society for Regenerative Medicine, 2007, 6, 263-268 Kitagawa et al., Journal of Japanese Society for Regenerative Medicine, 2008, 7, 14-18

  In particular, when proliferating 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. Moreover, it is necessary to proliferate stem cells until the number necessary for treatment is maintained while maintaining this undifferentiated state. If the undifferentiated state of the stem cells cannot be maintained during this proliferation and the differentiation has progressed, the ability (pluripotency) of the finally prepared stem cells has been lost, and the desired therapeutic effect ( It is difficult to reproduce organs and tissues.

  In addition, since stem cells have the ability to differentiate into various cells, when differentiation progresses during the proliferation of stem cells, cells other than stem cells may also be mixed and proliferated. In this case, the stem cells finally prepared contain cells other than the stem cells, and it is difficult to exert the intended therapeutic effect (eg, regeneration of organs and tissues).

  Based on the above, when considering the use of stem cells for regenerative medicine in the future, the stem cells can be proliferated to the number necessary for transplantation while maintaining the undifferentiated state of the stem cells. A technique to prepare is needed. This requires a technique for preparing stem cells that maintain the undifferentiated state of the stem cells, specifically proliferate only the stem cells, and have high engraftment in the tissue at the time of transplantation.

  To date, there have been several reports on techniques for growing stem cells while maintaining the undifferentiated state of stem cells, 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 Non-Patent Documents 8 to 10 and Patent Document 3). ). However, recently, an example of infection between different animals caused by endogenous virus derived from feeder cells has been reported (see Non-Patent Document 11), and is not suitable for culturing stem cells for medical purposes.

Thomson J.M. A. et al. , Proc. Natl. Acad. Sci. USA, 1995, 92, 7844-7848. Thomson J.M. A. et al. , Science, 1998, 282, 1145-1147. Reubinoff B.E. E. et al. , Nature Biotech, 2000, 18, 399-404. JP 2004-24089 A Van der Laan LucJ. W. et al. , Nature, 2000, 407, 90-94.

  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 in an undifferentiated state by adding LIF (Leukemia Inhibitory Factor) to the medium (see Non-patent Document 12 and Patent Document 4). 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 Document 5 and Non-Patent Document 13).

Smith A. G. et al. Dev. Biol, 1987, 121, 1-9 JP-T-2002-525042 Japanese Patent No. 3573354 Madlambayan G.M. J. et al. et al. , J. et al. Hematother. Stem Cell Res. , 2001, 10, 481-492

  Furthermore, in recent years, a method of adding a basic fibroblast growth factor (bFGF) or the like has been invented as a technique for artificially preparing stem cells or a technique for growing them (see Patent Document 6). In addition, as a technique for purifying stem cells, a method of proliferating while maintaining the undifferentiated state of stem cells by using a special culture solution (containing bFGF) has been invented (see Patent Document 7). In either case, a technique has been developed that promotes proliferation while maintaining the undifferentiated state of stem cells by adding growth factors such as bFGF and cytokines. However, the growth factors and cytokines used in such a technique are extremely expensive, and are difficult to use due to problems such as differences in quality and efficacy between manufacturers and lots. In addition, components that are effective for maintaining undifferentiation such as LIF, which have been widely used so far, are limited to specific cell lines, and in particular, in primate ES cells and somatic stem cells, only the addition of LIF Is not able to maintain an undifferentiated state (see Non-Patent Document 9).

Japanese Patent No. 4411362 Special table 2010-500047 gazette

  As described above, the conventional techniques are not particularly effective in maintaining the undivided state of the stem cells, and the proliferation of the stem cells is possible, but at the same time other than the cells differentiated from the stem cells or the stem cells mixed in the initial stage. The cells also proliferate, and the finally prepared stem cells are mixed with cells other than the stem cells.

  In other words, none of the techniques for maintaining and proliferating the undifferentiated state of stem cells that have been reported so far can produce satisfactory effects when preparing stem cells for transplantation and actually transplanting them. It is considered to be. In addition, the conventional techniques require complicated operations or have a low effect of maintaining the undifferentiated state of the stem cells, so that eventually the cells other than the stem cells proliferate, and when transplanted, Since cells other than stem cells are transplanted, the engraftment ratio of stem cells to the tissue is extremely reduced. Therefore, when preparing stem cells for treatment in future regenerative medicine, a technique for maintaining the undifferentiated state of the stem cells and a technique for proliferating only the stem cells to the required number while maintaining the undifferentiated state. Development is desired. For this purpose, it is necessary to develop components and culture techniques that selectively exhibit undifferentiation maintenance and proliferation promoting effects on stem cells. Further, it is necessary to efficiently engraft stem cells prepared by these techniques in living tissues. In other words, it is safe, convenient, and efficient, proliferating while maintaining an undifferentiated state specific to stem cells, showing no proliferative effect on other cells, and the rate of engraftment in living tissue at the time of transplantation High stem cell preparation technology has been demanded.

  In view of such circumstances, the present invention solves the problems in the prior art as described above, and exhibits a proliferation-promoting effect specific to stem cells while maintaining the undifferentiated state of mammalian stem cells. An object of the present invention is to develop a stem cell undifferentiated maintenance and / or growth promoter, and to provide a stem cell having a high engraftment rate in a living tissue at the time of transplantation and a preparation technique thereof.

  As a result of intensive studies aimed at solving the above-mentioned problems, the present inventors have found that an extract of makomotake has a specific undifferentiation maintaining effect and a growth promoting effect on stem cells, and further, It has been clarified that the engraftment rate to the tissue is extremely improved when the stem cells for transplantation prepared using the contained culture solution are actually transplanted, and the present invention has been completed.

That is, the present invention is as follows.
(1) An agent for maintaining undifferentiation of stem cells, which comprises an extract of Makotake.
(2) An agent for promoting proliferation of stem cells, comprising an extract of Makomotake.
(3) A method for preparing a stem cell, which comprises culturing using a medium containing an extract of Makomotake.
(4) Stem cells prepared using the stem cell preparation method according to (3).
(5) A medium for use in stem cell culture, characterized by containing an extract of Makomotake.

  Hereinafter, the present invention will be described in more detail.

  The present invention uses an extract of Mokomyotake to specifically proliferate only stem cells while maintaining the undifferentiated state of stem cells, and further to improve the survival rate of stem cells to tissues at the time of transplantation By using a culture solution containing an undifferentiated maintenance and / or growth promoter specific for stem cells capable of undergoing growth and the extract, a stem cell having a very high engraftment rate in a tissue and a method for preparing the stem cell are provided.

  The makomotake used in the present invention is a fungal gill formed by parasitism of a fungus (Ustilago esculenta), which is a kind of smut, on the stem part of macomo (scientific name: Zizania latifolia). This fungus can be classified into two types, depending on the degree of formation of the spore layer. Macomo's fungi grown and grown in China and Taiwan are of the type of Makotake, which is extremely enlarged and looks like “bamboo shoots”. This fungus is used as an edible or medicinal product as a vegetable. In Japan, makomotake is also cultivated and can be easily obtained.

  The extract of Makotake used in the present invention may be extracted raw with a solvent, or a dried product may be extracted with a solvent. Moreover, you may use what was fermented as follows. The fermentation of makomotake is characterized by freezing and thawing raw makomotake and then fermenting it with the fungus itself. As freezing conditions, it is only necessary to freeze at 0 ° C. or lower, and it is preferable to freeze at −20 ° C. to 0 ° C. The melting condition may be a temperature at which ice melts, and it may be heated at room temperature or warm. As fermentation conditions, it is preferable to ferment under the temperature conditions of 10 to 80 ° C., preferably 20 to 60 ° C. The heating time at this time is 10 hours or more, preferably 15 to 48 hours. When heated for more than 48 hours, the enhancement of the effect is hardly recognized and it is uneconomical. As the above fermentation treatment method, the plant body of makomotake may be used as it is, or it may be made into slices, may be chopped or chopped through a mixer, and is appropriately determined in consideration of fermentation efficiency. It can be carried out. Furthermore, it is preferable to heat-dry the fermented material of the makomotake of the present invention in addition to the above fermented treatment. As heating temperature, 50 to 150 degreeC is preferable and 60 to 120 degreeC is the most preferable. The heating time is preferably 1 hour to 48 hours, and most preferably 6 hours to 24 hours. In the present invention, an extract of a processed product of makomotake is preferable in terms of effects.

  Examples of the solvent to be extracted 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. The extraction method is not particularly limited, but extraction by heating is preferable. Furthermore, the pH may be adjusted by adding an acid or alkali to the extraction solvent.

  The extract of makomotake may be used as an extracted solution, and may be used after treatment such as concentration, dilution, filtration, and decolorization or deodorization with activated carbon or the like, if necessary. In particular, the extracted solution is preferably subjected to treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

  The component content in the case of using the extract of Makotake is not particularly limited, but it is preferably 0.00001 to 10% by weight as a dry product, and used at a concentration of 0.0001 to 1% by weight. Most preferred. If it is less than 0.00001% by weight, the effects of the present invention may not be sufficiently exerted, and if it exceeds 10% by weight, it is uneconomical.

  In addition, an extract showing a specific undifferentiation maintenance effect and proliferation promoting effect on the stem cell of the present invention, a stem cell prepared thereby, a preparation method thereof, and the like, an additive for cell culture, a reagent for research, It can be formulated and applied to medical reagents, cell transplants, pharmaceuticals, quasi drugs, cosmetics, foods and the like.

  The stem cell of the present invention is an embryonic stem cell (ES cell) or bone marrow, blood, skin, fat, brain, liver, pancreas, kidney, muscle, and other tissues as long as the purpose of the present invention is met. In addition, somatic stem cells, stem cells artificially prepared by gene transfer or the like, and primary culture cells, subculture cells, or frozen cells of the cells may be used. Preferably, it is more effective against bone marrow, blood, skin, and adipose tissue-derived stem cells. Moreover, if it has the same characteristic about the direction of differentiation of a stem cell in a mammal, the process of differentiation, etc., it can apply to all mammals. For example, the effect can be exerted on stem cells of mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats and pigs.

  As the medium for culturing the stem cells of the present invention, or the additive used at the same time, for example, the following can be used, but is not limited.

  Specifically, a basic medium containing components necessary for cell survival (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids), such as Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixure F-12 (D-MEM / F-12), Gss In the liquid (Hank's balanced salt solution), basic fibroblast growth factor ( A medium supplemented with at least one of bFGF) and leukocyte migration inhibitory factor (LIF) is used, and preferably contains all of these growth factors. If necessary, epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplements, ITS-supplements, antibiotics, and the like may be included.

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

  Commercially available products include Mesenchymal stem cell basal medium manufactured by Invitrogen, Mesenchymal stem cell basal medium manufactured by Sanko Junyaku, MF medium manufactured by TOYOBO, Hank's balanced salt solution manufactured by Sigma, 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. It is done.

  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 with 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.

Culture conditions can be set as appropriate. For example, the culture temperature is not particularly limited, but is about 30 to 40 ° C, preferably about 37 ° C. The CO ₂ gas concentration is, for example, about 1 to 10%, preferably about 2 to 5%.

  The extract of the mushroom of the present invention showed a very high undifferentiated state maintaining effect on stem cells. Furthermore, with regard to the growth promoting effect, the present inventors have found a very specific growth promoting effect only on stem cells without showing a growth promoting effect on cells other than stem cells. In addition, when stem cells prepared using this Momotake extract were transplanted into living tissue, the rate of engraftment in the tissue was significantly improved as compared to conventional methods. As described above, the present invention is useful for tissue regeneration treatment and regeneration beauty, and can greatly contribute to the field of tissue regeneration.

  Hereinafter, in order to describe the present invention in detail, specific and detailed examples will be given, but the present invention is not limited thereto.

  First, as an example, the production example of the extract of Mokomyotake used in the present invention and the experimental example showing the effect of maintaining the undifferentiated state of the stem cells are given, but the present invention is not limited to this.

  Below, the manufacture example of the extract of Makomotake is shown.

Production Example 1 Processed Fermented Makotake Bamboo 2 kg of raw Momotake was frozen at −20 ° C., then melted at room temperature and sliced to about 5 mm. Then, it was fermented for 24 hours under a temperature condition of 60 ° C., and then heat-dried at 100 ° C. for 12 hours to obtain 84 g of a processed product of makomotake. This was used in Production Examples 2-4.

Production Example 2 Hot Water Extract of Processed Fermented Makotake Bamboo Add 400 ml of purified water to 20 g of dried product of Produced Processed Makotake (Production Example 1), extract at 95-100 ° C. for 2 hours, filter, and concentrate the filtrate. Then, it was freeze-dried to obtain 7.8 g of a hot water extract of the processed product of the makomotake fermented product.

Production Example 3 50% Ethanol Extract of Processed Fermented Makotake Bamboo Addition of 200 mL of purified water and 200 mL of ethanol to 20 g of dried product of fermented Makotake (Production Example 1), followed by extraction at room temperature for 7 days, followed by filtration, Concentration to dryness yielded 6.3 g of a 50% ethanol extract of the processed product of Momotake.

Production Example 4 Ethanol Extract of Processed Fermented Mushroom Bamboo Add 400 ml of ethanol to 20 g of dried product of Processed Fermented Mushroom (Production Example 1), extract at room temperature for 7 days, filter, concentrate the filtrate to dryness, 1.1 g of an ethanol extract of the processed makomotake fermentation product was obtained.
Production Example 5 Hot water extract of Momotake bamboo Add 500 mL of purified water to 60 g of fresh Momotake, extract at 95-100 ° C. for 2 hours, filter, concentrate the filtrate, freeze-dry and extract hot water extract of Momotake 3.7g was obtained.

  Below, the experiment example and result of the undifferentiated state maintenance effect of a stem cell and the cell growth promotion effect using the extract of the makomotake of the manufacture examples 2-4 shown in Example 1 are shown.

Experimental Example 1 Evaluation of effect of maintaining undifferentiated state on stem cells In Dulbecco's Modified Eagle Medium culture solution (Gibco), fetal bovine serum (FBS, 15%, Sigma), nucleoside solution (100-fold dilution, Dainippon) Manufactured by Pharmaceutical Co., Ltd.), non-essential amino acid solution (100-fold dilution, manufactured by Dainippon Pharmaceutical), β2-mercaptoethanol solution (100-fold diluted, manufactured by Dainippon Pharmaceutical), L-glutamine solution (100-fold diluted, Dainippon Pharmaceutical) Mouse embryonic stem cells (mouse ES cells: manufactured by Cosmo Bio) using a medium prepared by adding penicillin (100 units / mL, manufactured by Sigma) and streptomycin (100 μg / mL, manufactured by Sigma). , 1 × 10 ⁵ seeds were sown in a 6 cm dish, and the final concentration of the extract of Momotake (Production Examples 2-4) It added so that it might become 0.001%, and culture | cultivation was continued for 3 days. Next, the cells were washed 3 times with PBS (−), collected with a rubber policeman, counted with a hemocytometer, and then extracted with CelLytic (Sigma), undifferentiated state. Was measured according to the report of Toyooka et al. (Reference: Yayoi Toyooka, Extraordinary issue of Molecular Medicine, Regenerative Medicine, 2003, 106-115). That is, stem cells (1 × 10 ⁵ ) seeded at the start of culture were expressed using the expression level of octamer binding protein 3/4 protein (Oct3 / 4 protein) indicating the undifferentiated state of stem cells as an index. The amount of Oct3 / 4 protein was quantitatively analyzed by Western blotting after the amount of Oct3 / 4 protein was 100% undifferentiated, the extract of Macomatake (Production Examples 2 to 4) was added and cultured for 3 days. The effect of maintaining the undifferentiated state was evaluated by comparing the amount of Oct3 / 4 protein at the start of culture and after 3 days of culture. In addition, the same evaluation was performed using the basic fibroblast growth factor (bFGF) (Japanese translations of PCT publication 2010-500047 gazette) currently reported as a substance which shows the undifferentiation maintenance effect of a stem cell as a positive control until now. .

As a specific evaluation method, the amount of Oct3 / 4 protein (referred to as A) expressed in the single cell number (1 × 10 ⁵ ) of stem cells at the start of culture was measured by Western blotting, and then started. The undifferentiated state (100%). Furthermore, the amount of Oct3 / 4 protein (referred to as B) expressed in the number of single cells (1 × 10 ⁵ cells) after adding the extract of Momotake (Production Examples 2 to 4) and culturing for 3 days was measured. The undifferentiated state (%) after 3 days of culture was calculated from the following formula. Calculation formula of undifferentiated state (%) after 3 days of culture = B / A × 100 (%). The maintenance effect of the undifferentiated state was evaluated using the undifferentiated state (%) after 3 days as an index.

  The test results are shown in Table 1. As a result, as compared with the positive control substance (bFGF), a significant stem cell undifferentiated state maintaining effect was observed in all the extracts of Momotake (Production Examples 2 to 4). Based on the above, the extremely excellent stem cell undifferentiated state maintenance effect of the extract of Momotake was clarified. In addition to the stem cells used in this experimental example, similar tests were performed on somatic stem cells and stem cells artificially prepared by gene transfer, and a remarkable effect of maintaining the undifferentiated state of stem cells was observed. In addition, the extract of Production Example 5 also showed the effect of maintaining the undifferentiated state of stem cells.

Experimental Example 2 Evaluation of Specific Cell Proliferation Promoting Effect on Stem Cells Biological tissues are broadly classified into ectoderm, mesoderm, and endoderm tissues, and it is considered that there are about 200 types of cells constituting these tissues. Among them, keratinocytes (DS Pharma Biomedical), which are cells of ectoderm tissue, fibroblasts (DS Pharma Biomedical), which are cells of mesoderm tissue, and endoderm tissue are representative of them. Using the liver cells (HEPG2) and human somatic stem cells, the cell-specific proliferation-promoting effect of the extract of Makotake (manufacture examples 2 to 4) on these cells was evaluated. That is, among these cells, it was evaluated whether or not the extract of Makomotake (Manufacturing Examples 2 to 4) specifically showed a growth promoting effect only on stem cells. Specifically, each of these cells was seeded at 1 × 10 ⁶ cells in a 6 cm dish, and the extract of Makomotake (Manufacturing Examples 2 to 4) was added so that the final concentration was 0.001%. The culture was continued. Next, the cells were washed three times with PBS (−), and then each cell was collected with a rubber policeman, and the number of cells was counted.
When the total number of cells when no extract is added to each cell is taken as a control (100%), the increase / decrease (%) in the number of cells when the extract of Momotake (Production Examples 2 to 4) is added The cell proliferation promoting effect on each cell was calculated. The same evaluation was performed using a basic fibroblast growth factor (bFGF) (Japanese Patent Publication No. 2010-500047), which has been reported so far as a substance exhibiting an effect of promoting the proliferation of stem cells, as a positive control.

The test results are shown in Table 2. As a result, the extract of Makomotake (Production Examples 2 to 4) showed a remarkable cell growth promoting effect only on stem cells. This effect was not seen for fibroblasts, keratinocytes or liver cells, but was specific for stem cells. In addition, bFGF, which has been used as a stem cell proliferation promoting substance so far, showed a significant proliferation effect on keratinocytes, fibroblasts and liver cells in addition to stem cells. From the above, the extract of Momotake showed a very excellent specific growth promoting effect on stem cells. In addition to the stem cells used in this experimental example, embryonic stem cells (ES cells) and stem cells artificially prepared by gene transfer were subjected to the same test. The promotion effect was recognized. In addition, the extract of Production Example 5 similarly recognized the effect of promoting stem cell proliferation.

  For transplantation to tissues, stem cells prepared by conventional techniques and stem cells prepared by adding the extract of makomotake were used for actual transplantation, and the survival rate (%) after transplantation was compared. .

Experimental Example 4 Preparation of Stem Cells for Transplantation Human somatic stem cells (DS Pharma Biomedical) were seeded at 1 × 10 ⁶ cells in a ⁶ cm dish, and the extract of Makomotake (Production Examples 2 to 4) had a final concentration of 0. It was added to 01% and the culture was continued for 3 days. Next, each cell was washed three times with PBS (−), collected aseptically with a rubber policeman, spun down, and dispersed in a Hanks solution containing 5% FBS (Hunk's balanced salt solution). Used as stem cells for transplantation. Thereafter, skin transplantation was performed by the following method, and the survival rate (%) was measured.

Experimental Example 5 Stem Cell Transplantation and Measurement of Engraftment Rate (%) The stem cells prepared in Experimental Example 4 were sampled 1 × 10 ⁶ times again, and the number of cells was adjusted. After transplantation, the survival rate (%) after transplantation was measured. As a specific method for measuring the survival rate (%), 1 × 10 ⁶ stem cells sampled for transplantation are fluorescently labeled in advance with Cell Tracker (manufactured by Molecular Probes), and the fluorescence intensity at that time is determined. The total fluorescence intensity (100%) of the transplanted cells was measured. The cells were transplanted subcutaneously into nude mice (male, 4 weeks old) using a syringe. The transplant site is marked with magic, the transplant site is removed 3 days and 7 days after transplantation, the cells are dispersed by enzyme treatment, the total fluorescence intensity of the obtained cells is measured, and the total fluorescence intensity at the time of transplantation (100 %), The survival rate (%) was calculated. That is, as the number of engrafted stem cells increases, the fluorescence intensity equivalent to the fluorescence intensity initially transplanted is detected. Conversely, if the engraftment does not occur, the fluorescence intensity is not detected from the transplanted site.

  These test results are shown in Table 3. As a result, both 3 days and 7 days after transplantation showed a very high engraftment rate when stem cells prepared using the extract of Makomotake (Production Examples 2 to 4) were transplanted compared to stem cells prepared by conventional methods. It was. The extract of Production Example 5 also showed a high engraftment rate.

  From the above results, it was confirmed that the engraftment rate of stem cells to the tissue was remarkably improved by preparing stem cells for transplantation using the extract of Makomotake rather than the stem cells prepared by the conventional method. In addition to the stem cells used in this experimental example, similar tests were conducted on embryonic stem cells (ES cells) and stem cells artificially prepared by gene transfer. The effect was recognized.

  By using the extract of the present invention for stem cells, it becomes possible to promote the proliferation of only stem cells while maintaining the undifferentiated state of the stem cells. Stem cells with a high adherence rate can be easily prepared.

As an application example of the present invention, application to regenerative medicine and regenerative beauty is expected. For example, by using the present invention, when preparing stem cells for transplantation used in regenerative medicine and regenerative beauty, the number of stem cells necessary for transplantation can be selectively and efficiently maintained while maintaining the undifferentiated state of stem cells. It is possible to grow. Furthermore, the stem cells prepared by the technique of the present invention have the property of exhibiting a high engraftment rate in the tissue at the time of transplantation, and it becomes possible to prepare stem cells that are extremely useful in regenerative medicine and regenerative beauty. In addition, for stem cells existing after transplantation or in tissues as a use other than transplantation, the extract of the present invention is introduced directly into the tissue by injection or oral administration, application, sticking, etc. It is possible to find an effect of maintaining a stem cell-specific undifferentiated state and an effect of promoting proliferation.
That is, the present invention provides applicability as an undifferentiated maintenance agent and / or growth promoter specific to stem cells in regenerative medicine and regenerative beauty, and the rate of engraftment in tissues by using the extract. It is a technology that enables a method for preparing high stem cells.

Claims (5)

An agent for maintaining undifferentiation of stem cells, which comprises an extract of makomotake. An agent for promoting proliferation of stem cells, comprising an extract of makomotake. A method for preparing a stem cell, comprising culturing using a medium containing an extract of makomotake. Stem cells prepared using the method for preparing stem cells according to claim 3. A medium for culturing stem cells, comprising an extract of makomotake.