JP5197998B2 - Stem cells cultured by specific method and method for producing the same - Google Patents

Description

  The present invention relates to a stem cell and / or a method for producing the stem cell, which are characterized by culturing using an extract of a mushroom belonging to the genus Mushroom and a medium containing the component. Specifically, by containing an extract of a mushroom belonging to the genus Mushroom, it has a function of maintaining the undifferentiated state of mammalian stem cells, and further, while maintaining the effects of the functions, an effect of promoting proliferation of stem cells, The present invention relates to an undifferentiated stem cell having an effect of increasing the engraftment rate of a stem cell to a living tissue at the time of stem cell transplantation and / or a production method thereof.

  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 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 tissues. 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 the bone marrow contains 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).
Pittenger M.M. F. , Et al. , Science, 1999, 284, 143-147.

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 the regeneration and homeostasis of each organ / tissue. (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 that have been impossible to self-replicate until now. In order to fully exploit the potential of these stem cells and use them for cell transplantation treatment and tissue engineering (regenerative medicine and regenerative beauty), stem cells are isolated from living tissue and then transformed into an undifferentiated state by in vitro culture. A culture technique with a high engraftment rate is important when the cells are grown while maintained and transplanted.
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.

  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 6 to 8 and Patent Document 1). ). However, recently, cases of infection between different animals caused by endogenous cells derived from feeder cells have been reported (see Non-Patent Document 9), which 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 10 and Patent Document 2). 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 3 and Non-Patent Document 11).
Smith A. G. et al. Dev. Biol, 1987, 121, 1-9 JP-T-2002-525042 Patent registration 35733354 Madlambayan G.M. J. et al. et al. , J. et al. Hematother. Stem Cell Res. , 2001, 10, 481-492

  However, cytokines are expensive and difficult to use easily due to problems such as collection raw materials and storage stability. 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 7).

  All of the currently reported methods for maintaining the undifferentiated state of stem cells require cumbersome operations and have a low effect of maintaining the undifferentiated state. Therefore, when stem cells are used for regenerative medicine, they are undifferentiated. There has been a demand for stem cells with a high engraftment rate when proliferated to a number sufficient for transplantation while maintaining the state and transplanted to a target tissue. That is, there has been a demand for a stem cell having a high engraftment rate and a technique and a substance related thereto, which are proliferated while maintaining the undifferentiated state of the stem cell safely, simply and efficiently.

  In view of such a situation, the present invention solves the problems in the prior art as described above, efficiently proliferating while maintaining the undifferentiated state of mammalian stem cells, and a stem cell with a high engraftment rate and its It is to provide a manufacturing method and the like.

  As a result of intensive studies aimed at solving the above-mentioned problems, the present inventors have conducted culture using a medium containing an extract of a mushroom belonging to the genus Mushroom, which has excellent stem cell undifferentiation maintenance effects and proliferation. The inventor found a promoting effect, and further revealed that the engraftment rate in the living body is greatly improved when the stem cells are transplanted, and the present invention has been completed.

  That is, the present invention is as follows.

  A stem cell which is cultured using a medium containing an extract of a mushroom belonging to the genus Mannentake.

  A stem cell characterized by maintaining an undifferentiated state by culturing using a medium containing an extract of a mushroom belonging to the genus Mannentake.

  A stem cell, wherein the mushroom belonging to the genus Mannentake is at least one selected from the group consisting of black ganoderma, red ganoderma and maggot.

  A stem cell, wherein the stem cell is derived from a mammal.

  A method for producing a stem cell, comprising culturing in a medium containing an extract of a mushroom belonging to the genus Mannenmus.

  An agent for maintaining an undifferentiated state of stem cells, comprising an extract of a mushroom belonging to the genus Mannentake.

  A method for maintaining an undifferentiated state of a stem cell, comprising an extract of a mushroom belonging to the genus Mannentake (hereinafter referred to as a solvent extract of Mannentake).

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

  The present invention promotes cell growth while maintaining an undifferentiated state of stem cells by culturing using a medium containing a solvent extract of garlic mushroom, and further, the survival rate of stem cells to the tissue at the time of transplantation A stem cell and a method for producing the same are provided.

Mushrooms belonging to the genus Amanita used in the present invention are mushrooms belonging to the family Ganodermaaceae and Ganoderma, and are basidiomycetes used in the herbal medicine “Ganoderma”. The scientific names are: red ganoderma (Akashiba, scientific name: Ganoderma lucidum), black ganoderma (Kuroshiba, scientific name: Ganoderma synthum), purple ganoderma (Ganoderma Sinese), magajakuma (scientific name: Ganoderma Japan), etc. The classification method is not limited to these, but the Chinese pharmacology books “Hakusa Tsuname” and “Shinnohonsaku Sutra” include red ganoderma (Akashiba) and black ganoderma (Kuroshiba). ), Purple ganoderma (purple turf), blue ganoderma (blue turf), yellow ganoderma (yellow turf), and white ganoderma (white turf) (see Non-Patent Document 12). Moreover, the mushroom which belongs to the genus Bamboo genus used for this invention can use what is distribute | circulated widely in China, the Japanese market, etc., and may use a native product or a cultivated product. A mycelium culture can also be used. These can be dried or pulverized. In either case, the portion to be used is selected from fruit bodies, mycelium, mycelium culture, etc., and preferably fruit bodies are used.
Acta Microbiological Sinica. 1979, 19 (3), 265-279.

  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 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 solvent extract of Mannentake may be used as it is, or may be used after treatment such as concentration, dilution, filtration, and decolorization or deodorization with activated carbon as 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.

  There is no particular limitation on the content of the component when using the solvent extract of Mannentake in the form of a solution, but it is preferably 0.00001 to 10% by weight as a dried product, and used at a concentration of 0.0001 to 1% by weight. Most preferably. If it is less than 0.00001% by weight, the effect of the present invention is not sufficiently exhibited.

In addition, stem cells of the present invention, production methods thereof, undifferentiated maintenance agents, undifferentiation maintenance methods, and the like include cell culture additives, research reagents, medical reagents, cell transplants, pharmaceuticals, quasi drugs. It can be used in cosmetics and foods.
It is also possible to directly introduce the solvent extract of Mannentake into the body.

  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. Somatic stem cells, and primary cells, subculture cells, and frozen cells of the cells. 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 simultaneously, 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), for example, Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modify Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), GlasgominMimE s balanced salt solution), basic fibroblast growth factor (bFG) as a growth factor. ), Media supplemented with at least one kind of leukocyte migration inhibitory factor (LIF) is used, preferably those that all of these growth factors is contained. 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 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 Co., Ltd., MF medium manufactured by TOYOBO, Hank's solution (hank's balanced salt solution) manufactured by Sigma, etc. Can be used.

  Stem cells cultured using a medium containing the solvent extract of Mannentake of the present invention maintained an extremely high undifferentiated state. Further, when the proliferation of stem cells was promoted while maintaining this undifferentiated state and transplanted to a living body, the engraftment rate to the tissue was remarkably improved. 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, specific and detailed examples will be given to describe the present invention in detail, but the present invention is not limited thereto.

  Next, in order to explain the present invention in detail, examples of the production of the solvent extract of Mannentake used in the present invention and the experimental examples showing the effect of maintaining the undifferentiated state of stem cells are given as examples, but the present invention is not limited thereto. It is not something.

  In the following, a production example of a solvent extract using black ganoderma, red ganoderma, and maggots as mannentake is shown.

Production Example 1 Preparation of hot water extract of Kuro Ganoderma 400 mL of purified water was added to 20 g of Kuro Ganoderma dried product, extracted at 95-100 ° C. for 2 hours, filtered, and the filtrate was concentrated and freeze-dried. 1.4 g of hot water extract of black reishi was obtained.

Production Example 2 Preparation of Kuro Ganoderma Ethanol Extract 900 g ethanol was added to 100 g of Kuro Ganoderma dried product, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness. 1.5 g of ethanol extract was obtained.

Production Example 3 Preparation of hot water extract of red ganoderma 400 ml of purified water was added to 20 g of dried red ganoderma turf, extracted at 95-100 ° C. for 2 hours, filtered, and the filtrate was concentrated and lyophilized. As a result, 2.0 g of hot water extract of red ganoderma was obtained.

Production Example 4 Preparation of red ganoderma ethanol extract 900 g of ethanol was added to 100 g of red ganoderma dried product, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness. 2.5 g of ethanol extract was obtained.

Production Example 5 Preparation of hot water extract of pearl oyster 2 L of purified water was added to 100 g of dried pearl oyster, extracted at 95-100 ° C. for 2 hours, filtered, and the filtrate was concentrated, freeze-dried, 5.4 g of water extract was obtained.

Production Example 6 Preparation of ethanol extract of maggots 900 mL of ethanol was added to 100 g of dried products of magpies, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness. 0 g was obtained.

  Below, the experimental example and result of the maintenance agent (undifferentiation state maintenance effect, cell growth promotion effect) of the undifferentiated state of the stem cells using the solvent extract of Mannentake of Production Examples 1 to 6 shown in Example 1 Indicates.

Experimental Example 1 Evaluation of effect of maintaining undifferentiated state on stem cells Dulbecco's Modified Eagle Medium medium (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). , 6 cm dish 1x10 to ^five seeding, each extract (production example 1-6) a final concentration of 0.00 Was added to a%, it was continued for 3 days of culture. 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, using the expression level of octamer binding protein 3/4 protein (Oct3 / 4 protein) indicating the undifferentiated state of stem cells as an indicator, the stem cells (1 × 10 ⁵ cells) seeded at the start of culture were expressed in Oct3 / After the amount of 4 proteins was 100% undifferentiated, each extract (Production Examples 1 to 6) was added and cultured for 3 days, the amount of Oct3 / 4 protein was quantitatively analyzed by Western blotting and cultured. The effect of maintaining the undifferentiated state was evaluated by comparing the amount of Oct3 / 4 protein at the start and after culturing for 3 days.
At the time of this evaluation, the amount of Oct3 / 4 protein expressed in the number of single cells (1 × 10 ⁵ cells) at the start of the culture was taken as 100% undifferentiated state, and the undifferentiated state (% undifferentiated) at the time of each extract addition. State) was calculated, and the effect of maintaining the undifferentiated state was evaluated.

The test results are shown in Table 1. As a result, a remarkable effect of maintaining the undifferentiated state of stem cells was observed in all the solvent extracts of Mannentake (Production Examples 1 to 6). From the above, the extremely excellent stem cell undifferentiated state maintenance effect of the solvent extract of Mannentake was clarified. In addition to the stem cells used in this experimental example, a similar test was performed on somatic stem cells. As a result, a remarkable effect of maintaining the undifferentiated state of stem cells was observed.

Experimental Example 2 Evaluation of Cell Proliferation Promoting Effect on Stem Cells Using a human stem cell culture solution (manufactured by TOYOBO), 1 × 10 ⁵ human somatic stem cells (DS Pharma Biomedical) are seeded in a 6 cm dish, and the solvent of each mannentake The extract (Production Examples 1 to 6) was added to a final concentration of 0.001%, and the culture was continued for 3 days. Next, the cells were washed three times with PBS (−) and then collected with a rubber policeman, and the number of each cell was counted.
Calculate the increase / decrease (%) in the number of cells when each extract is added, and evaluate the effect of promoting the proliferation of stem cells when the total number of cells when no extract is added is the control and the control is 100 (%). went.

  The test results are shown in Table 2. As a result, a remarkable effect of promoting cell proliferation of stem cells was observed in all the solvent extracts of Mannentake (Production Examples 1 to 6). From the above, the extremely superior stem cell proliferation promoting effect of the solvent extract of Mannentake 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 cell proliferation of stem cells was observed.

  The stem cells cultured by adding the solvent extract of mannentake according to the present invention were actually transplanted, and the respective survival rates (%) after transplantation were compared.

Experimental Example 3 Preparation of Stem Cells for Transplantation Human somatic stem cells (DS Pharma Biomedical) were seeded at 1 × 10 ⁶ in a 6 cm dish, and each extract (Production Examples 1 to 6) had a final concentration of 0.01%. 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 4 Stem Cell Transplantation and Measurement of Engraftment Rate (%) ^Six 1 × 10 ⁶ stem cells prepared in Experimental Example 3 were transplanted subcutaneously into nude mice (male, 4 weeks old). Specifically, 1 × 10 ⁶ cells to be transplanted were fluorescently labeled in advance with Cell Tracker (manufactured by Molecular Probes), and the fluorescence intensity at that time was measured to obtain the total number of transplanted cells (100%). Mark the transplant site with magic, extract the transplant site 3 days and 7 days after transplantation, disperse the cells by enzyme treatment, measure the fluorescence intensity of the obtained cells, and the fluorescence intensity at the time of transplantation (100%) The survival rate (%) was calculated.

  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 using the solvent extract of Mannentake (Production Examples 1 to 6).

  From the above results, it was confirmed that the solvent extract of Mannentake significantly improves the engraftment rate in stem cell transplantation. 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 improving the survival rate in stem cell transplantation was observed.

  The use of the solvent extract of the garlic mushroom of the present invention for stem cells promotes the proliferation of stem cells while maintaining an undifferentiated state as compared with conventional techniques, and when transplanted, the engraftment rate is extremely high. It became possible to easily prepare high stem cells.

Applications of the present invention are expected to be applied to regenerative medicine and regenerative beauty. For example, by utilizing the present invention, it becomes possible to easily and efficiently prepare undifferentiated stem cells having a high engraftment rate used for regenerative medicine and regenerative beauty. In addition, the stem cell existing after transplantation or in the tissue is allowed to maintain the undifferentiated state of the stem cell by directly injecting the oral extract, coating, sticking, etc. It can be grown as is.
That is, the present invention can be used as a method for preparing stem cells and / or a maintenance agent for the undifferentiated state of stem cells in regenerative medicine and regenerative beauty.

Claims (1)

An agent for maintaining an undifferentiated state of stem cells, comprising an extract of a mushroom belonging to the genus Mannentake.