JP6840376B2 - Stem cell undifferentiated state maintainer and growth promoter - Google Patents

Description

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

In vertebrate (especially mammal) tissues, when cell / organ damage occurs due to injury or disease, aging, etc., the regenerative system works to recover the cell / organ damage. 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 it is thought that this property leads to recovery by compensating for damaged parts of cells and organs. Expectations are high for regenerative medicine, which is a next-generation medical treatment that applies such stem cells.

The most advanced tissue in stem cell research in mammals is the bone marrow. Hematopoietic stem cells of the living body are present in the bone marrow, and it has been clarified that they are the source of all blood cell regeneration. Further, it has been reported that bone marrow contains stem cells capable of differentiating into organs and tissues (for example, bone, cartilage, muscle, fat, etc.) in addition to hematopoietic stem cells (see Non-Patent Document 1).

Furthermore, in recent years, it has been clarified that stem cells exist in all organs and tissues such as skin, liver, pancreas, and fat in addition to bone marrow, and it is responsible for the regeneration and maintenance of homeostasis of each organ and tissue. It has become clear (see Non-Patent Documents 2 to 5). In addition, the stem cells existing in each organ or tissue have excellent plasticity, and may be used for regeneration of organs or tissues that could not be self-replicating until now.

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

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

From the above, when stem cells are used for cell transplantation therapy and tissue engineering (regenerative medicine and regenerative beauty) and the regeneration of organs and 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 growing stem cells while maintaining their undifferentiated state, but they are still under development. For example, embryonic stem cells (ES cells) and hematopoietic stem cells can be maintained undifferentiated by co-culturing with supporting cells (stromal cells or feeder cells) (see Patent Document 1 and Non-Patent Documents 9 to 11). ). However, recently, cases of infection between heterologous animals by endogenous viruses derived from feeder cells have been reported (see Non-Patent Document 12), and culturing of stem cells using supporting cells is a stem cell for medical purposes. Not suitable for culturing.

Another method is to maintain the undifferentiated state of stem cells by combining cytokines in a complex manner. 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 undifferentiated status in the presence of the early-acting cytokine thrombopoietin (TPO), interleukin 6 (IL-6), FLT-3 ligand, and stem cell factor (SCF) is an embryonic stem cell. It has been reported in somatic stem cells and the like (see Patent Document 3 and Non-Patent Document 14).

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

As described above, all of the currently reported methods for maintaining the undifferentiated state of stem cells require complicated operations, and the effect of maintaining the undifferentiated state is low. Therefore, in order to utilize stem cells for regenerative medicine, there has been a demand for a technique for proliferating stem cells while maintaining an undifferentiated state. That is, there has been a demand for a technique capable of proliferating stem cells safely, easily and efficiently while maintaining an undifferentiated state.

On the other hand, elastin is a structural protein that is abundantly present together with collagen in tissues that require elasticity, such as the dermis, ligaments, tendons, blood vessel walls, and cartilage of the skin. It is known that elastin has a physical function of improving elasticity of skin and blood vessels, and a biological function of cell migration and platelet aggregation inhibitory action. Further, since elastin is an insoluble protein, a method for solubilization and a method for obtaining a high-purity elastin peptide containing a large amount of peculiar amino acids constituting elastin such as desmosine and isodesmosine have been developed. It has been reported that the elastin peptide has a growth promoting action on dermal fibroblasts and vascular endothelial cells (Patent Document 4) and exhibits a smooth muscle growth inhibitory activity in blood vessels (Patent Document 5). Further, it has been reported that when an elastin peptide is used in combination with a collagen peptide, it promotes the proliferation of fibroblasts, but the elastin peptide alone suppresses the proliferation of fibroblasts (Non-Patent Document 15). As described above, the effect of the elastin peptide on cell proliferation differs depending on the cell type and conditions. Moreover, the effect of elastin peptide on promoting proliferation of stem cells and the effect of maintaining an undifferentiated state has not been known so far.

Japanese Unexamined Patent Publication No. 2004-24089 Special Table 2002-525402A Japanese Patent No. 35733554 Japanese Unexamined Patent Publication No. 2007-151453 JP-A-2015-42688

Pittenger M. et al. F. Et al., Science, 1999, Vol. 284, pp. 143-147 Goodell M. A. Et al., Nat. Med. , 1997, Vol. 3, pp. 1337-1345 Zulewski H. Et al., Diabetes, 2001, Vol. 50, pp. 521-533 Suzuki A. Et al., Hepatology, 2000, Vol. 32, pp. 1230-1239 Zuk P. A. Et al., Tissue Engineering, 2001, Vol. 7, pp. 211-228 Yasushi Hasegawa, Aesthetic Dermatology, 2013, Vol. 23, pp. 1-11 Hiroshi Mabuchi et al., Journal of the Japanese Society of Regenerative Medicine, 2007, Vol. 6, pp. 263-268 Zen Kitagawa et al., Journal of the Japanese Society of Regenerative Medicine, 2008, Vol. 7, pp. 14-18 Thomason J. A. Et al., Proc. Natl. Acad. Sci. USA, 1995, Vol. 92, pp. 7844-7884 Thomason J. A. Et al., Science, 1998, Vol. 282, pp. 1145-1147 Reubinoff B. E. Et al., Nature Biotechnology. , 2000, Vol. 18, pp. 399-404 Van der Laan L. J. Et al., Nature, 2000, Vol. 407, pp. 90-94 Smith A. G. Et al., Dev. Biol. , 1987, Vol. 121, pp. 1-9 Madlambayan G.M. J. Et al., J. et al. Hematother. Stem Cell Res. , 2001, Vol. 10, pp. 481-492 Ayako Motomura et al., Journal of Japanese Society of Fisheries Science, 2009, Vol. 75, pp. 86-88

In view of the above-mentioned circumstances, the present invention finds a new substance capable of efficiently proliferating stem cells while maintaining the undifferentiated state, and provides the stem cells as an undifferentiated state maintaining agent or a growth promoting agent. Is the subject.

As a result of diligent research to solve the above problems, the present inventors have found that an elastin peptide has an excellent undifferentiated state-maintaining effect and a growth-promoting effect on stem cells, which have not been known so far. The invention was completed.

That is, the present invention includes the following.
(1) An agent for maintaining an undifferentiated state of stem cells containing an elastin peptide as an active ingredient.
(2) A stem cell growth promoter containing an elastin peptide as an active ingredient.
(3) The agent according to (1) or (2), wherein the stem cell is a hematopoietic stem cell.
(4) The agent according to (1) or (2), wherein the elastin peptide is an elastin peptide derived from a fish arterial bulb.
(5) A method for producing stem cells, which comprises a step of culturing the stem cells in a medium containing an elastin peptide.
(6) A method for maintaining an undifferentiated state of stem cells, which comprises a step of culturing the stem cells in a medium containing an elastin peptide.
(7) A method for promoting proliferation of stem cells, which comprises a step of culturing the stem cells in a medium containing an elastin peptide.
(8) The method according to any one of (5) to (7), wherein the stem cell is a hematopoietic stem cell.
(9) The method according to any one of (5) to (7), wherein the elastin peptide is an elastin peptide derived from a fish arterial bulb.

According to the present invention, stem cells can be efficiently proliferated while maintaining an undifferentiated state. Therefore, the present invention can make a great contribution in the fields of regenerative medicine and regenerative cosmetology.

Hereinafter, the present invention will be described in detail.
The undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention contains an elastin peptide as an active ingredient.

The "elastin peptide" used in the present invention means a "water-soluble elastin peptide" in which elastin existing in a living tissue as an insoluble protein is decomposed into a peptide and made soluble. The water-soluble elastin peptide is obtained, for example, by decomposing living tissues such as fish, mammals, and birds containing elastin by acid treatment or alkali treatment, or by enzymatic treatment. The elastin peptide used in the present invention may be one in which all of the elastin contained in the living tissue is decomposed into peptides, or may be partially decomposed into peptides. The elastin peptide used in the present invention is a composition containing peptides having various molecular weight sizes, and the molecular weight is not particularly limited. Further, it may be modified by N-acetylation or the like by a chemical or enzymatic method.

Examples of biological tissues include arterial spheres obtained from fish such as bonito, tuna, hamachi, and salmon, term ligaments obtained from mammals such as pigs, cows, horses, and sheep, and aortic blood vessels. Among them, fish arteries are preferable because they are rich in elastin composed of peculiar amino acids such as desmosine and isodesmosine, and arteries of large fish such as bonito, tuna, marlin, cod, hamachi, bristle, salmon, and trout. Spheres are more preferred.

The elastin peptide used in the present invention is any method known as a method for preparing a water-soluble elastin (peptide), as long as it is a method including a solubilization treatment for fragmenting the polypeptide chain of elastin to improve water solubility. (For example, refer to JP-A-2007-151453, JP-A-2009-219422, JP-A-2010-155820, etc.).

The elastin peptide can be prepared according to, for example, the following method. First, in order to facilitate solubilization, a pretreatment is performed on a living tissue as a raw material. Pretreatment includes washing of raw materials, immersion in alkaline solution, pulverization and homogenization, removal of unnecessary substances (blood, lipid, soluble protein, collagen), degreasing and the like. This pretreatment gives an insoluble protein mixture containing elastin as the main component.

The resulting insoluble protein mixture is then solubilized. The solubilization treatment can be performed using any known method, and examples thereof include acid treatment, alkali treatment, and enzyme treatment. The acid treatment is carried out by immersing the insoluble protein in an acid solution for a certain period of time to dissolve it. Examples of the acid used include oxalic acid, formic acid, acetic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, difluoroacetic acid, trifluoroacetic acid, phosphoric acid, sulfamic acid, perchloric acid, trichloroacetic acid and the like. However, citric acid is preferred. As a specific method, an acid decomposition method in which heat treatment is performed in an oxalic acid solution is known. After the acid treatment, neutralization is performed with an alkali, and sodium hydroxide and calcium hydroxide are preferable as the alkali to be used. The alkaline treatment is carried out by immersing the insoluble protein in an alkaline solution for a certain period of time to dissolve it. Examples of the alkali include sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate and the like, but sodium hydroxide is preferable. As a specific method, an alkali-ethanol method for treating with an alkaline hydrous ethanol solution is known. Further, after the alkali treatment, it is preferable to neutralize with an acid (for example, hydrochloric acid, acetic acid, sulfuric acid, etc.). The enzyme treatment can use any proteolytic enzyme used in the manufacture of pharmaceuticals, cosmetics and foods. The proteolytic enzyme is not particularly limited as long as it is an enzyme capable of degrading elastin, and for example, elastase, pepsin, trypsin, chymotrypsin, papain, bromelain and the like can be used. The enzyme treatment is carried out by adding the protease to the insoluble protein-containing solution and incubating the protease at the optimum temperature for an appropriate time (for example, about 24 hours), and after the enzyme treatment, heating (for example, about 10 minutes at 90 ° C.). ) To inactivate the enzyme.

If necessary, the water-soluble elastin peptide obtained by any of the above treatments is subjected to pH adjustment of the solution and desalting treatment according to the mode of use. The desalting treatment can be carried out by any method such as an ultrafiltration method and an ion exchange method. The removal of impurities and residues can be performed by treatment with diatomaceous earth, activated carbon, etc., and purification is performed by conventional methods such as ultrafiltration, gel filtration, ion exchange column chromatography, and reverse phase high performance liquid chromatography. be able to. The obtained water-soluble elastin peptide can be used as it is as a solution, but it can be pulverized by drying by a conventional method such as freeze-drying or ventilation drying.

In the present invention, commercially available elastin peptides may be used, for example, tuna arterial cell-derived elastin (manufactured by Wako Pure Chemical Industries, Ltd.), bonito arterial cell-derived elastin (manufactured by Hayashikane Sangyo Co., Ltd.), and porcine aorta-derived elastin (Japanese). (Made by Kojunyaku Kogyo Co., Ltd.), elastin derived from bovine ligament (manufactured by Wako Pure Chemical Industries, Ltd.), etc. can be used. In addition, one type of elastin peptide may be used, or two or more types of elastin peptides having different origins, degree of hydrolysis, molecular weight distribution, etc. may be used in combination.

Since the elastin peptide has an action of efficiently proliferating stem cells while maintaining an undifferentiated state at a biological level or a culture level, it can be used as an undifferentiated state maintaining agent or a growth promoting agent for stem cells. Furthermore, the undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention is also used as a medium additive for stem cell culture and a research reagent for efficiently proliferating stem cells while maintaining the undifferentiated state. be able to.

By applying the undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention to mammalian stem cells including humans, it is possible to maintain the undifferentiated state of stem cells and promote the growth of stem cells. .. The stem cells to which the undifferentiated state-maintaining agent or growth-promoting agent of the stem cells according to the present invention are 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 skin (epidermal, dermal, subcutaneous tissue), fat, hair follicles, brain, nerves, liver, pancreas, kidneys, muscles and other tissues; stem cells artificially produced by gene transfer, etc. (Artificial pluripotent stem cells: iPS cells). Preferably, the undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention exerts more effect on stem cells derived from bone marrow, blood, skin or adipose tissue. For example, hematopoietic stem cells are mentioned as stem cells derived from bone marrow, and in the present invention, "hematopoietic stem cells" include pluripotent myeloid stem cells and pluripotent lymphoid stem cells, and myeloid cells (erythrocytes, platelets, eosinophils). , Stem cells, neutrophils, monospheres, etc.) and cells capable of differentiating into lymphoid cells (B cells, T cells, NK cells, etc.). Hematopoietic stem cells are characterized by being positive for the CD34 antigen and negative for the CD38 antigen (CD34 + CD38-). Examples of ES cells include ES cells established by culturing early embryos before implantation, and ES cells established by culturing early embryos prepared by nuclear transplantation of somatic cell nuclei. And ES cells in which genes on the chromosomes of those ES cells are modified by using genetic engineering techniques. Such ES cells can be produced, for example, by a method known per se, but can be obtained from a predetermined institution, and a commercially available product can also be purchased. In addition, these stem cells may be either primary cultured cells, subcultured cells, or frozen cells.

Furthermore, the undifferentiated state-maintaining agent or proliferation-promoting agent for stem cells according to the present invention can be applied to stem cells derived from all mammals as long as they have the same characteristics regarding the direction of stem cell differentiation and the process of differentiation. It is possible. For example, the undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention is a mammalian stem cell such as human, monkey, mouse, rat, guinea pig, rabbit, cat, dog, horse, cow, sheep, goat, or pig. Can be effective against.

The application of the undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention to stem cells may be in vitro or in vivo, and in either case, the action can be exerted. Therefore, the undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention can be administered by culturing stem cells in a stem cell culture medium to which an effective amount thereof has been added, or by administering to mammals including humans. , Can maintain the undifferentiated state of stem cells and promote proliferation.

In the stem cell undifferentiated state-maintaining agent or growth-promoting agent according to the present invention, since the elastin peptide as an active ingredient has an excellent stem cell undifferentiated state-maintaining action and a growth promoting action, skin, osteoblast, cartilage, muscle, etc. It is effective in treating, ameliorating, and preventing damage or damage to a tissue or organ in the living body such as nerve, fat, or liver by acting on the stem cells of the tissue or organ. In addition, since stem cells decrease or decrease in function with aging, the undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention causes the decrease or function of stem cells in the above-mentioned tissues 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, reduction of stem cells or functional deterioration, for example, in the case of skin, wrinkles, tarmi, stains, dullness, rough skin, thickening of the skin, open pores, acne scars, etc. , Wounds, scars, keloids, etc., and also includes scalp and hair damage such as thinning hair and hair loss. In addition, osteoporosis and fractures (spine compression fractures, femoral neck fractures, etc.) are involved in bone-related diseases, and periodontal disease, rheumatoid arthritis, and disc hernia are involved in cartilage diseases. Spinal cord injury and facial nerve palsy are involved in nerve-related diseases. , Alzheimer's disease, muscular atrophic lateral sclerosis, Parkinson's disease, age-related memory loss, etc., blood-related, regenerative anemia, leukemia, etc., cardiovascular-related, myocardial infarction, obstructive arteriosclerosis, etc. Related items include periodontal disease and alveolar bone damage due to alveolar pyorrhea, ophthalmology-related items include retinal pigment degeneration, age-related yellow spot degeneration, and glaucoma, and liver / pancreas-related items include hepatitis, liver cirrhosis, and diabetes. Not limited to.

In addition, since the elastin peptide has a growth promoting action and an undifferentiated state maintaining action of hematopoietic stem cells, it is used to treat, improve, and prevent a disease or pathological condition of blood and hematopoietic organs associated with a decrease or functional decline of hematopoietic stem cells. It is effective as a medicine. Here, as the diseases or pathological conditions of blood and hematopoietic organs related to the decrease or functional deterioration of hematopoietic stem cells, for example, iron deficiency anemia, giant erythroblastic anemia, hemolytic anemia, erythroblastemia, aplastic anemia, etc. Anemia, congenital anemia (eg, sickle erythrocytosis), paroxysmal nocturnal pigmenturia, secondary anemia (anemia associated with infectious diseases, malignant tumors, chronic diseases, renal diseases, liver diseases, endocrine diseases, etc.), Leukemia, myelodysplastic syndrome, malignant lymphoma, multiple myeloma, myeloid proliferative disorders (true polyemia, essential thromboemia, myeloid fibrosis, etc.), idiopathic thrombocytopenic purpura, autoimmune hemolytic disease In addition to anemia, general anemia conditions (movement, shortness of breath, dizziness, dizziness, dizziness, aplastic anemia, easy fatigue, malaise, loss of appetite, nausea, headache, pale face, skin stains and bears, ringing in the ears, stiff shoulders, etc. (Mouth hornitis, etc.), but are not limited to these. In addition, "decrease or functional decline of hematopoietic stem cells" includes not only those caused by the above-mentioned diseases but also those caused by administration of anticancer agents and immunosuppressive agents and radiotherapy for cancer.

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

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

In the method according to the present invention, for culturing stem cells, a medium generally used for maintaining and proliferating the undifferentiated state of stem cells may be used. For example, a basal medium containing components necessary for stem cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids), specifically Dulvecco's Modified Eagle Medium (D-MEM). ), Minimum Essential Medium (MEM), RPMI 1640, Basic Medium Eagle (BME), Dulvecco's Modern Eagle's Medium: Natural MixEmeMend Hank's balanced salt solution and the like can be mentioned. In addition, basic fibroblast growth factor (bFGF) and / or leukocyte migration inhibitory factor (LIF) may be added to the medium as growth factors. In addition, if desired, the medium is epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferase, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27. -Supplement, N2-supplement, ITS-supplement, antibiotics, etc. may be contained.

In addition to the above components, it is preferable that serum is contained in the medium at a content of 1 to 20%. However, since the components of serum differ depending on the lot and the effect varies, it is preferable to use the serum after performing a lot check.

Commercially available media include Invitrogen's mesenchymal stem cell basal medium, Sanko Pure Chemical's mesenchymal stem cell basal medium, TOYOBO's MF medium, and Sigma's Hanks solution (Hank's balanced salt solution). ) Etc. can be used.

The incubator used for culturing stem cells is not particularly limited as long as it can cultivate stem cells, and examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. ..

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

The concentration of the elastin peptide added to the medium used for culturing the stem cells can be appropriately determined according to the content of the elastin peptide in the above-mentioned undifferentiated state-maintaining agent or growth promoter of the stem cells according to the present invention. In terms of the amount of peptide, for example, a concentration of 10 to 10000 μg / mL, preferably 100 to 5000 μg / mL can be mentioned. Elastin peptides may also be added to the medium on a regular basis during the stem cell culture period.

The stem cell culture conditions may follow the usual 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 1-10%, preferably about 2-5%. The medium is preferably changed once every 2 to 3 days, and more preferably every day. The culture conditions can be appropriately varied and set within a range in which stem cells can survive and proliferate.

For maintaining the undifferentiated state of stem cells, for example, the stem cells are cultured in the presence of the undifferentiated state maintaining agent of the stem cells according to the present invention, as compared with the stem cells cultured in the absence of the undifferentiated state maintaining agent of the stem cells according to the present invention. It can be evaluated whether or not the expression level of the stem cell undifferentiated marker gene is significantly maintained at the mRNA level or the protein level at the same level as the expression level at the start of culture in the stem cells. Examples of the stem cell undifferentiated marker gene include the Nanog gene (Cell Res. 2007 Jan; 17 (1): 42-9. Review. Nanog and transcriptional network) in embryonic stem cell Ghonci. ..

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

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

Further, the promotion of stem cell proliferation is carried out, for example, as compared with the stem cells cultured in the absence of the stem cell proliferation promoter according to the present invention, the stem cells cultured in the presence 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 cell number can be measured by using a commercially available cell number measurement kit by, for example, the MTT method or the WST method. As a result of the measurement, the relative ratio between the number of stem cells at the start of culturing and the number of stem cells after culturing for a predetermined time in the presence of the stem cell growth promoter of the present invention is not the stem cell growth promoter of the present invention. When it is larger than the same relative ratio (control) when cultured in the presence, it can be determined that the proliferation of stem cells could be promoted.

The stem cells prepared by the above method according to the present invention can be used as a transplant material (cell transplant agent), and can be carried out by the same method as conventional bone marrow transplantation or cord blood transplantation.

According to the above-mentioned undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention or the method according to the present invention, the elastin peptide is added to the undifferentiated state of stem cells alone or separately from the medium or mixed with the medium. It can also be provided as a reagent kit for maintenance or growth promotion. The kit may include an instruction manual or the like, if necessary. Alternatively, the elastin peptide can be mixed with a medium and provided as a medium for maintaining the undifferentiated state of stem cells or promoting proliferation.

When the above-mentioned undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention is administered in vivo, it can be administered as it is, but together with an appropriate additive as long as the effect of the present invention is not impaired. It can be blended and provided in various compositions such as cosmetics, quasi-drugs, pharmaceuticals, and foods and drinks. The pharmaceutical product of the present invention also includes a drug used for animals, that is, a veterinary drug.

When the undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention is blended in cosmetics or non-pharmaceutical products, the dosage form is an aqueous solution system, a solubilization system, an emulsification system, a powder system, a powder dispersion system, It may be any of an oil solution type, a gel type, an ointment type, an aerosol type, a water-oil two-layer system, a water-oil-powder three-layer system, and the like. In addition, for the cosmetics and quasi-drugs, various components, additives, bases and the like usually used in the external composition for skin are selected according to the type, together with the undifferentiated state maintaining agent or growth promoting agent for stem cells. However, it can be appropriately blended and produced according to a method known in the art. The form may be any of liquid, emulsion, cream, gel, paste, spray and the like. Examples of the components of the composition for external use on the skin include fats and oils (olive oil, palm oil, evening primrose oil, jojoba oil, castor oil, hardened castor oil, etc.), waxes (lanolin, honeybee, carnauba wax, etc.), and hydrocarbons (carnauba wax, etc.). Liquid paraffin, squalane, squalane, vaseline, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.) , Esters (isopropyl myristate, isopropyl palmitate, cetyl octanoate, glycerin trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citrate, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid) Acids, etc.), sugars (martitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and hydrolyzates of proteins, amino acids and their salts, vitamins, plant / animal extracts, various surface activities Examples include agents, moisturizers, ultraviolet absorbers, antioxidants, stabilizers, preservatives, bactericides, fragrances and the like.

Types of cosmetics and non-pharmaceutical products include, for example, lotion, milky lotion, gel, beauty essence, general cream, sunscreen cream, facial mask, mask, face wash, cosmetic soap, foundation, face powder, bathing agent, body lotion, etc. Examples include body shampoos, hair shampoos, hair conditioners, and hair restorers.

When the undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention is blended with a pharmaceutical product, it is mixed with a pharmacologically and pharmaceutically acceptable additive and is suitable for application to an affected area. Can be formulated into various formulations of. Pharmacologically and pharmaceutically acceptable additives include formulation substrates and carriers, excipients, diluents, binders, preservatives, and coatings that are appropriately selected according to the dosage form and application. Agents, disintegrants or disintegrants, stabilizers, preservatives, preservatives, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH regulators, propellants , Coloring agents, sweeteners, flavoring agents, fragrances and the like may be appropriately added to prepare various formulations that can be orally or parenterally administered systemically or topically by various known methods. When the pharmaceutical product of the present invention is provided in each of the above forms, it can be produced by a manufacturing method usually used by those skilled in the art, for example, the manufacturing method shown in each article of the general formulation [2] formulation of the Japanese Pharmacopoeia.

The form of the pharmaceutical product of the present invention is not particularly limited, but for example, tablets, sugar-coated tablets, capsules, troches, granules, powders, liquids, pills, emulsions, syrups, suspending agents, and elixirs. Oral agents such as drugs, injections (for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), infusions, suppositories, ointments, lotions, eye drops, sprays, trans. Examples include parenteral agents such as skin absorbents, transmucosal absorbents, and patches. It may also be a dry product that is redissolved upon use, and in the case of injectable formulations, it is provided in the form of unit dose ampoules or multidose containers.

A suitable form for using the undifferentiated state-maintaining agent or growth-promoting agent for stem cells according to the present invention as a pharmaceutical product for treating, ameliorating, or preventing the skin-related damage or disease is an external preparation, for example. Examples include ointments, creams, gels, liquids, patches (pups, plasters), foams, sprays, sprays 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. A gel agent refers to an external preparation in which a water-holding compound, which is a water-insoluble component, is suspended in an aqueous solution. The liquid agent refers to a liquid external preparation, and includes a lotion agent, a suspension agent, an emulsion, a liniment agent, and the like.

The pharmaceutical product of the present invention functions as a preventive agent for suppressing the onset of the above-mentioned diseases and / or as a therapeutic agent for improving a normal state. Since the active ingredient of the pharmaceutical product of the present invention is derived from a natural product, it is extremely safe and has no side effects. Therefore, when it is used as a therapeutic, ameliorating, or preventive drug for the above-mentioned diseases, humans, mice, rats, and rabbits. , Dogs, cats and other mammals can be administered orally or parenterally in a wide range of doses.

The content of the undifferentiated state-maintaining agent or growth-promoting agent for stem cells in the cosmetics, pharmaceuticals, and quasi-drugs of the present invention is not particularly limited, but is converted into the amount of elastin peptide with respect to the total weight of the preparation (composition). Therefore, 0.001 to 30% by weight is preferable, and 0.01 to 10% by weight is more preferable. The above amounts are merely examples, and may be appropriately set and adjusted in consideration of the type and form of the composition, general usage amount, efficacy / effect, and the like. Further, the method of adding the active ingredient in the formulation may be added in advance or may be added during the production, and may be appropriately selected in consideration of workability.

In addition, the stem cell undifferentiated state maintaining agent or growth promoting agent according to the present invention can also be blended in foods and drinks. Further, in the present invention, the food and drink are not only general foods and drinks but also foods other than pharmaceuticals that can be ingested for the purpose of maintaining or improving health, such as health foods, functional foods, health functional foods, or special uses. It is used to include food. Health foods include foods provided under the names of dietary supplements, dietary supplements, supplements and the like. Health functional foods are defined by the Food Hygiene Law or the Health Promotion Law, and are based on specified health foods and nutritionally functional foods that can display specific health effects, functions of nutritional components, reduction of disease risk, etc., and scientific evidence. Includes foods with functional claims that can display the contents notified to the Commissioner of the Consumer Affairs Agency. In addition, special-purpose foods include foods for the sick, foods for the elderly, foods for babies, foods for pregnant women, etc. that indicate that they are suitable for a specific target person or a patient having a specific disease. If the stem cells are hematopoietic stem cells, this drug may cause anemia or symptoms associated with anemia, especially in elderly people, pregnant women, and diseases associated with menstruation and bleeding tendency (gastric ulcer, duodenal ulcer, gastrointestinal polyps and cancer, hemorrhoids, etc.). It can be suitably used for health foods and the like for improvement and prevention of. Here, the indications such as specific health effects and functions of nutritional components attached to foods and drinks are indications such as product containers, packaging, manuals, package inserts, product leaflets and pamphlets, newspapers and magazines, etc. It can be used as an advertisement for a product of.

The form of the food or drink may be any of edible forms such as solid, liquid, granular, granular, powdery, capsule-like, cream-like, and paste-like. In particular, in the case of the above-mentioned health foods, for example, tablets, rounds, capsules, powders, granules, fine granules, troches, and liquids (including syrup, milky, and suspension) Etc. are preferable.

The types of foods and drinks include breads, noodles, confectionery, dairy products, processed marine and livestock foods, fats and oils, processed fats and oils, seasonings, and various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks). , Milk beverages, etc.), concentrated stock solutions of the beverages, preparation powders, etc., but are not limited thereto.

The food and drink of the present invention may appropriately contain additives that are usually used depending on the type of food and drink. As the additive, any additive that is acceptable under the Food Sanitation Law can be used, and for example, sweeteners such as starch, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; citric acid, malic acid, etc. , Acidulants such as citric acid; Excipients such as dextrin and starch; Binding agents, diluents, fragrances, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspensions Examples include turbidants and preservatives.

When the food or drink of the present invention is a general food or drink, it can be produced by including a step of adding an elastin peptide in a normal manufacturing process of the food or drink. In the case of health foods, the above-mentioned method for manufacturing pharmaceutical products may be followed. For example, in the case of tablet-shaped supplements, elastin peptides are added and mixed, and the products are manufactured by applying pressure with a tableting machine or the like. be able to. Further, other materials (e.g., vitamin C, vitamin B _2, vitamin such as vitamin B _6, minerals such as calcium, fiber, etc.) optionally may be added.

The amount of the elastin peptide to be blended in the food or drink of the present invention may be an amount capable of exerting the undifferentiated state maintaining effect and the growth promoting effect of stem cells, but the general intake amount of the target food or drink, the form of the food or drink, and the efficacy -It may be appropriately set in consideration of the effect, taste, taste, cost, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these Examples.

[Example 1] Evaluation of growth promoting effect and undifferentiated state maintaining effect of elastin peptide on stem cells The following shows experimental examples of the growth promoting effect and undifferentiated state maintaining effect of elastin peptide on stem cells and their results.
As elastin peptides, tuna arterial cell-derived elastin (trade name "elastin, water-soluble, tuna arterial cell-derived", manufactured by Wako Pure Chemical Industries, Ltd.), katsuo arterial cell-derived elastin (trade name "katsuo elastin"; manufactured by Hayashikane Sangyo Co., Ltd.) , Elastin derived from porcine aorta (trade name "elastin, water-soluble, derived from porcine aorta"; manufactured by Wako Pure Chemical Industries, Ltd.), elastin derived from bovine ligament (trade name "elastin, water-soluble, derived from bovine ligament"; Made by Kogyo Co., Ltd.) was used.

(Experimental Example 1) Evaluation of Proliferation-Promoting Effect on Stem Cells Adult adult stem cells (manufactured by DS Pharma Biomedical) cultured using a human stem cell culture medium (manufactured by TOYOBO) ^{were seeded in 3x10 5} pieces in a 6 cm dish, and the test substance was used. (Elastin peptide) was added to a final concentration of 1250 μg / mL, 2500 μg / mL, 5000 μg / mL, and the culture was continued for 3 days.

After culturing for 3 days, the cells were washed 3 times with PBS (−) and then collected with a rubber policeman, and the number of each cell was counted.
When the total number of cells when the test substance was not added was used as the control and the control was set to 100 (%), the increase / decrease (%) in the number of cells when the test substance was added was calculated, and the stem cell proliferation promoting effect was evaluated. The results of these tests are shown in Table 1 below.

As shown in Table 1, all of the elastin peptides had a remarkable effect of promoting stem cell proliferation. When the above-mentioned control value was set to 100%, the number of human somatic stem cells at the start of culturing was 25%. From the above, it was clarified that the elastin peptide has an extremely excellent effect of promoting stem cell proliferation. In addition to the stem cells used in this experimental example, commercially available human hematopoietic stem cells and mouse embryonic stem cells (ES cells) were also subjected to similar tests, and a remarkable effect of promoting stem cell proliferation was observed.

(Experimental Example 2) Evaluation of undifferentiated state maintenance effect on stem cells The effect of the test substance (elastin peptide) on the maintenance of undifferentiated state on hematopoietic stem cells is indexed by the expression of CD34 (CD34 engine), which is an undifferentiated marker of hematopoietic stem cells. Evaluated to.

Using DMEM / F12 medium as the basal medium, A- using a medium prepared to be fetal bovine serum (1%), Insurin (10 ng / ml), Transferrin (10 ng / ml), and 2-mercaptoethanol (100 μM). 6 cells (mouse hematopoietic stem cells) (RIKEN) were cultured. Incidentally, A-6 cells were seeded by 1 × 10 ⁶ cells in 12 well plate. At that time, a test substance (elastin peptide) was added so that the final concentration was 2500 μg / mL. After 4 days of culturing, the cells were collected, washed twice with PBS (−), and RNA was extracted from the cells by Trizol Reagent (Invitrogen). After reverse transcribing the extracted RNA into cDNA using the 2-STEP real-time PCR kit (Applied Biosystems), real-time PCR (95 ° C: 15 seconds, 60 ° C.) using the following primer set using ABI7300 (Applied Biosystems). : 40 cycles) was carried out for 30 seconds, and the expression of the CD34 gene was confirmed. Other operations were performed according to the prescribed method.

Primer set for CD34 gene (undifferentiated marker))
Forward primer: 5'-CTTCTGCTCCGAGTGCCATT-3'(SEQ ID NO: 1)
Reverse primer: 5'-ATACCCTGGGCCAACCTCAC-3'(SEQ ID NO: 2)
Primer set for Gapdh (internal standard):
Forward primer: 5'-CCGTGTTCCTACCCCCAAT-3'(SEQ ID NO: 3)
Reverse primer: 5'-TGCCTGCTTCACCACCTTCT-3'(SEQ ID NO: 4)

The undifferentiated state maintenance effect is the gene relative expression level of CD34 (CD34 gene expression level / Gapdh gene) calculated as the ratio of the expression level of CD34 mRNA in A-6 cells at the start of culture to the expression level of Gapdh mRNA, which is an internal standard. The value of (expression level) was set to 100% undifferentiated state, and the value of the gene relative expression level of CD34 in A-6 cells 4 days after culture was calculated and evaluated. In addition, bFGF was used as a positive control. The results are shown in Table 2.

As shown in Table 2, all of the elastin peptides were found to have a remarkable effect of maintaining the undifferentiated state of stem cells. In addition to the hematopoietic stem cells used in this experimental example, a similar test was performed on commercially available mouse embryonic stem cells (ES cells), and a remarkable effect of maintaining the undifferentiated state of the stem cells was observed.

As shown in each of the above experimental examples, by applying the elastin peptide to stem cells, it is possible to promote the proliferation of stem cells while maintaining the undifferentiated state more easily and efficiently than in the conventional technique. Became.

As an example of utilization of the present invention, application to regenerative medicine and regenerative beauty is expected. For example, by using the present invention, it becomes possible to easily and efficiently prepare undifferentiated stem cells used for regenerative medicine and regenerative cosmetology. Furthermore, the stem cells are undifferentiated by directly injecting the elastin peptide according to the present invention into the stem cells after transplantation of the stem cells or existing in the tissue, or by applying the elastin peptide according to the present invention by oral administration, application, application, or the like. It is possible to grow while maintaining the state.
That is, the present invention can be used as a method for producing stem cells and / or as an undifferentiated state-maintaining agent or a growth-promoting agent for stem cells in regenerative medicine and cosmetology.

Claims (2)

An undifferentiated state-maintaining agent for hematopoietic stem cells containing an elastin peptide derived from fish arterial cells as an active ingredient. A method for maintaining an undifferentiated state of hematopoietic stem cells, which comprises a step of culturing hematopoietic stem cells in a medium containing an elastin peptide derived from fish arterial spheres.