Composition for increasing biological activity of stem cells using mixture 4F

Abstract

The present invention relates to a composition for increasing the biological activity of stem cells using mixture 4F. Stem cells treated with mixture 4F according to the present invention can not only obtain undifferentiated characteristics (stemness), but also have the effect of improving cell proliferation ability and migration ability, so that after the stem cells are transplanted into the body, cell survival and implantation survival rate can be improved, and blood vessel and tissue regeneration ability can be further improved, and can be applied in various ways to the fields of stem cell differentiation, prevention or treatment of ischemic diseases.

Description

Technical Field

[0001] The present invention relates to a composition for increasing the biological activity of stem cells using mixture 4F. Background Art

[0002] Ischemic vascular diseases, such as heart disease, cerebrovascular disease, and peripheral vascular disease, have a prevalence rate as high as 40% and are among the highest mortality rates worldwide. Current treatment options include medical therapy, primarily medication, mechanical vascular revascularization, and surgical bypass procedures. However, even with all of these treatments, satisfactory treatment often remains unsatisfactory due to limited regeneration of ischemic tissue, which lacks a smooth supply of oxygen and nutrients. Therefore, angiogenesis therapy, which promotes angiogenesis in the tissue surrounding the ischemic area to ensure blood flow and minimize tissue damage, has been proposed as a treatment strategy for a wide range of vascular diseases that are currently untreatable.

[0003] To form vascular tissue composed of cells, a supply of angiogenic cells is necessary. Therefore, various approaches have been explored as therapeutic strategies, including direct administration of angiogenesis-promoting factors or administration of genes encoding these factors to promote the proliferation and development of endothelial cells. However, these approaches have been limited in effectiveness and have raised various side effects and safety concerns. Therefore, further research is needed to practically introduce angiogenesis-promoting factors.

[0004] In addition, it has been found that vascular endothelial progenitor cells are effective cells for the repair of damaged blood vessels and the regeneration of ischemic tissues, and various studies are being attempted to promote angiogenesis by directly administering cells that generate blood vessels. At present, in the field of angiogenesis and regeneration using stem cells, adult stem cells are used for clinical applications, and their advantage is that they can be extracted from the patient's body and autologously transplanted into the desired tissue, and there are no ethical issues compared to embryonic stem cells. However, due to the aging and functional reduction of patient cells caused by Westernized diet and reduced exercise, aging, etc., it is difficult to enter the practical stage. Therefore, research for amplifying stem cells and enhancing biological activity has become a core factor in the practical application of cell therapy. For this reason, it is necessary to develop function-enhancing factors that are safe for the human body and analyze the mechanism of action of each factor to provide a system of stem cells with more excellent functions. Due to this need, recently, actively conducting cell function enhancement research for the practical application of stem cell therapeutic agents is becoming a trend. Research using gene introduction is a method for improving cell function by amplifying and deleting specific genes, and its stability needs to be further studied. Summary of the Invention

[0005] Technical issues

[0006] Therefore, in order to solve the problems of the prior art as described above, the present inventors developed a mixture 4F comprising fucoidan, tauroursodeoxycholic acid, oleuropein, and vascular endothelial growth factor, thereby completing the present invention.

[0007] Therefore, an object of the present invention is to provide a composition for inhibiting stem cell aging, promoting stem cell proliferation or inducing stem cell differentiation, wherein the composition comprises fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor as active ingredients.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating ischemic diseases, wherein the pharmaceutical composition comprises fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor as active ingredients.

[0009] Another object of the present invention is to provide a culture medium composition for stem cell culture, comprising fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor as active ingredients.

[0010] Another object of the present invention is to provide a xeno-free culture method, comprising the step of treating stem cells with a stem cell culture medium composition comprising fucoidan, tauroursodeoxycholic acid, oleuropein, and vascular endothelial growth factor as active ingredients.

[0011] Another object of the present invention is to provide a stem cell therapy adjuvant, which comprises fucoidan, tauroursodeoxycholic acid, olive oil and vascular endothelial growth factor as active ingredients.

[0012] Another object of the present invention is to provide stem cells cultured by the xeno-free culture method.

[0013] Another object of the present invention is to provide a pharmaceutical composition comprising the stem cells for preventing or treating ischemic diseases.

[0014] Technical Solution

[0015] To achieve the above objectives, the present invention provides a composition for inhibiting the aging of stem cells, promoting the proliferation of stem cells, or inducing the differentiation of stem cells, wherein the composition comprises fucoidan, tauroursodeoxycholic acid, oleuropein, and vascular endothelial growth factor as active ingredients.

[0016] In addition, the present invention provides a pharmaceutical composition for preventing or treating ischemic diseases, wherein the pharmaceutical composition comprises fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor as active ingredients.

[0017] In addition, the present invention provides a culture medium composition for stem cell culture, which contains fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor as active ingredients.

[0018] In addition, the present invention provides a xeno-free culture method, which includes the step of treating stem cells with a stem cell culture medium composition containing fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor as active ingredients.

[0019] In addition, the present invention provides a stem cell treatment adjuvant, which contains fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor as active ingredients.

[0020] Furthermore, the present invention provides stem cells cultured by the xeno-free culture method.

[0021] Furthermore, the present invention provides a pharmaceutical composition comprising the stem cells for preventing or treating ischemic diseases.

[0022] Technical Effects

[0023] Stem cells treated with the mixture 4F according to the present invention not only acquire undifferentiated characteristics (stemness), but also have the effect of improving cell proliferation and migration capabilities. Therefore, after stem cells are transplanted into the body, cell survival and implantation survival rates can be improved, and blood vessel and tissue regeneration capabilities can be further enhanced. Therefore, they can be widely used in the fields of stem cell differentiation, prevention or treatment of ischemic diseases. <110> YOUTH BIO GLOBAL CO., LTD. <120> Composition for increasing biological activity of stem cells using mixture 4F <130> PCT2020-031 <150> KR 10-2019-0082712 <151> 2019-07-09 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> VEGF-A_Forward Primer <400> 1 gctcggtgct ggaatttgat 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> VEGF-A_reverse primer <400> 2 gcccgattca agtggggaat 20 <210> 3 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> IL-8_Forward Primer <400> 3 caccggaagg aaccatctca ct 22 <210> 4 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> IL-8_reverse primer <400> 4 tcagccctct tcaaaaactt ctcc 24 <210> 5 <211> 25 <212> DNA <213> Artificial sequence <220> <223> b-FGF_Forward Primer <400> 5 ggagaagagc gaccctcaca tcaag 25 <210> 6 <211> 27 <212> DNA <213> Artificial sequence <220> <223> b-FGF_reverse primer <400> 6 ccagttcgtt tcagtgccac ataccaa 27 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Ang2_Forward Primer <400> 7 gggaagggaa tgaggcttac 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Ang2_reverse primer <400> 8 aagttggaag gaccacatgc 20 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> b-actin_forward primer <400> 9 agcgagcatc ccccaaagtt 20 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> b-actin_reverse primer <400> 10 gggcacgaag gctcatcatt 20 BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the results of confirming the effect of the mixture 4F according to the present invention on the phenotype of cells.

[0025] Figure 2 This is a graph showing the results of confirming the migration ability of cells treated with the mixture 4F of the present invention.

[0026] Figure 3aThis figure shows the results of confirming the in vivo angiogenesis effect after transplantation of cells treated with the mixture 4F according to the present invention into a lower limb ischemia animal model.

[0027] Figure 3b This figure shows an image of a vascular suture site three days after transplantation of GFP-labeled cells, using in vivo imaging technology.

[0028] Figure 4a The results were obtained by evaluating cardiac function by measuring the ejection fraction and fractional shortening 28 days after inducing myocardial infarction in an animal model of severe myocardial infarction.

[0029] Figure 4b The results are based on the results of Masson's trichrome staining performed 28 days after induction of myocardial infarction in an animal model of severe myocardial infarction to analyze the degree of myocardial fibrosis.

[0030] Figure 4c The graph shows the results of transplanting GFP-labeled cells into a severe myocardial infarction model, and analyzing the engraftment and proliferation rate of the cells in the myocardium by immunostaining for PCNA 3 days later.

[0031] Figure 5a This is a graph showing the results of analysis of changes in cell proliferation rate following treatment with the mixture 4F of the present invention.

[0032] Figure 5b This is a graph showing the results of analysis of changes in the cell cycle following treatment with the mixture 4F of the present invention.

[0033] Figure 5c This is a graph showing the results of analyzing senescence of cells treated with the mixture 4F of the present invention.

[0034] Figure 5d This is a graph showing the results of analysis of the colony-forming ability of cells treated with the mixture 4F of the present invention.

[0035] Figure 6a This is a graph showing the results of measuring changes in gene expression of markers related to undifferentiated stem cells following treatment with the mixture 4F of the present invention.

[0036] Figure 6b This figure shows the analysis results of gene expression of markers related to undifferentiated stem cells treated with the mixture 4F of the present invention in the form of principal component analysis and heat map.

[0037] Figure 7 A graph showing the results of analysis of the mortality rate of undifferentiated stem cells treated with the mixture 4F of the present invention.

[0038] Figure 8a This is a graph showing analysis of the proliferation rate of undifferentiated stem cells treated with the mixture 4F of the present invention.

[0039] Figure 8b and Figure 8c This is a graph showing the results of analysis of marker expression in undifferentiated stem cells treated with the mixture 4F of the present invention.

[0040] Figures 9a to 9e Graphs showing the results of analysis of marker expression following treatment with the mixture 4F of the present invention or with a single component.

[0041] Figure 10 This is a graph showing the results of analysis of the angiogenesis ability of cells treated with the mixture 4F of the present invention or with a single component.

[0042] Figure 11 A diagram schematically illustrates a xeno-free culture method using the mixture 4F according to the present invention.

[0043] Figure 12 This figure shows the results of confirming the morphological characteristics of vascular stem cells cultured using the xeno-free culture method using the mixture 4F according to the present invention.

[0044] Figure 13 This figure shows the results of analysis of gene expression of undifferentiated stem cell-related markers in vascular stem cells cultured using the xeno-free culture method using the mixture 4F of the present invention.

[0045] Figure 14a The graph shows the results of analysis of the proliferation ability and colony-forming ability of vascular stem cells cultured using the xeno-free culture method using the mixture 4F according to the present invention.

[0046] Figure 14b This figure shows the results of analyzing cell cycle changes of vascular stem cells cultured using the xeno-free culture method using the mixture 4F according to the present invention.

[0047] Figure 15 This figure shows the results of analyzing the genetic similarity between vascular stem cells cultured using the xeno-free culture method using the mixture 4F according to the present invention and cells of a control group.

[0048] Figure 16Graphs showing the results of gene ontology and gene category analysis of vascular stem cells cultured using the xeno-free culture method using the mixture 4F according to the present invention and control cells.

[0049] Figure 17a This is a graph showing the results of analysis of gene expression levels in vascular stem cells cultured using the xeno-free culture method using the mixture 4F of the present invention and in control cells.

[0050] Figure 17b It is shown as a heat map Figure 17a A graph showing changes in gene expression levels in .

[0051] Figure 18 This figure shows the results of confirming the angiogenesis ability of vascular stem cells cultured using the xeno-free culture method of the mixture 4F according to the present invention.

[0052] Figure 19 This figure shows the results of confirming the effect of peripheral secretory blood vessel formation on vascular stem cells cultured using the xeno-free culture method using the mixture 4F according to the present invention.

[0053] Figure 20 The graph shows the results of confirming the in vivo angiogenesis effect after transplantation of vascular stem cells cultured using the xeno-free culture method using the mixture 4F according to the present invention into an animal model of lower limb ischemia.

[0054] Figure 21 This figure shows an image of a vascular suture site after transplantation of GFP-labeled cells in the lower limb ischemia animal model according to the present invention, using in vivo imaging technology.

[0055] Optimal Factual Method

[0056] Hereinafter, the present invention will be described in detail.

[0057] According to one example of the present invention, the present invention provides a composition for inhibiting stem cell aging, promoting stem cell proliferation or inducing stem cell differentiation, wherein the composition comprises fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor as active ingredients.

[0058] In the present invention, "mixture 4F" refers to a mixture of fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor.

[0059] In the present invention, "stem cell" refers to a cell with self-replication ability and the ability to differentiate into two or more cells, which can be divided into omnipotent stem cells, pluripotent stem cells, and multipotent stem cells. The stem cell can be appropriately selected according to the purpose without restriction, and can be from mammals including humans, preferably adult cells from all known tissues, cells, etc. of humans, such as bone marrow, umbilical cord blood, placenta (or placental tissue cells), fat (or adipose tissue cells), etc. For example, the stem cell can be a stem cell obtained from bone marrow, adipose tissue, muscle tissue, self-tissue mesenchymal stem cells cultured in vitro, allogeneic mesenchymal stem cells, umbilical cord blood, yolk sac, placenta, umbilical cord, periosteum, fetal and adolescent skin, and blood, but is not limited thereto, and can be a stem cell from a fetus or soon after birth or an adult.

[0060] In a specific example of the present invention, the stem cell is selected from the group consisting of endothelial progenitor cells, angiogenic stem cells, mesenchymal stem cells, embryonic stem cells, myoblasts and cardiac stem cells, more preferably endothelial progenitor cells or angiogenic stem cells, and most preferably endothelial progenitor cells.

[0061] In the present invention, "senescence" refers to the cessation or intentional delay of cell growth and cell division by stem cells in response to various internal or external stresses (e.g., high oxygen concentration conditions during continuous subculture and in vitro culture).

[0062] In the present invention, "proliferation" refers to an increase in the number of a cell type from an initial cell population of a characteristic cell type or cells that may be the same or different. The initial cells used for proliferation may be different from the cells generated during proliferation.

[0063] In the present invention, "differentiation induction" includes not only the case of inducing complete differentiation from stem cells to specific cells, but also the formation of embryonic bodies achieved as an intermediate step before stem cells are completely differentiated into specific cells.

[0064] In embodiments of the present invention, the differentiation may be angiogenesis.

[0065] In the present invention, "angiogenesis" refers to the process of neovascularization, that is, the generation and differentiation of new blood vessels in cells, tissues or organs.

[0066] The angiogenesis of the present invention includes vascular regeneration, vascular restoration, and vascular differentiation associated with the angiogenesis process, such as endothelial cell initiation, migration, proliferation, matrix remodeling, and cell stabilization.

[0067] Neoangiogenesis is essential for the recovery of ischemic tissue caused by vascular injury. Since neoangiogenesis and restoration are not sufficient solely through the proliferation of existing vascular endothelial cells, the most important step in the angiogenesis process is to mobilize bone marrow-derived vascular stem cells to the ischemic area and contribute to vascular repair.

[0068] According to the present invention, the composition enhances the mobilization of vascular stem cells from bone marrow to ischemic tissues, thereby improving the ability of stem cells to bind to vascular endothelial cells and the ability of stem cells to differentiate into blood vessels.

[0069] In the present invention, "fucoidan" refers to a polysaccharide composed of the basic sugar fucose and sulfate groups, found in large quantities in brown algae. Fucoidan is known to have the following effects: blood coagulation prevention, anti-tumor effects, gastric ulcer treatment promotion, antibacterial effects, blood pressure suppression, induction of hepatocyte growth factor (HGF) production, blood sugar suppression, immune cell regulation, anti-allergic effects, and antiviral effects.

[0070] In a specific example of the present invention, fucoidan is preferably represented by the following Chemical Formula 1, but is not limited thereto.

[0071] [Chemical Formula 1]

[0072]

[0073] In a specific example of the present invention, the concentration of fucoidan in the composition according to the present invention ranges from 1 nM to 300 nM, more preferably from 50 nM to 250 nM, and most preferably is 150 nM.

[0074] In the present invention, the term "tauroursodeoxycholic acid (TUDCA)" refers to a taurine conjugate of ursodeoxycholic acid (UDCA), which is known to be beneficial for treating gallstones, liver cirrhosis, Huntington's disease, Parkinson's disease, cerebral stroke, and the like.

[0075] In a specific example of the present invention, tauroursodeoxycholic acid is preferably represented by the following Chemical Formula 2, but is not limited thereto.

[0076] [Chemical Formula 2]

[0077]

[0078] In a specific example of the present invention, the composition according to the present invention comprises tauroursodeoxycholic acid in a concentration range of 2.5 μM to 250 μM, preferably in a concentration range of 12.5 μM to 50 μM, more preferably in a concentration range of 17.5 μM to 35 μM, and most preferably in a concentration of 25 μM.

[0079] In the present invention, "oleuropein" refers to a polyphenol present in olive fruits and olive leaves.

[0080] In a specific example of the present invention, oleuropein is preferably represented by the following Chemical Formula 3, but is not limited thereto.

[0081] [Chemical Formula 3]

[0082]

[0083] In a specific embodiment of the present invention, the composition according to the present invention comprises oleuropein in a concentration of 0.05 μM to 5 μM, preferably in a concentration range of 0.25 μM to 1 μM, more preferably in a concentration range of 0.35 μM to 0.7 μM, and most preferably in a concentration of 0.5 μM.

[0084] In the present invention, "vascular endothelial growth factor (VEGF)" refers to a signaling protein produced by cells that stimulates angiogenesis. The VEGF is involved in vasculogenesis and angiogenesis.

[0085] In a specific example of the present invention, the vascular endothelial growth factor is preferably at least one selected from the group consisting of VEGF-A, VEGF-B, VEGF-C and PIGF, but is not limited thereto.

[0086] In a specific example of the present invention, the composition according to the present invention comprises vascular endothelial growth factor at a concentration of 0.1 nM to 10 nM, preferably at a concentration ranging from 1 nM to 5 nM, and most preferably at a concentration of 2.5 nM.

[0087] According to another example of the present invention, the present invention provides a pharmaceutical composition for preventing or treating ischemic diseases, wherein the pharmaceutical composition comprises fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor as active ingredients.

[0088] Furthermore, the present invention provides a method for preventing or treating an ischemic disease, comprising the step of treating an individual with the pharmaceutical composition.

[0089] In addition, the present invention provides use of the pharmaceutical composition for preventing or treating ischemic diseases.

[0090] In the present invention, " ischemic disease " refers to the disease that the blood supply to body organs, tissues or parts is reduced and is caused by the contraction or occlusion of blood vessels. After the tissue or part ischemic, even if the reperfusion of blood occurs, various sequelae will be caused by the damage of nerve cells, eventually leading to irreversible damage (that is, necrosis of cells and tissues). The ischemic disease can be selected from the group consisting of ischemic heart disease, ischemic myocardial infarction, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, ischemic alopecia, ischemic stroke and ischemic lower limb disease, more preferably, can be selected from the group consisting of ischemic heart disease, ischemic myocardial infarction, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic stroke and ischemic lower limb disease, most preferably, can be ischemic myocardial infarction or ischemic lower limb disease.

[0091] According to another example of the present invention, the present invention provides a culture medium composition for stem cell culture, wherein the culture medium composition for stem cell culture comprises fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor as active ingredients.

[0092] In the present invention, "media" refers to a culture medium that can support the growth and survival of stem cells under in vitro culture conditions, including all common culture media used in the art that are suitable for the culture of stem cells. In addition, the culture medium and culture conditions can be selected according to the cell type. The culture medium used for culture is preferably a cell culture minimum medium (CCMM), and generally contains a carbon source, a nitrogen source, and trace elements. Such cell culture minimum medium includes, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI1640, F-10, F-12, Glasgow's Minimal Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium, etc., but is not necessarily limited to these.

[0093] In a specific example of the present invention, preferably, the culture medium composition of the present invention is a xeno-free culture medium excluding cytokines, growth factors, or animal serum (FBS) added to existing culture media, but is not limited thereto.

[0094] In addition, the present invention provides a xeno-free culture method, which includes the step of treating stem cells with a stem cell culture medium composition containing fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor as active ingredients.

[0095] According to another example of the present invention, the present invention provides stem cells cultured by the culture method.

[0096] In a specific example of the present invention, the stem cell is selected from the group consisting of endothelial progenitor cells, angiogenic stem cells, mesenchymal stem cells, embryonic stem cells, myoblasts and cardiac stem cells, more preferably endothelial progenitor cells or angiogenic stem cells, and most preferably endothelial progenitor cells.

[0097] Stem cells cultured using the xeno-free culture method according to the present invention not only acquire the effect of undifferentiated characteristics (stemness), but also have the effects of generating blood vessels, improving cell proliferation and migration abilities. Therefore, after endothelial progenitor cells are transplanted into the body, cell survival and implantation survival rates can be increased, and the ability of blood vessels and tissue regeneration can be further improved.

[0098] Therefore, according to another example of the present invention, the present invention provides a pharmaceutical composition comprising the stem cells for preventing or treating ischemic diseases.

[0099] Furthermore, the present invention provides a method for preventing or treating an ischemic disease, comprising the step of treating an individual with the stem cells.

[0100] In addition, the present invention provides use of the stem cells for preventing or treating ischemic diseases.

[0101] In the present invention, "subject" refers to a subject in need of disease treatment, and more specifically, refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cows.

[0102] The pharmaceutical composition of the present invention can be formulated and used in various dosage forms according to conventional methods. For example, it can be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can also be formulated into external preparations, suppositories, and sterile injectable solutions.

[0103] The composition of the present invention may contain, together with the above-mentioned active ingredient, one or more known active ingredients having a preventive or therapeutic effect on ischemic diseases.

[0104] The composition of the present invention may further include pharmaceutically acceptable additives. In this case, the following ingredients can be used as pharmaceutically acceptable additives: starch, gelatinized starch, microcrystalline cellulose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium carboxymethyl starch, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, etc. Preferably, the pharmaceutically acceptable additive according to the present invention is included in an amount of 0.1 to 90 parts by weight relative to the composition, but is not limited thereto.

[0105] The composition of the present invention can be administered in various oral or parenteral dosage forms during actual clinical administration. When formulated, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be used for the formulation. Suitable formulations known in the art are preferably those disclosed in the literature of Leamington.

[0106] The solid preparation for oral administration includes tablets, pills, powders, granules, capsules, etc., and this solid preparation mixes at least one excipient (for example starch, calcium carbonate (Calcium car bonate), sucrose (Sucrose) or lactose (lactose), gelatin, etc.) to prepare. In addition, except simple excipient, lubricant (such as magnesium stearate, talc) is also used. In addition, the liquid preparation for oral administration includes suspending agents, solutions, emulsions, syrups, etc., and except water, liquid paraffin of commonly used simple diluent, various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. can also be included.

[0107] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, and injectable esters such as ethyl oleate, etc., can be used as suspending solvents. Synthetic fatty acid esters (witepsol), polyethylene glycol, Tween 61, cocoa butter, glyceryl laurate, glyceryl gelatin, etc., can be used as suppository preparations.

[0108] The fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor of the present invention can be used in the form of pharmaceutically acceptable salts, and include all salts, hydrates and solvates prepared by conventional methods.

[0109] As salts, acid addition salts formed from pharmaceutically acceptable free acids are more useful. Acid addition salts are prepared by conventional methods, for example, by dissolving the compound in an excess of an acidic solution and precipitating the salt using a miscible organic solvent (e.g., methanol, ethanol, acetone, or acetonitrile). Equimolar amounts of the compound and an acid or alcohol (e.g., ethylene glycol monomethyl ether) in water can be heated, and the mixture can then be evaporated and dried, or the precipitated salt can be filtered. At this time, organic acids and inorganic acids can be used as free acids. Hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, etc. can be used as inorganic acids. Methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc. can be used as organic acids, but are not limited thereto.

[0110] In addition, a basic group can be used to prepare a pharmaceutically acceptable metal salt. Alkali metal salts or alkaline earth metal salts can be obtained, for example, by dissolving the compound in an excess of an alkali metal hydroxide solution or an alkaline earth metal hydroxide solution, then filtering the insoluble compound salt, and evaporating and drying the filtrate. In this case, among the metal salts, the preparation of sodium salts, potassium salts or calcium salts is particularly pharmaceutically acceptable, but is not limited thereto. In addition, corresponding silver salts can be obtained by reacting an alkali metal salt or an alkaline earth metal salt with an appropriate amount of a silver salt (e.g., silver nitrate).

[0111] Unless otherwise indicated, pharmaceutically acceptable salts of the compounds include salts of acidic groups or basic groups that may be present in the compounds. For example, pharmaceutically acceptable salts may include sodium, calcium, and potassium salts of hydroxyl groups, and other pharmaceutically acceptable salts of amino groups may include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate), and p-toluenesulfonate (toluenesulfonate), and the like, and may be prepared by methods for preparing salts known in the art.

[0112] The dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time and / or administration route of the pharmaceutical composition, and may vary according to various factors including the type and degree of the reaction achieved by the administration of the pharmaceutical composition, the type of individual serving as the administration subject, age, weight, general health status, symptoms or degree of the disease, gender, diet, excretion, the components of other compositions of the drug used simultaneously or at different times for the corresponding individual, and similar factors known in the medical field. A technician with ordinary knowledge in this technical field can easily determine the dosage effective for the target treatment and prescribe it.

[0113] The administration route and administration method of the pharmaceutical composition of the present invention can be independent of each other and are not particularly limited. As long as the pharmaceutical composition can reach the corresponding part of the target, it can be administered according to any administration route and administration method.

[0114] The pharmaceutical composition can be administered orally or non-orally, including intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration.

[0115] The pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiotherapy, hormone therapy, chemotherapy, and the use of biological response modifiers for the prevention or treatment of ischemic diseases.

[0116] According to another example of the present invention, the present invention provides a stem cell therapy adjuvant, which contains fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor as active ingredients.

[0117] In the present invention, "cell therapy agents" refer to pharmaceuticals used for therapeutic, diagnostic, or preventive purposes to restore the functions of cells and tissues by proliferating and screening surviving autologous, allogenic, or xenogenic cells in vitro, or by altering the biological characteristics of cells through other methods. Specifically, they can be divided into "stem cell therapy agents," embryonic stem cell therapy agents, and adult stem cell therapy agents.

[0118] In the present invention, a "stem cell therapy adjuvant" refers to an agent that can be used to enhance the efficacy of stem cell therapeutics commonly used in the art. The compositions of the present invention can be used to inhibit aging and promote proliferation of stem cells in stem cell therapeutics, thereby improving the therapeutic effect. In a preferred embodiment of the present invention, the stem cell therapy is for the treatment of ischemic diseases.

[0119] As long as the stem cell therapy adjuvant can reach the target tissue, it can be administered to the human body through any conventional route.

[0120] Parenteral administration may be, for example, intraperitoneal administration, intravenous administration, intramuscular administration, or subcutaneous administration, but is not limited thereto.

[0121] The stem cell therapy adjuvant can also be administered via any device capable of transferring active substances to target cells and can be administered together with a pharmaceutical carrier commonly used in stem cell therapy, such as physiological saline.

[0122] Hereinafter, the present invention will be described in more detail by way of examples. However, it should be understood that these examples are merely examples of the present invention, and the scope of the present invention is not limited by these examples, which should be apparent to those skilled in the art.

[0123] [Example]

[0124] Example 1. Preparation of mixture 4F

[0125] Fucoidan (150 nM), tauroursodeoxycholic acid (25 μM), oleuropein (0.5 μM), and vascular endothelial growth factor (2.5 nM) (recombinant human VEGF165) were mixed to prepare mixture 4F. In the examples described below, b-FGF, IGF, EGF, and ascorbic acid added to the existing culture medium (EndoGo XF medium, Biological Industries) were not used, and only 4F was added to the basal medium for the experiments.

[0126] Example 2. Confirmation of morphological characteristics of endothelial progenitor cells treated with mixture 4F

[0127] To investigate the effects of 4F treatment on cell phenotypes, major cell markers were roughly divided into undifferentiated markers (stem cell markers) and endothelial lineage markers (endothelial lineage markers), and analyzed using flow cytometry. Specifically, a mixture of 4F (fucoidan, tauroursodeoxycholic acid (TUDCA), oleuropein, and vascular endothelial growth factor (VEGF)) was added to basal medium (EBM2, Lonza) for culturing vascular endothelial cells along with 5% FBS and 1% penicillin / streptomycin. The cells were then treated for various periods of time, and the expression of undifferentiated markers (CD34, CD90, c-kit, CXCR4) and endothelial lineage markers (Tie2, CD31) was analyzed using flow cytometry. To verify the reproducibility of the results, L-EPCs were isolated and cultured from umbilical cord blood from five pregnant women (n=5) to confirm the expression of each marker. Figure 1 The results of the confirmation based on the morphological characteristics of endothelial progenitor cells treated with the mixture 4F are shown.

[0128] like Figure 1 As shown, it was confirmed that the cell population expressing undifferentiated markers and the cell population expressing distinct differentiation markers of the endothelial cell lineage while having undifferentiated properties increased significantly in a manner dependent on the number of days treated with mixture 4F. Thus, it was confirmed that treatment with mixture 4F further acquired the following characteristics of vascular stem cells: while maintaining the endothelial cell differentiation properties inherent to the cells, by increasing only their undifferentiated properties (stemness), they possessed phenotypes and functions similar to those of endothelial cells, and possessed the ability to differentiate into endothelial cells at a higher level.

[0129] Example 3. Confirmation of the migration ability of cells treated with mixture 4F

[0130] CXCR4 is known to regulate the migration ability of stem cells in a manner that depends on the number of days of 4F treatment, and the expression of CXCR4 has been confirmed to increase. Based on this, the cell migration ability, which is an essential process for angiogenesis and regeneration, was analyzed using SDF-1α, a ligand of CXCR4. Specifically, a Transwell migration assay was performed to analyze the cell migration ability caused by chemokine stimulation. Cells (5×10 4An 8.0μm mesh membrane insert containing cells was inserted into a culture plate, and stromal cell-derived factor (SDF-1α) (100ng / ml), a chemokine that stimulates cell migration, was added to the bottom of the insert and cultured. After 6 hours, cells that had escaped from the pores of the mesh membrane due to stimulation with SDF-1α were stained with crystal violet, and the number of migrated cells was counted. SDF-1α is a major cytokine secreted from ischemic tissue and is involved in remodeling cells involved in vascular regeneration and tissue regeneration. Figure 2 Shown are the results of confirmation based on the migration ability of cells treated with mixture 4F.

[0131] like Figure 2 As shown, the migration ability of cells was significantly increased by priming with mixture 4F compared to the control group. In addition, although the negative control group, which was performed without specific stimulation, was observed to have a similar level of migration ability as the positive control group, the migration ability of cells stimulated by stromal cell-derived factor (SDF-1α) treated with mixture 4F was confirmed to have increased by more than three times. These results indicate that priming with mixture 4F enhances the responsiveness of cells to stromal cell-derived factor (SDF-1α) and their ability to migrate specifically to ischemic tissue.

[0132] Example 4. Confirmation of the angiogenesis effect of the mixture 4F according to the present invention in an animal model of lower limb ischemia

[0133] An animal model of lower limb ischemia was prepared as a model of severe vascular disease to evaluate the therapeutic effect of vascular stem cells whose function was enhanced by treatment with mixture 4F. Specifically, a laser Doppler flowmeter was used to analyze the degree of improvement in blood flow after cell transplantation from the perspective of hemodynamics. Specifically, in order to confirm the therapeutic effect of cells triggered by mixture 4F in a severe ischemic animal model, a lower limb ischemia animal model was prepared using nude mice (male) aged 7-8 weeks. The lower limb ischemia animal model was prepared by blocking the blood flow to the lower limbs by femoral artery ligation, and after vascular resection, cells (5×10 5 cells / PBS 50μl), and the degree of recovery of blocked blood flow was analyzed by laser Doppler flowmetry on a daily basis (day 3, day 7, and day 14). In addition, the engraftment and survival of the transplanted cells and the angiogenesis effect were confirmed by histological analysis using live cell imaging. The results are shown in the figure. Figure 3a and 3b .

[0134] like Figure 3a As shown, it was confirmed that in vivo angiogenesis was significantly enhanced when 4F-treated cells were transplanted compared to the control group.

[0135] like Figure 3b As shown, using in vivo imaging technology, the vascular suture site was imaged in a living state 3 days after the transplantation of GFP-labeled cells. The results showed that many transplanted GFP cells could be observed around the sutured artery. + These results suggest that the vascular stem cells whose survival capacity was improved by the treatment with the mixture 4F were remodeled to be used for vascular regeneration.

[0136] Example 5. Confirmation of the effect of enhancing cardiac function by treatment with mixture 4F in a severe myocardial infarction animal model

[0137] The therapeutic efficacy of cells derived from the mixture 4F was confirmed using a severe myocardial infarction animal model. Specifically, a severe myocardial infarction model was established using 7-8 week old nude mice. The severe myocardial infarction model was established by excising the left coronary artery, and 5×10 cells were directly transplanted into each individual. 5 Cells were delivered to the ischemic site of the heart. Echocardiography was used to measure cardiac function 28 days after induction of myocardial infarction, including left ventricular mass and volume (left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), and left ventricular ejection fraction (LVEF). Cardiac output and left ventricular diastolic function were also assessed in the animal model. Figure 4a Results of echocardiographic, cardiac output, and left ventricular diastolic function analyses are shown.

[0138] like Figure 4a As shown, cardiac function evaluation results 28 days after myocardial infarction showed significant increases in ejection fraction (EF) and fractional shortening (FS) in the 4F-treated group compared to the control group. These results suggest that 4F further enhances the function of existing vascular stem cells.

[0139] In addition, 28 days after myocardial infarction, the degree of myocardial fibrosis (lighter color part) was analyzed by Masson's trichrome staining, which can characteristically stain donkey-hide gelatin fibers. The results are shown in the figure. Figure 4b middle.

[0140] like Figure 4b As shown, it was confirmed that the degree of left ventricular fibrosis was significantly reduced by treatment with the mixture 4F.

[0141] Furthermore, 3 days after the transplantation of green fluorescent protein (GFP)-labeled cells into a severe myocardial infarction model, the engraftment of cells in the myocardium and the proliferation rate were confirmed by immunostaining of a cell proliferation marker (i.e., proliferating cell nuclear antigen (PCNA)). The results are shown in the figure. Figure 4c .

[0142] exist Figure 4c In the mixture 4F-treated group, multiple expressions of proliferating cell nuclear antigen (PCNA) merged with green fluorescent protein (GFP) were observed.

[0143] These results suggest that in an animal model of severe myocardial infarction, vascular stem cells primed with the transplanted mixture 4F promote myocardial recovery and cardiac function through proliferation.

[0144] Example 6. Analysis of proliferation ability, cell cycle changes, cell senescence and colony formation ability according to mixture 4F treatment

[0145] To analyze cell proliferation, 10,000 cells were seeded in 96-well plates and treated with the 4F mixture for several days. Cell proliferation was then confirmed by MTS analysis. Furthermore, to confirm protein expression of CDK inhibitors (p21 and p16), cell proliferation regulators, intracellular proteins in the control group were isolated, extracted, and quantified using protein lysis buffer. Western blotting was then performed using equal amounts of protein. Proteins were subjected to SDS-PAGE and transferred to a PVD F membrane, which was blocked in 5% skim milk for 30 minutes. Following blocking, the membrane was reacted with primary antibodies against p53 (abcam), p27 (abcam), p21 (abcam), p16 (abcam), and β-actin (santacruz) at a 1:1000 dilution overnight at 4°C. After washing, the membrane was reacted with an HRP-conjugated secondary antibody for one hour at room temperature, and protein expression was visualized using LAS3000 (Fujifilm). Furthermore, to confirm that the decrease in cell proliferation was due to cell cycle arrest induced by increased undifferentiated characteristics, cell cycle analysis was performed using PI staining and Hst / PY staining. Specifically, cells cultured at 60%-70% confluence were stained with PI (Santa Cruz), Hoechst 33342 (Santa Cruz), and PY (Santa Cruz), and the cell cycle was analyzed by flow cytometry. Figure 5aand 5b The results of the confirmation based on the changes in the proliferation rate and cell cycle of cells treated with the mixture 4F are shown.

[0146] like Figure 5a and 5b As shown, it was confirmed that the cell proliferation ability was significantly reduced in a manner dependent on the number of days of mixture 4F treatment. In addition, it was confirmed that in the cells treated with mixture 4F, the protein expression of CDK inhibitors (p21, p16) as cell proliferation regulatory factors significantly increased. In addition, according to the cell cycle analysis results, it was confirmed that when treated with mixture 4F (4 days), compared to a control group, the S group and the G2 / M group where DNA replication and cytoplasmic division occur significantly decreased, but the G0 / G1 group significantly increased. Not only that, it was also confirmed that compared to a control group, when treated with mixture 4F (4 days), the cell population of the G0 group in a dormant state significantly increased.

[0147] The degree of senescence of cells that had arrested the cell cycle and cells that had become senescent through repeated passages (old) was analyzed. Cell senescence analysis was performed using the SA-β-gal detection kit (Cell Signaling) according to the manufacturer's manual. The number of senescent cells (β-positive - green) in each group of cells was measured by microscopic imaging and graphed. The results are shown in the figure. Figure 5c .

[0148] like Figure 5c As shown, it was confirmed that the cell cycle arrest caused by the mixture 4F treatment was not caused by cell damage or senescence, but was caused by the increase of undifferentiated characteristics.

[0149] In addition, to confirm the colony-forming ability of the cells, the cells were seeded on 96-well plates coated with 1% gelatin and cultured for 14 to 20 days. The number of wells in which the cultured cells formed colonies was measured by microscopic imaging and graphed. Figure 5d .

[0150] like Figure 5d As shown, the results of the analysis of the colony-forming ability of single cells by analyzing single cell colonies confirmed that vascular stem cells fell into a dormant state due to induction with the mixture 4F, and their undifferentiated properties as stem cells were greatly increased.

[0151] Example 7. Analysis of stem cell differentiation gene expression after treatment with mixture 4F

[0152] The expression of genes related to undifferentiation or differentiation in vascular endothelial cells treated with mixture 4F was measured. Specifically, total RNA was extracted from vascular endothelial cells initiated with mixture 4F. 2 μg of total RNA was prepared and RNA sequencing was performed by Teragen. Differentially expressed genes (DEG) analysis was performed based on the Cuffdiff method using a q-value threshold of less than 0.05. The results of the analysis of gene expression related to stem cell differentiation are shown in the figure. Figure 6a and 6b .

[0153] like Figure 6a and 6b As shown, transcriptome analysis by RNA sequencing confirmed that cells primed with the 4F mixture exhibited significant genetic differences from fully differentiated HUVECs. Furthermore, expression of some undifferentiated stem cell markers (CD34, KIT, HEY1, and PODXL, among others) was significantly increased compared to those in OECs (outgrowing ECs, L-EPCs). Expression levels of genes specific to differentiated HUVECs were also similar to or slightly decreased compared to those in OECs (outgrowing ECs, L-EPCs). These results suggest that cells primed with the 4F mixture acquire immature characteristics.

[0154] Example 8. Analysis of apoptosis rate of undifferentiated stem cells treated with mixture 4F

[0155] Undifferentiated stem cells have a higher survival ability due to their ability to defend against pathological physiological environments such as ischemic stimulation, and thus have a higher survival rate after transplantation and implantation than differentiated cells. To confirm this, the apoptosis rate of cells was analyzed by realizing an ischemic state (nutrient-free and hypoxic state) in vitro. The apoptosis rate analysis was performed by culturing the cell group under 1% O2 hypoxic conditions for 24 hours in a nutrient-free medium from which FBS, growth factors, and cytokines were removed, thereby realizing the in vivo ischemic state in vitro. Subsequently, flow cytometric analysis was performed using the Annexin V / PI staining kit (BD Biosciences), and the results are shown in the figure. Figure 7 .

[0156] like Figure 7 As shown, it was confirmed that the viable cell population of cells primed with mixture 4F significantly increased compared to the control group. These results suggest that treatment with mixture 4F increases the undifferentiated characteristics of cells, which means that it enhances the defense mechanism against ischemic stimulation and thus contributes to improved cell viability.

[0157] Example 9. Proliferation Rate and Marker Expression Analysis of Undifferentiated Stem Cells Treated with Mixture 4F

[0158] The cell proliferation ability and the recovery of marker expression when the cells whose cell cycle was arrested by the treatment of mixture 4F were reperfused to the nutrient medium (adding b-FGF, EGF, IGF-1, ascorbic acid) for existing cell culture were analyzed. Specifically, the cells whose cell cycle was arrested by treatment with mixture 4F for 4 days were cultured (reperfusion) under the conditions of nutrient medium (adding b-FGF, EGF, IGF-1, ascorbic acid) for 5 days. On the 3rd and 5th days after changing to nutrient medium, the recovery of cell proliferation ability was confirmed using MTS analysis kit. MTS analysis was carried out according to the instructions of the manufacturer's manual. The cell proliferation rate is shown in FIG. Figure 8a The results of marker expression analysis are shown in Figure 8b and Figure 8c .

[0159] like Figures 8a to 8c As shown, it was confirmed that cells primed with Mixture 4F not only recovered their proliferative capacity, which had been reduced by reperfusion of the nutrient medium, but also that marker expression returned to levels similar to those of the control group. These results confirm that the cell cycle arrest and the resulting reduction in cell proliferative capacity caused by treatment with Mixture 4F are not irreversible phenomena such as cell damage or senescence, but are rather a general characteristic of resting cells that occurs along with an increase in the undifferentiated nature of the cells, and are reversible phenomena that can be fully restored with the necessary stimulation.

[0160] Example 10. Comparison of marker expression and angiogenesis ability according to mixture 4F or single component treatment

[0161] The marker expression and angiogenesis ability of cells treated with the mixture 4F or the individual components (fucoidan, tauroursodeoxycholic acid, oleuropein, and vascular endothelial growth factor) were compared. The experimental group was treated with the mixture 4F, while the control group used an untreated group (con), a fucoidan-treated group (Fu, 0.1 μg / ml), a tauroursodeoxycholic acid-treated group (TD, 25 μM), an oleuropein-treated group (OLP, 0.5 μM), and a vascular endothelial growth factor-treated group (VEGF, 100 ng / ml).

[0162] First, the expression of markers of cells primed with the mixture 4F, namely CD90, c-kit, CXCR4, Tie2 and CD144, was analyzed by the method of Example 1. The results are shown in the graphs: Figures 9a to 9e .

[0163] like Figures 9a to 9e As shown, compared with the single treatment conditions of each component, the mixture 4F treatment group had a greater increase in the cell population expressing undifferentiation markers (CD34, CD90, c-kit, CXCR4).

[0164] To analyze the angiogenesis ability, 60 μl of Matrigel GFR (BD Biosciences) was added to a 96-well plate for solidification, and then 10,000 cells were seeded and cultured. After 6 hours of culture, the degree of tubular network formation was observed under a microscope. The angiogenesis ability of the cells was plotted using image processing software (Image J) by counting the number of branches observed under the microscope. The results are shown in the figure. Figure 10 .

[0165] like Figure 10 As shown, it was confirmed that the angiogenesis ability of the mixture 4F-treated group was significantly increased compared to the single-component treatment condition.

[0166] The above results indicate that treatment with mixture 4F significantly increased the undifferentiated characteristics and angiogenesis ability of cells compared to treatment with a single component.

[0167] Example 11. Xeno-free culture method using mixture 4F

[0168] Figure 11 This is a diagram briefly showing the xeno-free culture method using mixture 4F. Specifically, for the isolation and culture of endothelial progenitor cells (EPCs), mononuclear cells (MNCs) were isolated and obtained by obtaining umbilical cord blood (IRB approval number 05-2017-053, 50cc or more) from pregnant women visiting the Department of Obstetrics and Gynecology at Pusan ​​National University Hospital in Yangsan, South Korea, using density difference centrifugation using Ficoll. After lysis of the red blood cells, the cells were inoculated (7×10 6 cells / well) on a plate coated with fibronectin (1 μg / cm 2) in a 6-well plate and cultured for 5 days under the conditions of each culture medium (a xeno-free culture medium containing mixture 4F or a known existing vascular endothelial cell differentiation medium (EGM2 medium)) at 37°C and 5% CO2. The xeno-free culture medium containing the mixture 4F is a culture medium that excludes cytokines or growth factors and animal serum (FBS) added to the existing culture medium and adds only mixture 4F and human serum (2%) to the basal culture medium. After cultivation, the culture medium is replaced every day to confirm the attachment of the EPC colony, and subculture is performed when the confluence is more than 70%. For the cultured cells, the cells obtained by culturing in the existing vascular endothelial cell differentiation medium will be named "L-EPC" according to the type of culture medium, and the cells obtained by culturing in the xeno-free culture medium containing mixture 4F will be named "X-EPC".

[0169] Example 12. Confirmation of the morphological characteristics of vascular stem cells cultured by the xeno-free culture method using mixture 4F

[0170] In the morphological comparison of the cells, the morphological characteristics of the cells were confirmed by microscopic imaging at a confluence of 70% to 80%. Specifically, in the Ac-LDL absorption and UEA-1 binding analysis as an endothelial cell line-specific functional assessment, cells cultured at a confluence of 10%-20% were cultured in a serum starvation state for 2 hours. The cultured cells were treated with Ac-LDL (Invitrogen) at a concentration of 10 μg / ml and then reacted at 37°C for 4 hours. The reacted cells were fixed with 4% PFA at room temperature for 10-15 minutes and reacted with UEA-1 (SigmaAldrich) at a concentration of 10 μg / ml for 1 hour at room temperature. The reacted cells were washed with phosphate-buffered saline (PBS), stained with DAPI, and observed with a fluorescence microscope. Figure 12 Shown are the results of analyzing the morphological characteristics of vascular stem cells cultured by a xeno-free culture method using mixture 4F.

[0171] Figure 12 It was confirmed that cells cultured using the xeno-free culture method using mixture 4F (X-EPCs) had the same expression profile as cells cultured using conventional culture methods (E-EPCs, L-EPCs). Furthermore, it was confirmed that the xeno-free culture method using mixture 4F did not cause specific morphological changes in the cells.

[0172] Example 13. Analysis of Gene Expression of Undifferentiated Stem Cell Markers in Vascular Stem Cells Cultured by the Xeno-free Culture Method Using Mixture 4F

[0173] The expression of hematopoietic markers (CD11b, CD14, CD45), undifferentiated markers (CD34, c-kit, CXCR4), and endothelial cell markers (VEGFR2, PECAM, VE-cadherin) in the cells cultured in Example 11 was analyzed. Specifically, the cells cultured in Example 11 were reacted with trypsin-EDTA at 1:100 and 4°C for 30 minutes and peeled off from the culture plate. The expression of genes related to undifferentiated stem cell markers in the cells was analyzed by flow cytometry, and the results are shown in the figure. Figure 13 .

[0174] like Figure 13 As shown, it was confirmed that the cells (X-EPC) cultured using the xeno-free culture method using the mixture 4F showed little expression of hematopoietic markers (CD11b, CD14, CD45) compared to the cell group (E-EPC, L-EPC, HUVEC) obtained using the conventional culture method, and that the expression of undifferentiated markers and vascular endothelial cell markers remained high (three lots were secured for each umbilical cord blood donor, and even after continuous subculture, the expression of representative markers CD34, CXCR4, and VE-cadherin remained higher than that of L-EPC). Thus, it was confirmed that the cells (X-EPC) obtained using the xeno-free culture method using the mixture 4F are vascular stem cells with higher undifferentiated properties than cells cultured using the conventional culture method.

[0175] Example 14. Analysis of the Proliferation Ability, Cell Cycle Changes, and Colony Formation Ability of Vascular Stem Cells Cultured by the Xeno-free Culture Method Using Mixture 4F

[0176] The cells cultured by the xeno-free culture method using the mixture 4F according to the method of Example 11 were separated into single cells, and the proliferation ability, cell cycle changes and colony formation ability were analyzed by the same method as in Example 6. The control group of this example used cells cultured by the existing culture method (L-EPC) and cells cultured in a xeno-free culture medium containing each single component [fucoidan treatment group (Fu, 0.1 μg / ml), tauroursodeoxycholic acid treatment group (TD, 25 μM), oleuropein treatment group (OLP, 0.5 μM) and vascular endothelial growth factor treatment group (VEGF, 100 ng / ml)]. The results of the analysis of colony formation ability are shown in the figure. Figure 14a and 14b .

[0177] like Figure 14a and 14bAs shown, cells cultured using the xeno-free culture method using mixture 4F demonstrated excellent colony-forming ability and a short division time. However, no EPC colonies formed in the control group treated with the single components. While EPC colonies were observed in cells treated with vascular endothelial growth factor, VE-cadherin expression was low and cell division time was prolonged, impairing cell proliferation.

[0178] Example 15. Analysis of Gene Expression Related to Stem Cell Differentiation in Vascular Stem Cells Cultured by the Xeno-Free Culture Method Using Mixture 4F

[0179] The expression of genes related to stem cell differentiation in cells cultured using the xeno-free culture method using mixture 4F according to the method of Example 11 was analyzed using the method of Example 7. Gene ontology, category, and expression pattern were analyzed based on RNA sequencing results. The analysis results of gene expression related to stem cell differentiation are shown in the figure. Figure 15 to Figure 1 7.

[0180] like Figure 15 As shown, cells cultured by a xeno-free culture method using mixture 4F (X-EPC) were genetically different from L-EPCs obtained by conventional culture methods, and the results of PCA analysis of the correlation between each cell group confirmed that, unlike HUVEC / L-EPC, which showed high similarity, X-EPCs had low similarity with each cell group.

[0181] like Figure 16 As shown in the results, gene ontology and category analysis were performed on genes with expression differences of more than 2-fold in X-EPCs compared to L-EPCs. The results confirmed that the expression levels of genes related to endothelial cell activation, angiogenesis, and endothelial cell migration were significantly changed.

[0182] like Figure 17a and 17b As shown, it was confirmed that X-EPCs significantly increased the expression of some undifferentiated stem cell-related genes (CD34, KIT, HEY1, PODXL, etc.).

[0183] Example 16. Confirmation of the Angiogenic Ability of Vascular Stem Cells Cultured Using the Xeno-Free Culture Method of Mixture 4F

[0184] The angiogenesis ability of the cells cultured using the xenogeneic culture method of mixture 4F in the method of Example 11 was confirmed by the method of Example 10. Furthermore, the 3D microsphere (beads) sprouting analysis was to culture the culture microspheres and each cell for 4 hours, and then coat the microspheres with cells. 100 to 200 cell-coated microspheres were inoculated in the cell-coated culture fibrinogen / thrombin gel and cultured at 37°C. The degree of angiogenesis sprouting of the microspheres was observed during the culture process. The results of the 2D and 3D angiogenesis ability analysis are shown in the figure. Figure 18 .

[0185] like Figure 18 As shown, it was confirmed that cells cultured by the xeno-free culture method using the mixture 4F had improved blood vessel formation ability for a longer period of time compared to L-EPC and HUVEC.

[0186] Example 17: Confirmation of the effect of mixture 4F on peripheral secretory angiogenesis of vascular stem cells cultured using a xeno-free culture method

[0187] According to the method of Example 11, the effect of peripheral secretion of cells cultured by the non-xenogeneic culture method using mixture 4F on angiogenesis was analyzed. Specifically, total RNA was extracted from the cells of Example 11 using Trizol reagent, and cDNA was synthesized based on the extracted total RNA. In addition, real-time fluorescence quantitative polynucleotide chain reaction (qPCR) using the synthesized cDNA as a template was performed to analyze the mRNA expression of major angiogenesis stimulating factors (VEGF-A, IL-8, b-FGF, Ang2). The primers used for polymerase chain reaction (PCR) are shown in Table 1, and the real-time fluorescence quantitative polynucleotide chain reaction (qPCR) results are shown in Table 1. Figure 19 .

[0188] [Table 1]

[0189]

[0190] like Figure 19 As shown, it was confirmed that cells cultured by the xeno-free culture method using the mixture 4F (X-EPC) showed significantly higher expression of major growth factors and cytokines that stimulate angiogenesis compared to the control group (L-EPC, E-EPC, HUVEC).

[0191] Example 18: Confirmation of the angiogenesis effect of vascular stem cells cultured using the xeno-free culture method of mixture 4F in a lower limb ischemia animal model

[0192] The angiogenesis effect of cells cultured in a xeno-free culture method using mixture 4F according to the method of Example 11 was confirmed by the same method as in Example 4. The results are shown in FIG. Figure 20 and Figure 21 .

[0193] like Figure 20 and Figure 21 As shown in the results, it was confirmed that the cells (X-EPC) cultured by the xeno-free culture method using the mixture 4F were transplanted into the lower limb ischemia animal model, and the in vivo angiogenesis was significantly enhanced compared with the control group. In addition, in the lower limb ischemia animal model transplanted with cells (X-EPC) cultured by the xeno-free culture method using the mixture 4F, many transplanted GFP cells were observed around the sutured arterial vessels. + cells, and confirmed GFP + The cells enter existing blood vessels. In the L-EPC-transplanted hind limb ischemia animal model, only cell debris was observed 7 days after transplantation. In contrast, in the X-EPC-transplanted hind limb ischemia animal model, GFP signals of living cells were detected 21 days after transplantation. The detection of GFP signals indicates improved cell engraftment and survival rates. Furthermore, more CD31 (red: mouse vessels)-positive blood vessels were confirmed in the X-EPC-transplanted hind limb ischemia animal model compared to the L-EPC-transplanted group. These results indicate that X-EPCs have excellent in vivo angiogenesis effects.

[0194] In summary, the present inventors have discovered that when endothelial progenitor cells are treated with 4F, a mixture containing fucoidan, tauroursodeoxycholic acid, oleuropein and vascular endothelial growth factor, the endothelial progenitor cells not only acquire undifferentiated characteristics (stemness), but also have the effects of generating blood vessels, improving cell proliferation ability and migration ability. Therefore, after the endothelial progenitor cells are transplanted into the body, the cell survival and implantation survival rate can be improved, and the angiogenesis ability and tissue regeneration ability can be further improved, so that they can be applied in various ways to the field of stem cell differentiation, prevention or treatment of ischemic diseases.

[0195] The present invention will be described in more detail below by means of formulation examples, which are merely provided to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0196] Preparation Example 1. Preparation of pharmaceutical composition

[0197] 1-1. Preparation of powder

[0198] Mixture 4F 20 mg

[0199] Lactose 100mg

[0200] Talc 10mg

[0201] The above ingredients were mixed and filled into sealed bags to prepare powder.

[0202] 1-2. Preparation of Tablets

[0203] Mixture 4F 10 mg

[0204] Corn starch 100mg

[0205] Lactose 100mg

[0206] Magnesium stearate 2mg

[0207] After mixing the above ingredients, tablets are prepared by compression according to a conventional tablet preparation method.

[0208] 1-3. Preparation of capsules

[0209] Mixture 4F 10 mg

[0210] 3mg crystalline cellulose

[0211] Lactose 14.8mg

[0212] Magnesium stearate 0.2mg

[0213] The above ingredients are mixed according to a common capsule preparation method and filled into gelatin capsules to prepare capsules.

[0214] 1-4. Preparation of Injections

[0215] Mixture 4F 10 mg

[0216] Mannitol 180mg

[0217] Sterile distilled water for injection 2974mg

[0218] Sodium dihydrogen phosphate dihydrate (Na2HPO42H2O) 26 mg

[0219] The injection is prepared according to a common method for preparing injections so that the above-mentioned components are contained in one ampoule (2 ml).

[0220] 1-5. Preparation of liquid preparation

[0221] Mixture 4F 20 mg

[0222] Isomerized sugar 10g

[0223] Mannitol 5g

[0224] Appropriate amount of purified water

[0225] According to the conventional method for preparing a liquid preparation, each component is added to purified water and dissolved, an appropriate amount of lemon fragrance is added, the above components are mixed, and then purified water is added. After the whole is added with purified water and the total volume is adjusted to 100 ml, it is filled in a brown bottle and sterilized to prepare a liquid.

[0226] Although the characteristic parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such detailed description is only a preferred implementation example and the scope of the present invention is not limited thereto.

[0227] Industrial applicability

[0228] According to the present invention, stem cells treated with the mixture 4F not only acquire undifferentiated characteristics (stemness) but also have the effect of improving cell proliferation and migration ability. Therefore, by transplanting the stem cells into the body as a therapeutic agent for ischemic diseases, cell survival and engraftment, as well as the regeneration ability of blood vessels and tissues can be improved.

[0229] Stem cells can be cultured using a xeno-free culture method utilizing the mixture 4F. The stem cells cultured using this xeno-free culture method have higher undifferentiated properties, colony-forming ability, and proliferation ability than cells cultured using existing culture methods, and have higher expression of major growth factors and cytokines for stimulating angiogenesis. Furthermore, the mixture 4F and the stem cells can improve cell survival and engraftment in vivo, as well as the regenerative capacity of blood vessels and tissues. Therefore, the mixture 4F and the stem cells according to the present invention can be effectively used in basic research and clinical fields for the development of therapeutic agents for ischemic diseases.

Claims

1. A composition for inhibiting senescence, promoting proliferation or inducing differentiation of vascular endothelial progenitor cells, comprising fucoidan at a concentration of 1 nM to 300 nM, tauroursodeoxycholic acid at a concentration of 17.5 μM to 35 μM, oleuropein at a concentration of 0.35 μM to 0.7 μM, and vascular endothelial growth factor-165 at a concentration of 0.1 nM to 5 nM as active ingredients.

2. The composition according to claim 1, wherein The differentiation is angiogenesis.

3. A pharmaceutical composition for preventing or treating ischemic diseases, comprising as active ingredients fucoidan at a concentration of 1 nM to 300 nM, tauroursodeoxycholic acid at a concentration of 17.5 μM to 35 μM, oleuropein at a concentration of 0.35 μM to 0.7 μM, and vascular endothelial growth factor-165 at a concentration of 0.1 nM to 5 nM.

4. A culture medium composition for culturing endothelial progenitor cells, comprising as active ingredients fucoidan at a concentration of 1 nM to 300 nM, tauroursodeoxycholic acid at a concentration of 17.5 μM to 35 μM, oleuropein at a concentration of 0.35 μM to 0.7 μM, and vascular endothelial growth factor-165 at a concentration of 0.1 nM to 5 nM.

5. The culture medium composition according to claim 4, wherein The medium composition is a xeno-free medium.

6. An in vitro xeno-free culture method comprising the following steps: The culture medium composition according to claim 4 is used to treat endothelial progenitor cells. 7 . A vascular endothelial progenitor cell, wherein the vascular endothelial progenitor cell is cultured by the xeno-free culture method according to claim 6 .

8. Use of the vascular endothelial progenitor cells according to claim 7 in preparing a pharmaceutical composition for preventing or treating lower limb ischemic diseases or severe myocardial infarction diseases.

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