Method for selecting mesenchymal stem cells having improved self-maintenance ability, and mesenchymal stem cells selected thereby

Abstract

To provide a mesenchymal stem cell which has excellent self-propagation ability, can be obtained in a large amount, and can be utilized for the development of a cell therapeutic agent.SOLUTION: Provided are mesenchymal stem cells genetically engineered to secrete or overexpress DOCK2 or AURKA compared to parent cells, wherein the DOCK2 or AURKA is one of adhesion-related genes, differentiation-related genes, chemotactic-related genes, and proliferation-related genes, and wherein the mesenchymal stem cells have improved migration ability, colony forming ability, and proliferation ability, or shortened replication time, compared to the parent cells.SELECTED DRAWING: Figure 4C

Description

[Technical Field]

[0001] This application is Korean Patent Application No. 10-2020-0 filed on December 7, 2020. No. 169835, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to a method for selecting mesenchymal stem cells with improved self-maintenance ability. [Background technology]

[0003] Mesenchymal stem cells are multipotent cells that grow in the bone marrow. It has been isolated from various tissues such as adipose tissue, placenta, and umbilical cord blood, and is used to treat bone, cartilage, muscle, fat, and These stem cell properties can be utilized to create tissues that cannot be regenerated. By regenerating tissues and cells, we will utilize mesenchymal stem cells as a treatment for various diseases. However, mesenchymal stem cells are not widely available as cell therapy agents. The donor variation is large, the production yield is low, and the production cost is high. The problem is that the survival period in the body is short and it is not possible to demonstrate long-term therapeutic effects. There is an issue.

[0004] Therefore, in the production of stem cells for the development of stem cell therapeutic agents, it has excellent proliferation ability and therapeutic efficacy. It is necessary to select mesenchymal stem cells with good properties. Summary of the Invention [Problem to be solved by the invention]

[0005] In one embodiment, mesenchymal stem cells are cultured and then treated with self-maintenance factors (MSMFs). The method further comprises measuring the expression or activity level of a ance factor (ALF) in the host cell. A method for selecting mesenchymal stem cells is provided.

[0006] Another aspect provides mesenchymal stem cells selected by the above method.

[0007] Another aspect is the increased expression of MSC self-maintenance factors (MSMFs). These cells are genetically engineered to have increased expression of MSMF or to have increased expression of MSMF compared to the parent cells. The present invention provides a cell death inhibitor containing mesenchymal stem cells or MSMFs.

[0008] In another embodiment, the cells secrete MSCs self-maintenance factors (MSMFs). or mesenchymal stem cells genetically engineered to overexpress MSMF compared to parent cells. and a pharmaceutical composition for preventing or treating muscle diseases, comprising the cells or MSMF. .

[0009] In another embodiment, the self-maintenance factor (MSMF) is used as an active ingredient. The present invention provides a pharmaceutical composition for preventing or treating a muscle disease, comprising:

[0010] In another embodiment, the self-maintenance factor (MSMF) is used as an active ingredient. The present invention provides a functional health food composition for preventing or improving muscle diseases, which comprises:

[0011] In another embodiment, the cells secrete MSCs self-maintenance factors (MSMFs). or mesenchymal stem cells genetically engineered to overexpress MSMF compared to parent cells. A cell therapy agent comprising cells or MSMF is provided.

[0012] In another embodiment, the cells secrete MSCs self-maintenance factors (MSMFs). or mesenchymal stem cells genetically engineered to overexpress MSMF compared to parent cells. administering a pharmaceutically effective amount of the cells, or MSMF to an individual in need thereof. A method for preventing or treating muscle diseases is provided.

[0013] Another embodiment is the use of MSCs self-maintenance factors (MSMFs) for treating muscle diseases. These cells are genetically engineered to secrete MSMF or overexpress MSMF compared to their parent cells. The present invention provides uses of compositions comprising genetically engineered mesenchymal stem cells, or MSMFs.

[0014] Another embodiment is a method for producing a therapeutic agent for muscle diseases using MSC self-maintenance factors (MSMFs). secrete MSMF or overexpress MSMF compared to parent cells. The present invention provides uses of compositions comprising genetically engineered mesenchymal stem cells, or MSMFs. [Means for solving the problem]

[0015] In one embodiment, mesenchymal stem cells are cultured and then treated with self-maintenance factors (MSMFs). The method further comprises measuring the expression or activity level of a ance factor (ALF) in the host cell. A method for selecting mesenchymal stem cells is provided.

[0016] As used herein, the term "mesenchymal stem cells (MSCs)" refers to cells that are naturally occurring in the body. It maintains self-renewal and stemness, and can produce a variety of mesenchymal tissues, including For example, various mesodermal cells, including bone, cartilage, fat, and muscle cells, or ectodermal cells, such as nerve cells They are multipotent stem cells that have the ability to differentiate into sex cells. Mesenchymal stem cells are derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle nerve, skin, amniotic membrane, chorion, and decidua. The mesenchymal stem cells may be derived from human, fetal, or placenta. They may also be derived from mammals other than humans.

[0017] As used herein, the term "MSCs self-maintenance factor (MSMF)" refers to " refers to various regulatory factors secreted by mesenchymal stem cells, which are necessary for the self-maintenance of said mesenchymal stem cells. MSMF is essential for adhesion, and is expressed in a stage-specific and cell-specific manner. Involved in differentiation, chemotaxis, or proliferation The MSMF may be, for example, FBLN. 5, OLR1, TNFAIP6, ANXA3, IL6, POU2F2, TNFAIP2, SERPINE2, INHBA, VEGFA, HMGB1, CSF2, GATA3, PC SK6, SYN1, F2RL1, DOCK2, SLC9A4, STX1B, RARRES 2, CXCL1, FGF7, PLAU, SCG2, NR4A3, COR01A, CHRM 3, NPR3, BST2, GATA4, CREG1, FGF7, YPEL5, AURKA Specifically, proteins involved in adhesion include FBLN5, OLR1, TN FAIP6, ANXA3, etc., and proteins involved in differentiation include IL6, POU2 F2, TNFAIP2, SERPINE2, INHBA, VEGFA, HMGB1, CS F2, GATA3, PCSK6, SYN1, F2RL1, DOCK2, SLC9A4, S TX1B, RARRES2, etc. Proteins involved in chemotaxis include IL6 , CXCL1, VEGFA, FGF7, PLAU, HMGB1, SCG2, NR4A3, GATA3, DOCK2, COR01A, RARRES2, etc., and are involved in proliferation The proteins are CXCL1, DOCK2, CHRM3, NPR3, NR4A3, and F2RL. 1, BST2, GATA4, CREG1, FGF7, YPEL5, AURKA, etc. do.

[0018] In one embodiment, the method further comprises culturing mesenchymal stem cells and then administering an inhibitor of MSMF expression or and treating the mesenchymal stem cells in which the expression or activity of MSMF is suppressed with an inhibitor of the expression or activity of MSMF. measuring the level of MSMF expression or activity in the cells; and The level of expression or activity of the MSMF is compared to a control group not treated with an inhibitor or inhibitor of activity. and isolating mesenchymal stem cells with high activity. It is also.

[0019] The expression inhibitor or activity inhibitor of the self-maintenance factor is an siRNA (small interference RNA), shRNA (short hair pin RNA), miRNA (micro RNA), Ribozyme, DNAzyme, PNA (peptide nucleic acids), Antisense oligonucleotides, antibodies, aptamers, and antibodies directed against the MSMF protein A compound selected from the group consisting of compounds and natural extracts that bind to and inhibit the activity of In one embodiment, the expression inhibitor or activity inhibitor of the self-maintenance factor is The first primer set is represented by SEQ ID NO: 5 and SEQ ID NO: 6, and the second primer set is represented by SEQ ID NO: 9 and SEQ ID NO: 10. This is also the second primer set represented.

[0020] The expression or activity level of the MSMF can be measured by reverse transcription polymerase reaction, competitive reverse transcription polymerase reaction, or the like. Enzyme reaction, real-time reverse transcription polymerase reaction, RNase protection assay, Northern blot The present invention also includes a protein chip and a DNA chip. Analysis, immunoassay, ligand binding assay, MALDI-TOF (matrix-assay) isted laser desorption / ionization time-of-flight mass spectrometry) analysis, SEL DI-TOF(surface-enhanced laser desorption / ionization time-of-flight mass s spectrophotometric analysis, radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion, location Immunoelectrophoresis, tissue immunostaining, complement fixation analysis, two-dimensional electrophoresis, liquid chromatography Liquid chromatography-mass spectrometry (LC-MS), LC -MS / MS(liquid chromatography-mass spectrometry / mass spectrometry), This consists of stain blotting and ELISA (enzyme linked immunosorbent assay). It may also be selected from the group:

[0021] The expression of MSMF was significantly increased compared to a control group not treated with an inhibitor of MSMF expression or activity. Alternatively, by isolating mesenchymal stem cells with increased levels of activity, it is possible to It is possible to select mesenchymal stem cells with improved maintenance ability. There are also.

[0022] In one embodiment, the expression of MSMF is reduced, thereby improving the migratory ability of mesenchymal stem cells. It was confirmed that the colony forming ability and cell proliferation ability of the mesenchymal stem cells were reduced. Therefore, it was confirmed that the self-sustaining ability of the mesenchymal stem cells was improved. The cells have increased expression or activity of MSMF, but the expression or activity of said MSMF is maintained. Alternatively, desired cells can be obtained by isolating the expanded mesenchymal stem cells. According to this method, mesenchymal stem cells with high proliferation potential can be selected at an early stage, thereby reducing production costs. In addition, it has the advantage of enabling mass production of mesenchymal stem cells.

[0023] Another aspect provides mesenchymal stem cells selected by the above method. , the expression of self-maintenance factors (MSMFs) is increased, Alternatively, mesenchymal stem cells genetically engineered to enhance expression of MSMF compared to parent cells The present invention provides a cell death inhibitor comprising stem cells and / or MSMF. The expression of SMF is increased or the expression of MSMF is enhanced compared to the parent cells. The genetically engineered mesenchymal stem cells and / or MSMFs are then cultured in vitro. ) or administering to an experimental animal in vivo. The present invention provides a method for inhibiting cell death, comprising the steps of: The expression of SMF is also increased. That is, the mesenchymal stem cells have the stem cell potential (st The cells also have improved serotonin, migration ability, colony formation ability and / or cell proliferation ability. The mesenchymal stem cells also have a shortened cell doubling time. In other words, MSMF expression and replication time show a negative correlation.

[0024] As used herein, the term "genetic engineering" or "genetically engineered" refers to "Genetically engineered" means that a cell has undergone one or more genetic modifications. It refers to the act of introducing a modification into a cell or the cells produced by such an act. The mesenchymal stem cells or host cells have increased expression or activity of MSMF or an active fragment thereof. Genetically engineered, for example, to encode MSMF or an active fragment thereof, The increased activity may also include exogenous genes that activate a given genetically engineered gene. endogenous proteins that the uninfected mother cell (e.g., wild-type) may or may not have The activity of an enzyme is higher than that of a protein or enzyme of the same type. The exogenous gene may be expressed in the mesenchymal stem cells or the host cells. expressed in amounts sufficient to increase the activity of the referenced protein compared to the parent cell of the The exogenous gene may be introduced into the mother cell via an expression vector. In addition, the exogenous gene is introduced into the mother cell in the form of a linear polynucleotide. In addition, the exogenous gene is expressed in a cell by an expression vector (e.g., a plasmid). ) for stable expression. They may also be inserted into genetic material (e.g., chromosomes) within the cell and expressed.

[0025] Another aspect is the increased expression of MSC self-maintenance factors (MSMFs). They are either enlarged or genetically engineered to have enhanced expression of MSMF compared to parent cells. Pharmaceuticals for preventing or treating muscle diseases comprising isolated mesenchymal stem cells and / or MSMFs In yet another embodiment, the mesenchymal stem cells and / or MSMFs are administered to an individual. The present invention also provides a method for preventing or treating a muscle disease, comprising administering to a subject a muscle-secreting MSMF. or mesenchymal cells genetically engineered to overexpress MSMF relative to parent cells. The specific details of the stem cells or MSMF are as described above. Muscle cells in which apoptosis was induced were classified as mesenchymal cells, which have relatively high expression of AURKA and DOCK2. It was confirmed that cell death was suppressed when co-cultured with stem cells. In another example, the muscle cells in which cell death was induced were treated with siAURKA and siDO By co-culturing with CK2-treated mesenchymal stem cells, the effect of suppressing cell death was reduced. Therefore, it was confirmed that AURKA mRNA and DOCK2 mRNA, as well as The protein is believed to prevent or treat muscle diseases by inhibiting the death of muscle cells. It can also be used for treatment.

[0026] The muscle disease may be, for example, Charcot-Marie-Tooth disease. disease), spinal muscular atrophy (SMA), Lou Gehrig's disease (ALS: amyotrophic lateral sclerosis), Duchenne muscular dystrophy nne muscular dystrophy, myotonic dystrophy, sarcopenia (sarcopenia), muscular atrophy, myasthenia, muscular dystrophy (muscular dystrophy), myotonia, hypotonia, muscle weakness (muscular weakness), muscle atrophy (muscular dystrophy), amyotrophic lateral sclerosis (a myotrophic lateral sclerosis, inflammatory myopathy, etc. do.

[0027] In another embodiment, the self-maintenance factor (MSMF) is used as an active ingredient. The present invention provides a pharmaceutical composition for preventing or treating a muscle disease, comprising: The MSMF may be isolated from mesenchymal stem cells.

[0028] In another embodiment, the composition further comprises mesenchymal stem cells. The mesenchymal stem cells secrete MSMF or an active fragment thereof, or It is also genetically engineered to secrete the active fragment. Cells are engineered to alter or enhance cell function for structural or therapeutic purposes. DNA insertion or injection in cell culture via the method of replenishment Therefore, the MSMF and the mesenchymal stem cells may be administered in combination. It is possible that.

[0029] The pharmaceutical composition for preventing or treating a muscle disease according to one embodiment can be prepared by a conventional method. Powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols It is formulated into oral preparations such as acetaminophen, topical preparations, suppositories, and sterile injection solutions. For the formulation, suitable pharmaceutical compositions generally used for the manufacture of pharmaceutical compositions may be used. It may include a carrier, excipient or diluent.

[0030] Such carriers, excipients or diluents include lactose, dextrose, sucrose, , sorbitol, mannitol, xylitol, erythritol, maltitol, starch, Acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydrogen Hydroxybenzoate, Propylhydroxybenzoate, Talc, Magnesium Stearate Examples of suitable compounds or mixtures include those containing cellulose and mineral oils.

[0031] When formulating, commonly used fillers, weighting agents, binders, wetting agents, disintegrating agents, It may be prepared using a diluent or excipient such as a surfactant.

[0032] The solid preparation for oral administration contains the above-mentioned bean extract and at least one or more excipients, e.g. It is made by mixing starch, calcium borate, sucrose or lactose, gelatin, etc. In addition to simple excipients, magnesium stearate, talc, etc. Such lubricants can also be used.

[0033] Oral liquid preparations include suspensions, oral liquids, emulsions, and syrups. However, in addition to the frequently used simple diluents water and liquid paraffin, various excipients, e.g. For example, they may contain wetting agents, sweetening agents, flavoring agents, preservatives, and the like.

[0034] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, frozen preparations, and the like. Dry preparations and suppositories are included. Non-water-soluble preparations and suspensions include propylene glycol, polyethylene ... polyethylene glycol, vegetable oils such as olive oil; Injectable esters and the like can be used. As a base for suppositories, witebuzole ( Witepsol®, Macrogol, Tween 61, Cocoa Butter, Lauryl Sulfate Fat, glycerol gelatin, etc. can be used.

[0035] The desired dosage of the pharmaceutical composition for preventing or treating a muscle disease according to one embodiment is The dosage will vary depending on the patient's condition, weight, severity of the disease, drug form, route of administration, and duration, but it is well within the skill of the art. However, for the desired effect, a daily dose of 0.0001 mg / day is recommended. to 2,000 mg / kg, preferably 0.001 to 2,000 mg / kg The dosage can be administered once a day or in several divided doses. However, the scope of the present invention is not limited to the above-mentioned dosage. do not have.

[0036] The pharmaceutical composition for preventing or treating muscle diseases according to one embodiment is for use in rats, mice, livestock, and the like. The compounds can be administered to mammals, including humans, by a variety of routes. For example, oral administration; or rectal, intravenous, intramuscular, subcutaneous, intrauterine, intradural or intracerebrovascular (intravenous) administration It can be administered by injection into the ventricle.

[0037] Yet another embodiment is directed to the expression of MSCs self-maintenance factors (MSMFs). The expression of MSMF is increased or genetically altered to enhance the expression of MSMF compared to the parent cells. and methods for preventing or ameliorating muscle diseases, including engineered mesenchymal stem cells, or MSMFs. The functional food composition is provided. The specific content of the mesenchymal stem cells or MSMFs is as described above. That's right.

[0038] In one embodiment of the health functional food for preventing or improving muscle diseases, the compound is When used as an additive in health functional foods, it can be added as it is or mixed with other foods. or with food ingredients and can be used appropriately in accordance with common practices. The amount of active ingredients to be mixed can be determined appropriately depending on the purpose of use, such as prevention, health, or treatment. It can be determined.

[0039] The dosage forms of health functional foods are not limited to powders, granules, pills, tablets, and capsules, It can be in the form of either a regular food or drink.

[0040] There is no particular limitation on the type of food, and examples of foods to which the substance can be added include: These include meat, sausages, bread, chocolate, candy, snacks, and confectionery. , pizza, ramen, other noodles, gum, dairy products including ice cream, various soups , drinking water, tea, energy drinks, alcoholic beverages and vitamin complexes, etc. It also includes any food in the sense of "food."

[0041] Generally, when producing food or beverages, the compound is added in an amount of 15 parts by weight per 100 parts by weight of raw material. It can be added in an amount of 10 parts by weight or less, preferably 10 parts by weight or less. In the case of long-term intake for the purpose of health and hygiene or health regulation, The amount may be less than the above range, and safety is ensured by using fractions from natural products. Since there are no problems with the surface, amounts greater than the above range can be used.

[0042] The beverage among the health functional foods according to one embodiment contains various flavoring agents or The natural carbohydrates may be added as additional ingredients. Monosaccharides such as sugar and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin, cyclodextrin; and xylitol It is also a sugar alcohol such as sorbitol and erythritol. Natural sweeteners such as maltodextrin and stevia extract, and synthetic sweeteners such as saccharin and aspartame The ratio of the natural carbohydrates in the beverage of the present invention is Approximately 0.01 to 0.04 g, preferably approximately 0.02 to 0.03 g per 100 mL of water. can.

[0043] In addition to the above, a health functional food for preventing or improving muscle diseases according to one embodiment includes: Various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and its salts, alginate Phosphoric acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycols It may also contain serine, alcohol, and carbonation agents used in carbonated drinks. The composition of the present invention is suitable for the preparation of natural fruit juices, fruit juice drinks and vegetable drinks. Such ingredients may be used individually or in admixture. The ratio of such additives is not limited, but may be adjusted to the range of the functional health food of the present invention. It is generally selected from the range of 0.01 to 0.1 parts by weight. do.

[0044] Another aspect provides a cell therapy agent comprising the mesenchymal stem cells as an active ingredient. The specific details of the stem cells are as described above.

[0045] As used herein, the term "cellular therapeutic agent" refers to a cell that has been isolated from an individual, cultured, and specially manipulated. Medicines for therapeutic, diagnostic and preventive purposes using cells and tissues produced through the method. It is a product (US FDA regulation) that is used to restore the function of living cells or tissues. Autologous, allogeneic, or xenogeneic cells can be expanded in vitro or otherwise purified to produce cells. Through a series of actions such as altering the biological properties of cells, "Treatment" refers to the treatment of a disease by administering the cell therapy agent. The term "medechocystic stem cells" refers to any action that improves or alleviates the symptoms of a patient. FBL, which has increased expression of MSMF and is associated with inflammatory diseases, immune diseases or cancer N5, TNFAIP6, ANXA3, IL6, POU2F2, TNFAIP2, INHB A, VEGFA, CSF2, GATA3, CXCL1, HMGB1, BST2, AURK Therefore, in one embodiment, the cell therapy agent is The therapeutic agent may be for treating a muscle disease, an inflammatory disease, an immune disease, or cancer, etc.

[0046] The inflammatory diseases include, for example, atopy, psoriasis, dermatitis, allergies, arthritis, rhinitis, and middle ear inflammation. inflammation, sore throat, tonsillitis, cystitis, nephritis, pelvic inflammation, Crohn's disease, ulcerative colitis, ankylosing Spondylitis, systemic lupus erythematosus (SLE), asthma, edema, delay Sexual allergy (type IV allergy), transplant rejection, graft-versus-host disease, autoimmune encephalomyelitis, Multiple sclerosis, inflammatory bowel disease, cystic fibrosis, diabetic retinopathy, ischemia-reperfusion injury, vascular regeneration It may be due to stenosis, glomerulonephritis, gastrointestinal allergy, etc.

[0047] The immune diseases include, for example, rheumatoid arthritis, type I diabetes, multiple sclerosis, and systemic leukemia. Autoimmune diseases such as lupus erythematosus; asthma, encephalitis, inflammation enterocolitis, chronic obstructive pulmonary disease, allergy, septic shock, pulmonary fibrosis, undifferentiated vertebrae Arthrosis, undifferentiated joint disease, arthritis, inflammatory osteolysis, chronic viral or bacterial infections The cause may be an inflammatory disease such as chronic inflammation caused by inflammatory bowel disease.

[0048] The cancers include, for example, multiple myeloma, lung cancer, liver cancer, stomach cancer, colon cancer, skin cancer, bladder cancer, and the like. Bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, thyroid cancer, kidney cancer, fibrosarcoma, melanoma, blood It may be a liquid cancer.

[0049] The cell therapy agent is prepared in a unit dose suitable for administration into the body of a patient by conventional methods in the pharmaceutical field. The compounds can be administered in a single or multiple doses in a pharmaceutical formulation. Suitable dosage forms for such purposes include parenteral administration formulations. Examples of the formulation include injections such as injection ampoules, infusions such as infusion bags, and aerosols. The injection ampoule is preferably mixed with an injection solution just before use. It can be prepared as an injection, and the injection solution can be saline, glucose, mannitol, Ringer's The infusion bag can be made of polyvinyl chloride or polyethylene. Baxter, Becton Dickinson, and other manufacturers of plastic materials are available. Becton Dickinson, Medcep, National Hospital Products Examples of infusion bags include National Hospital Products or Terumo. can be done.

[0050] The pharmaceutical preparation may further contain one or more pharmaceutically acceptable salts in addition to the active ingredient. In the case of injections, for example, preservatives, soothing agents, solubilizers or In the case of a topical formulation, it may further contain a stabilizer, an excipient, a lubricant, a preservative, etc. It can include:

[0051] The cell therapy agents or pharmaceutical formulations of the present invention so produced can be used in a variety of ways, including those commonly used in the art. and other stem cells for transplantation and other uses. The therapeutic agent may also be administered in the form of a mixture with such stem cells, preferably to a patient in need of treatment. It is either directly engrafted or transplanted into the affected area of ​​the patient, or directly transplanted or injected into the abdominal cavity. The administration can be performed by, but is not limited to, a catheter. Non-surgical administration using a syringe, and surgical administration such as injection or implantation after incision at the diseased site. Although both methods are possible, non-surgical administration using a catheter is preferred. In addition to parenteral administration, for example, administration directly to the lesion, the drug can also be administered by the general method. Transplantation by intravascular injection is also possible.

[0052] The daily dose of the mesenchymal stem cells is 1.0×10 4 to 1.0×10 10 cells / kg. body weight, preferably 2.5 × 10 5 to 5 × 10 7 cells / kg body weight, once or several times However, the actual dose of the active ingredient may vary depending on the treatment regimen. Various factors, such as the disease being treated, the severity of the disease, the route of administration, and the patient's weight, age, and sex, are considered. It should be understood that the dosage should be determined in light of the associated factors. are not intended to limit the scope of the present invention in any manner.

[0053] The mesenchymal stem cells are used to treat bodily tissues or organs that are susceptible to the engraftment, transplantation or regeneration of a desired cell population. It can also be used in a wide variety of therapeutic protocols to enhance, treat, or replace the effects of injections. do.

[0054] The cell therapy composition may be used unfrozen or frozen for later use. If frozen, it should be frozen in a standard cryopreservative (e.g., DMSO). , glycerol, Epilife™ Cell Freezing Medium (Cascade Biologics) may also be added to the cell population prior to freezing.

[0055] As mentioned above, mesenchymal stem cells or MSMFs according to one embodiment inhibit cell death of muscle cells. It acts as a preventive or therapeutic agent for various muscle disorders, including sprains, strains, and cramps. It can also be used. [Effects of the Invention]

[0056] In one embodiment, mesenchymal stem cells with excellent self-proliferation ability are selected, and donor bias is detected. r variation) can be reduced, but it is possible to secure a large amount of mesenchymal stem cells. The mesenchymal stem cells selected by the above method can be used for the development of cell therapy agents. Cells have the advantage of being able to survive longer in the body and improve therapeutic efficacy. be. [Brief explanation of the drawings]

[0057] [Figure 1A] 1 is a graph showing the doubling time between the P-high group and the P-low group. [Figure 1B] This is the result of comparing the expression levels of genes related to self-maintenance in the P-low group and the P-high group. [Figure 1C] 1 is a graph comparing the expression levels of AURKA and DOCK2, selected as MSMF gene candidates, in the P-low group and the P-high group. [Figure 2A] 1 is a graph comparing the relative mRNA expression of AURKA and DOCK2 by selected siRNA sequences. [Figure 2B] This shows the results of transfection of mesenchymal stem cells with siAURKA and siDOCK2 to knock down the MSMF gene, and then confirming the protein expression levels of AURKA and DOCK2. [Figure 2C]This is a graph showing the results of confirming stem cell potential via FACS after transfection of mesenchymal stem cells with siAURKA and siDOCK2 to knock down the MSMF gene. [Figure 2D] This is a graph showing the results of confirming stem cell potential via FACS after transfection of mesenchymal stem cells with siAURKA and siDOCK2 to knock down the MSMF gene. [Figure 2E] This is a graph showing the results of confirming stem cell potential via FACS after transfection of mesenchymal stem cells with siAURKA and siDOCK2 to knock down the MSMF gene. [Figure 2F] This is a graph showing the results of confirming stem cell potential via FACS after transfection of mesenchymal stem cells with siAURKA and siDOCK2 to knock down the MSMF gene. [Figure 3A] This is the result of transfection of mesenchymal stem cells with siAURKA and siDOCK2 to knock down the MSMF gene, and then confirming the migration ability of the mesenchymal stem cells. [Figure 3B] 10 is a graph showing the results of confirming the presence or absence of colony formation of mesenchymal stem cells after transfection of mesenchymal stem cells with siAURKA and siDOCK2 to knock down the MSMF gene. [Figure 3C] 1 is a graph showing the measurement of the doubling time of mesenchymal stem cells after transfection with siAURKA and siDOCK2 to knock down the MSMF gene. [Figure 3D] This is the result of transfection of mesenchymal stem cells with siAURKA and siDOCK2 to knock down the MSMF gene, and then confirming the cell proliferation ability of the mesenchymal stem cells. [Figure 3E] These results confirm the effects of suppressing the expression of the AURKA and DOCK2 genes on the phosphorylation of AKT and ERK, which are kinases involved in cell proliferation, and FAK and JNK, which are kinases involved in migration. [Figure 3F] These results confirm the effects of suppressing the expression of the AURKA and DOCK2 genes on the phosphorylation of AKT and ERK, which are kinases involved in cell proliferation, and FAK and JNK, which are kinases involved in migration. [Figure 4A] 1 is a graph comparing the relative expression levels of AURKA and DOCK2 mRNA in 10 lots of mesenchymal stem cells. [Figure 4B] 1 is a graph showing the replication times of 10 lots of mesenchymal stem cells in sequence. [Figure 4C] 1 is a graph showing the correlation between the expression levels of AURKA and DOCK2 mRNA and replication time in 10 lots of mesenchymal stem cells. [Figure 4D] This shows the results of confirming the protein expression levels of AURKA and DOCK2 in mesenchymal stem cells. [Figure 4E] These are the results of confirming the degree of phosphorylation of AKT, ERK, FAK, and JNK in mesenchymal stem cells. [Figure 4F] These are the results of confirming the degree of phosphorylation of AKT, ERK, FAK, and JNK in mesenchymal stem cells. [Figure 5A] Mesenchymal stem cells were co-cultured with muscle cells in which apoptosis had been induced, and the expression of apoptosis-related proteins was confirmed based on the mRNA expression levels of AURKA and DOCK2. [Figure 5B] Mesenchymal stem cells were co-cultured with muscle cells in which apoptosis had been induced, and the extent to which apoptosis was reduced due to the expression levels of AURKA and DOCK2 mRNA was confirmed. [Figure 6A] Mesenchymal stem cells in which the AURKA gene and DOCK2 gene were knocked down were co-cultured with muscle cells in which apoptosis had been induced, and the expression of apoptosis-related proteins due to the suppression of AURKA and DOCK2 gene expression was confirmed. [Figure 6B]Mesenchymal stem cells in which the AURKA gene and DOCK2 gene were knocked down were co-cultured with muscle cells in which cell death had been induced, and the extent to which cell death was reduced by suppressing the expression of the AURKA gene and DOCK2 gene was confirmed. [Figure 6C] This is the result of confirming the amount of XCL1 protein expression in mesenchymal stem cells in which the AURKA gene was knocked down. [Figure 7A] This result confirmed the effectiveness of suppressing cell death in muscle tissue due to differences in AURKA mRNA expression levels in mesenchymal stem cells in a mouse model of muscle dystrophies. [Figure 7B] This result confirmed the difference in inhibitory efficacy against muscle tissue cell death due to differences in AURKA mRNA expression levels in mesenchymal stem cells in a mouse model of muscle dystrophies. [Figure 8A] 10 is a graph confirming the degree of cell survival depending on the AURKA mRNA expression level in mesenchymal stem cells in a mouse model of muscle dystrophy. [Figure 8B] This result confirms the inhibitory effect of AURKA mRNA expression levels in mesenchymal stem cells on muscle tissue fibrosis in a mouse model of muscle dystrophies. [Figure 8C] These results confirm the degree to which muscle tissue fibrosis is suppressed by the expression level of AURKA mRNA in mesenchymal stem cells in a mouse model of muscle dystrophies. DETAILED DESCRIPTION OF THE INVENTION

[0058] In the following, preferred examples will be presented to aid in understanding the present invention. The following examples are provided so that the invention may be more readily understood and may be used in conjunction with the following examples. The examples are not intended to limit the scope of the present invention. [Example]

[0059] [Example 1. Isolation and culture of mesenchymal stem cells] The study was approved by the Institutional Review Board (IRB) of Samsung Seoul Hospital. B No. 2011-10-134), and all samples were collected with prior consent. Cells were isolated by a conventional method. n-Gibco) and 50 μg / mL gentamicin (Invitrogen-Gibco) in ME Mα (Minimum Essential Medium) (Invitrogen-Gibco, Rockville, MD) medium, The isolated cells were aliquoted at a density of 3 x 103 cells / cm2 and incubated at 37°C and 5% CO2. The cells were cultured under these conditions.

[0060] Example 2. Analysis of mesenchymal stem cell morphology, replication time, and gene expression comparison ] The mesenchymal stem cells isolated and cultured in Example 1 were treated at the Samsung Medical Center, It has passed the standard of Good Manufacturing Practice (GMP) for manufacturing and quality control. The mesenchymal stem cells were classified into those that passed the test and those that did not. The replication time of each mesenchymal stem cell classified according to the criteria was checked, and the time that passed the criteria was In the case of mesenchymal stem cells, the replication time is shorter than that of mesenchymal stem cells, which could not pass through. Based on these results, we confirmed that P-high (proliferative After dividing the cells into a P-low (proliferation-low) group and a P-high (proliferation-high) group, The doubling time was then confirmed. Specifically, we compared and analyzed the genes that were highly expressed in the P-low group and the P-high group. After extracting each RNA in the loop, the Agilent 2100 Bioanalyzer was used. The RNA quality was measured and the migration and peak patterns were analyzed. Microarray analysis was performed using seq 2500, and the P-low group was compared. In the P-high group, the log2 fold change was more than 3 times higher. The genes were comparatively analyzed.

[0061] Figure 1A shows the doubling time between the P-high and P-low groups. This is the graph.

[0062] As a result, as shown in Figure 1A, the replication time of mesenchymal stem cells in the P-high group was significantly shorter. The replication time of mesenchymal stem cells in the P-low group was 28.4±1.2 hours. , 47.9±1.6 hours, which was 1.6 times longer than in the P-high group.

[0063] Figure 1B shows the genes involved in self-maintenance in the P-low and P-high groups. The green line indicates the expression level of the P-low group compared to the P-high group. The red indicates genes that show lower expression in the P-low group compared to the P-high group. Figure 1C shows genes that showed higher expression in the P-low group and the P-low group. AURKA and DOCK2 were selected as MSMF gene candidates in the P-high and P-low groups. 1 is a graph comparing the expression levels of

[0064] As a result, as shown in Figure 1B, the P-low group showed a significantly higher oxidative stress response than the P-high group. Genes with an average log2 fold change of 3 or more in the group In adhesion, differentiation, chemotaxis or Thirty-three genes involved in proliferation were identified. Genes also showed higher expression in the P-high group compared to the P-low group. The genes are CORO1A, STX1B, HMGB1, DOCK2, AURKA, and SLC9. A4, CHRM3, NPR3, RARRES2, and ANXA3 were identified. As shown in Fig. 1, AURKA and DOCK2 were significantly elevated in the Ph group compared with the P-low group. Confirm that the expression level is approximately 4 times higher in the igh group, showing a statistically significant difference. I was able to do it.

[0065] Therefore, based on the above results, adhesion, differentiation, and migration The expression levels of genes involved in chemotaxis or proliferation were compared and analyzed. AURKA and DOCK2, which showed statistically significant differences, were selected as final candidates for MSMF genes. .

[0066] Example 3. Selection of optimal sequence through knockdown efficacy verification 3-1. Selection of siRNA sequence candidates for AURKA and DOCK2, and mRNA expression Check the quantity) In Example 2, the sequences of AURKA and DOCK2 selected as MSMF genes were After verifying knockdown efficacy using iRNA library, Specifically, mesenchymal stem cells cultured in a growth medium to about 50% were cultured in a bloodless medium. After transferring to clear medium, the siRNA library showed the activity of AURKA and DOCK2. Three candidate sequences (Bioneer) (see 1 below) were used for each of the three sequences. The cells were transfected at a concentration of 25 nM using a 25 nM syringe (Invitrogen). After 48 hours, Accu Extract RNA from mesenchymal stem cells using the Prep™ Universal RNA Extraction Kit qRT-PCR was performed using 2X Power SYBR Green Master Mix (AB). Then, the relative mRNA expression was compared to show the greatest knockdown effect. The sequences were selected as optimal sequences for inhibiting the expression of AURKA and DOCK2.

[0067] [Table 1]

[0068] As a result, in the case of siAURKA, the maximum knockdown (knoc In the case of siDOCK2, the sequence of candidate 2 showed the greatest knockdown effect. It was confirmed that the drug showed a knock-down effect.

[0069] Therefore, three candidate sequences of siAURKA and two candidate sequences of siDOCK2 were used. After transfection into mesenchymal stem cells, the relative expression of AURKA and DOCK2 by selected candidates was The mRNA expression rates were compared to confirm sequence specificity.

[0070] Figure 2A shows the relative mRNA expression of AURKA and DOCK2 by selected siRNA sequences. A is a graph comparing expression.

[0071] As a result, as shown in Figure 2A, in the case of AURKA, the siNC-treated group and the si mRNA expression was significantly lower in the siAURKA-treated group compared to the DOCK2-treated group. In the case of DOCK2, the siD It was confirmed that mRNA expression was significantly reduced in the OCK2-treated group. That is, the selected siRNA sequences had sequence specificity for each MSMF gene. It may be of the opposite sex.

[0072] (3-2. Confirmation of AURKA and DOCK2 protein expression levels) The siRNA sequence selected in Example 3-1 above was used to identify the link between AURKA and DOCK2. Whether or not the protein expression level was reduced was confirmed by Western blotting. Specifically, siAURKA (candidate 3) and siDOCK2 (candidate 2) were administered to mesenchymal stem cells. After transfection and culturing for 72 hours, the mesenchymal stem cells were washed with PBS. Protease Inhibitor Cocktail (Amresco, Solon, OH, USA) The cells were dissolved in RIPA buffer (BIOSESANG, Sungnam, Gyeonggi, Korea) containing 4 The mixture was centrifuged at 15,000 g for 30 minutes at ℃ to obtain the supernatant. After electrophoresis to separate the samples by size using SDS-PAGE, the samples were placed on PVDF (poly The membrane was then transferred to a 5% non-fat dry milk (PVDF) membrane. Blocking was performed for 1 hour at room temperature using TBST containing 5% skim milk powder. The primary antibody was diluted and incubated overnight at 4°C. The membrane was then washed with TBST for 10 minutes. After washing three times, the secondary antibody was further diluted in TBST containing 5% non-fat dry milk and incubated at room temperature for 1 After that, the membrane was washed three times with TBST for 10 minutes each, and then ECL solution was added. After processing with a gel imaging system (Amersham Imager 600, GE), The band image was verified by the Healthcare, Buckinghamshire, UK. The expression level of proteins was measured using Image J and normalized with β-actin. , AURKA (Invitrogen, CA), DOCK2 (Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) were used. .

[0073] Figure 2B shows the results of transfection of mesenchymal stem cells with siAURKA and siDOCK2, followed by MSMF After gene knockdown, the AURKA and DOCK2 proteins were This is the result of confirming the amount of quality expression.

[0074] As a result, as shown in Figure 2B, the siAURKA The protein expression levels of AURKA and DOC were significantly increased in the siDOCK2-treated and siDOCK2-treated groups. The protein expression levels of K2 were 0.51±0.06 times and 0.76±0.04 times, respectively. The reduction was statistically significant.

[0075] (3-3. Confirmation of stem cell potential) Using the sequences selected in Example 3-1 above, siAURKA and siDOCK 2 and 3 were transfected into mesenchymal stem cells, knocked down, and then analyzed by FACS. The mesenchymal stem cell markers were CD44, CD73, and CD9. Hematopoietic stem cell lineage markers, CD0, CD105, and CD166, were confirmed through expression of 14,CD11b,HLA-DR(MHCII),CD34,CD45,CD19(BD B Biosciences, USA) were used to compare the siNC-treated control group, siAURKA-treated group, and siDOC The stem cell potential was compared with that of the K2-treated group. 10,000 events were acquired and analyzed.

[0076] Figures 2C to 2F show the transfection of mesenchymal stem cells with siAURKA and siDOCK2. After knocking down the MSMF gene, stem cells were isolated via FACS. This is a graph confirming the performance.

[0077] As a result, as shown in Figures 2C to 2F, all of the cells showed positive markers. Positive markers CD44, CD73, CD90, CD105, and CD16 6 is expressed in over 90% of cases, but negative markers CD14 and CD1 1b, HLA-DR (MHCII), CD34, CD45, and CD19 were less than 5%. In other words, AURK was hardly expressed in mesenchymal stem cells by siRNA. Knockdown of A and DOCK2 does not alter stem cell potential You can see that.

[0078] Example 4: Suppression of AURKA and DOCK2 expression and migration, colony formation, and replication Correlation with time, cell proliferation, and kinase phosphorylation 4-1. Correlation between suppression of AURKA and DOCK2 expression and migration ability To confirm the correlation between the suppression of AURKA and DOCK2 expression and the migration ability of mesenchymal stem cells. To confirm this, a wound healing assay was performed. , and siAURKA and siDOCK2 selected in Example 3-1 above. The mesenchymal stem cells were cultured in MEMα (Minimum Essential Medium) supplemented with 10% FBS. m) (Invitrogen-Gibco, Rockville, MD) medium was used to prepare 12-well plates. × 105 cells / well and cultured for 48 hours. Then, rinse twice with MEMα medium without FBS, add 10 μg / ml of mitochondria to the MEMα medium, and Mycin C (Sigma-Aldrich, St. Louis, MO) was added and the mixture was incubated for 2 hours. Scratches were made using a 0 pipette tip. After rinsing five times with culture medium, After 24 hours and 30 hours, images were taken through a microscope at 40x magnification. Images were quantitatively analyzed via Java Image J software to assess wound closure. The migration ability of the cells was expressed as a percentage of the total number of cells per 1000 cells.

[0079] Figure 3A shows the results of transfection of mesenchymal stem cells with siAURKA and siDOCK2, followed by MSMF After knocking down the gene, the migration ability of mesenchymal stem cells was confirmed. is.

[0080] As a result, as shown in Figure 3A, the migration ability of the siNC-treated control group was 61.02 The migration ability of the siAURKA-treated group and the siDOCK2-treated group was ±1.0%. The AUR was 44.06±1.87% and 47.18±2.93%, respectively. The reduction of KA and DOCK2 gene expression increases the migration ability of mesenchymal stem cells. was confirmed to be significantly reduced.

[0081] (4-2. Correlation between suppression of AURKA and DOCK2 expression and colony formation ability) Correlation between suppression of AURKA and DOCK2 expression and colony-forming ability of mesenchymal stem cells Specifically, siNC and siAURK selected in Example 3-1 were confirmed. A, siDOCK2-transfected mesenchymal stem cells were cultured in 10% FBS-supplemented medium. MEM α (Minimum Essential Medium) (Invitrogen-Gibco, Rockville, MD) medium The cells were then plated onto a 6-well plate at 1 x 103 cells / well and cultured for 14 days. Afterwards, the cells were rinsed with PBS and fixed with cold 100% methanol for 20 minutes. Fixed cells were incubated in 1% Crystal Violet solution (Sigma-Aldrich, St. Louis, MO). After staining for 30 minutes using HCl, rinse with distilled water at least three times and then use a solution of 50 or more cells. Only colonies that were viable were counted.

[0082] Figure 3B shows the results of transfection of mesenchymal stem cells with siAURKA and siDOCK2, followed by MSMF After gene knockdown, mesenchymal stem cell colonies were formed. This is a graph confirming whether or not

[0083] As a result, as shown in Figure 3B, the number of colonies in the siNC-treated control group was 30. 7±4.0, while the colonies in the siRNA-treated and siDOCK2-treated groups The numbers of AU decreased to 13.0±2.7 and 11.0±1.4, respectively. The decreased expression of RKA and DOCK2 genes leads to the formation of mesenchymal stem cell colonies. It was confirmed that the formation of granules was significantly reduced.

[0084] (4-3. Correlation between suppression of AURKA and DOCK2 expression and replication time) A correlation was confirmed between the suppression of AURKA and DOCK2 expression and the replication time of mesenchymal stem cells. Specifically, siNC, siAURKA, and siD selected in Example 3-1 above were used. OCK2-transfected mesenchymal stem cells were cultured in MEMα (MEMα) medium supplemented with 10% FBS. In Vitro Culture Media (Invitrogen-Gibco, Rockville, MD) was used for 12 The cells were distributed to a well plate at a density of 3 × 103 cells / cm2, and then the cells were incubated at 0, 24, 72, At 144 hours, the cell number was measured and the replication time was confirmed.

[0085] Figure 3C shows the results of transfection of mesenchymal stem cells with siAURKA and siDOCK2, followed by MSMF After gene knockdown, the doubling time of mesenchymal stem cells was 1 is a graph showing the measurement of me.

[0086] As a result, as shown in Figure 3C, the replication time of the siNC-treated control group was 31.1 The mean time was ±0.4 hours for the siAURKA-treated group and the siDOCK2-treated group, respectively. The results showed that the AURKA gene By reducing the expression of DOCK2 gene, the replication time of mesenchymal stem cells is increased. I could accept that it would be long.

[0087] (4-4. Correlation between suppression of AURKA and DOCK2 expression and cell proliferation ability) Established a correlation between the suppression of AURKA and DOCK2 expression and the cell proliferation ability of mesenchymal stem cells Specifically, siNC and siAURKA selected in Example 3-1 above were used. The siDOCK2-transfected mesenchymal stem cells were cultured in ME containing 10% FBS. Mα (Minimum Essential Medium) (Invitrogen-Gibco, Rockville, MD) medium was used. Then, 2x103 cells / well were dispensed into a 96-well plate. After dispensing, the cells were incubated at 0, 24, and 7 2. At 144 hours, the cells were counted using Cell Counting Kit-8 (CCK-8) (DOJINDO LABORATORIE The absorbance at 450 nm was measured using a spectrophotometer (S, Tokyo, Japan).

[0088] Figure 3D shows the results of transfection of mesenchymal stem cells with siAURKA and siDOCK2, followed by MSMF After gene knockdown, the proliferation ability of mesenchymal stem cells was confirmed. This is the result.

[0089] As a result, as shown in Figure 3D, after 24 hours of cell culture, siAURKA and s The iDOCK2 treatment group showed a difference in cell proliferation ability compared to the siNC treatment control group. However, from 48 hours onwards, the siAURKA In the siDOCK2 and siDOCK2 treated groups, cell proliferation ability was reduced, and cell proliferation became more severe over time. It was found that the concentration of HCl was significantly reduced.

[0090] (4-5. Correlation between suppression of AURKA and DOCK2 expression and kinase phosphorylation) Suppression of AURKA and DOCK2 expression and phosphorylation of AKT, ERK, FAK, and JNK Specifically, the primary antibodies were AKT, p-AKT, and pE. RK (R&D, Minneapolis, MN, USA), ERK, FAK, p-FAK, JNK, p-JN K (Cell Signaling Technology, Danvers, MA), and β-actin (Santa Cruz Biotech). The same method as in Example 3-2 was used, except that a 1000-kJ / cm2 laser was used. Western blotting was performed.

[0091] Figures 3E and 3F show that suppression of AURKA and DOCK2 gene expression suppresses cell proliferation. AKT and ERK are kinases involved in migration, and FAK is a kinase involved in migration. The results confirmed the effects on phosphorylation with JNK.

[0092] As a result, as shown in Figures 3E and 3F, the expression of AURKA was suppressed. This suppresses the phosphorylation of AKT and FAK, and inhibits the expression of DOCK2. It was confirmed that the phosphorylation of AKT, ERK, and JNK was suppressed by Thus, AURKA affects cell proliferation and migration via AKT and FAK. DOCK2 affects cell proliferation and migration through AKT, ERK, and JNK. This shows that

[0093] Taking the results of Example 4 into consideration, the AURKA gene and the DOCK2 gene are closely related. It affects the migration, colony formation, and cell proliferation of mesenchymal stem cells, thereby promoting self-sustainability. I know that I'm involved.

[0094] Example 5: Expression levels of AURKA and DOCK2, replication time, and kinase phosphorylation [Confirming correlation between 5-1. AURKA and DOCK2 mRNA expression levels and replication time of mesenchymal stem cells correlation) Mesenchymal stem cells have the problem of large donor variation. Because of the topic, the expression levels of AURKA and DOCK2 mRNA and replication time of mesenchymal stem cells We attempted to select mesenchymal stem cells with reduced donor variation by confirming the correlation with the donor. Specifically, 10 mesenchymal stem cells obtained from 10 different donors were cultured in 10% FBS. MEMα (Minimum Essential Medium) (Invitrogen-Gibco, Rockville) supplemented with e, MD) medium was used to divide the cells into a 25T flask at a density of 3 × 103 cells / cm2. When the cells were about 80% grown, RNA was extracted and subjected to qRT-PCR. The relative mRNA expression levels of DOCK2 and DOCK1 were compared. After confirming the replication time, the AURKA and DOCK genes were identified through Pearson correlation analysis. We confirmed the correlation between the expression levels of the two genes and cell replication time.

[0095] Figure 4A shows the expression of AURKA and DOCK2 in 10 different mesenchymal stem cells. 1 is a graph comparing relative mRNA expression levels.

[0096] As a result, as shown in Figure 4A, MSC_A had the highest AU among the 10 stem cells. The expression level of RKA mRNA was the highest in MSC_C, and among the 10 stem cells, the expression level of DOCK In contrast, in the case of MSC_J, the expression level of AURKA and The mRNA expression level of DOCK2 was the lowest. Figure 4B shows the results of 10 different mesenchymal stem cells. This is a graph showing the sequential replication times of cells.

[0097] As a result, as shown in Figure 4B, the replication time of MSC_A was 20.9 ± 0.6 The replication time of MSC_J was the shortest at 41.9±0.6 hours, and that of MSC_J was the longest at 41.9±0.6 hours. The average replication time of 10 mesenchymal stem cells was 27.6±1.8 hours.

[0098] FIG. 4C shows the expression of AURKA and DOCK2 in 10 lots of mesenchymal stem cells. This is a graph showing the correlation between mRNA expression level and replication time.

[0099] As a result, as shown in Figure 4C, the expression level of AURKA mRNA and the replication time were significantly correlated. The correlation between DOCK2 and DOCK2 mRNA was r=-0.757, which is a very strong negative correlation. The correlation between the expression level of A and the replication time showed a strong negative correlation of r = -0.536 ( p<0.01).

[0100] Therefore, the expression levels of the AURKA gene and DOCK2 gene are high in mesenchymal stem cells. The higher the number of genes, the shorter the replication time. By determining this, mesenchymal stem cells with high self-proliferation ability can be easily selected.

[0101] 5-2. AURKA and DOCK2 mRNA expression levels in mesenchymal stem cells and AURKA and correlation with DOCK2 protein expression level) AURKA and DOCK2 mRNA expression levels in mesenchymal stem cells and AURKA and DOCK We confirmed the correlation between the amount of protein expression of 2 and the amount of protein expression of 80% in the growth medium. The same method as in Example 3-2 was used, except that mesenchymal stem cells cultured for about 10 minutes were used. Western blotting was performed using the α-glucan-1-phosphate dehydrogenase (AURKA) gene. MSC_A, which had the lowest mRNA expression of DOCK2, MSC_C, which had the highest mRNA expression of AURKA and D MSC_J, which has relatively low mRNA expression levels of both OCK2 and OCK2 (see Figure 4A), was selected. We then confirmed the difference in protein expression levels between AURKA and DOCK2.

[0102] Figure 4D shows the protein expression levels of AURKA and DOCK2 in mesenchymal stem cells. This is the result of acknowledging that.

[0103] As shown in Figure 4D, MSC_J exhibited a significantly higher A URKA and DOCK2 protein expression increased by 0.31±0.05-fold and 0.5 The mRNA expression of AURKA and DOCK2 was decreased by 9±0.02 times. It was also observed that mesenchymal stem cells exhibited reduced expression of proteins that indicate their function.

[0104] 5-3. AURKA and DOCK2 mRNA expression levels in mesenchymal stem cells and kinase activity (Correlation with phosphorylation) AURKA and DOCK2 mRNA expression levels in mesenchymal stem cells and AKT, ERK, and FA To confirm the correlation with the phosphorylation of K and JNK, the same method as in Examples 4-5 was used. Western blotting was performed.

[0105] Figures 4E and 4F show that AKT, ERK, FAK, and JNK are regulated in mesenchymal stem cells. This is the result of checking the degree of oxidation.

[0106] As a result, as shown in Figures 4E and 4F, in the cases of MSC_A and MSC_C, AKT and ERK are kinases involved in cell proliferation, and AKT is a kinase involved in migration. There was no significant difference in the phosphorylation of FAK and JNK. In the case of MSC_A and MSC_C, AKT was 0.46±0.08 times higher and ERK was 0.46±0.08 times higher than in MSC_A and MSC_C. The phosphorylation of FAK was reduced by 0.79±0.07 times. A reduction in phosphorylation was observed for IL-1 and JNK, with a reduction of 0.03-fold and 0.56±0.05-fold, respectively.

[0107] Therefore, the mRNA and protein expression of AURKA and DOCK2 in mesenchymal stem cells The difference in abundance affects the phosphorylation of kinases involved in cell proliferation and migration, Mesenchymal stem cells with high gene expression levels induce cell proliferation through phosphorylation of AKT and / or ERK. Promoting proliferation and migration through phosphorylation of FAK and / or JNK can be done.

[0108] Example 6. Correlation between AURKA and DOCK2 expression levels in mesenchymal stem cells and cell death [Confirmation of the person in charge] 6-1. Cell death due to AURKA and DOCK2 gene expression in mesenchymal stem cells Confirmation of expression of cell death-related proteins To confirm the efficacy of mesenchymal stem cells in suppressing cell death, the expression of AURKA and DOCK2 was examined. The correlation between the amount of cleaved PARP and the expression of cleaved caspase 3 was confirmed. Specifically, 10% FBS and 1 U / ml penicillin / streptomycin (Gibco BRL, Gr and Island, NY) supplemented with DMEM (Dulbecco's Modified Eagle's Medium) (Biow The muscle cells C2C1 were cultured in a 6-well plate using a medium (Est. SAS, Nuaille, France). 2 was dispensed at a density of 8 x 103 cells / cm2 and cultured for 24 hours, followed by another 24 hours of F BS was starvated and cell death (apoptosis) was induced. After that, the muscle cells in which apoptosis was induced were transformed into mesenchymal stem cells using an insert. The primary antibody was cleaved PARP (poly ADP ribose polymerase) (Cell Signalling Technology, Danvers, MA), cleaved caspase 3 (Cell Signalling Tech) Biotechnology, Danvers, MA) and β-actin (Santa Cruz Biotechnology, USA). Western blotting was performed to compare the degree of cell death.

[0109] Figure 5A shows the results of co-culture of mesenchymal stem cells with muscle cells in which apoptosis was induced. The results of confirming the expression of cell death-related proteins according to the expression level of DOCK2 mRNA. is.

[0110] As a result, as shown in Figure 5A, compared with the control group in which cell death was induced, the mesenchymal Expression of cleaved PARP and cleaved caspase 3 in the experimental group co-cultured with stem cells Specifically, it was confirmed that the amount of erythrocytes in the control group, in which cell death was induced, was reduced. In comparison with the experimental group co-cultured with MSC_A or MSC_C, cleaved caspers The expression levels of clea 3 were reduced to 0.41±0.07-fold and 0.38±0.07-fold, respectively. The PARP expression levels were significantly higher in the aged group (0.37±0.06-fold and 0.35±0.05-fold), respectively. However, in the experimental group co-cultured with MSC_J, The expression level of cleaved caspase 3 was 0.66±0.07 times, and the expression level of cleaved PARP was 0. The increase in the cell death rate was 0.78±0.01 times, which was slightly different from the control group in which cell death was induced. In mesenchymal stem cells, low expression levels of AURKA and DOCK2 mRNA may contribute to muscle It is clear that the ability of meat cells to suppress cell death is reduced.

[0111] 6-2. Cell death in mesenchymal stem cells due to AURKA and DOCK2 gene expression levels (Confirmation of the degree of reduction in In order to confirm the degree of reduction in cell death of C2C12 cells in Example 6-1, Apopto Live / Dead status was detected using the sis / necrosis detection kit (Abcam, Cambridge, MA, USA). Specifically, cell death was induced in the same manner as in Example 6-1. The experimental group in which the muscle cells were co-cultured with mesenchymal stem cells was washed twice with PBS, and then the cells were Apopxin Green Indicator can confirm death and Apopxin Green Indicator can confirm living cells. The reaction was carried out for 1 hour in the dark using CytoCalcein Violet 450, which can be used for the detection of cytocalcium phosphate. The cells were then washed twice with PBS and photographed under a fluorescence microscope to obtain images. The percentage of apoptotic cells was determined using Image J.

[0112] Figure 5B shows the results of co-culture of mesenchymal stem cells with muscle cells in which apoptosis was induced. The results show the degree of reduction in cell death due to the amount of DOCK2 mRNA expression.

[0113] As a result, as shown in Figure 5B, compared with the control group in which cell death was induced, the mesenchymal In the experimental group co-cultured with stem cells, cell death was suppressed in all cases. In the control group where cell death was induced, the percentage of cells in which cell death occurred was 52.04 ± 0.01. While the figure was 1.93%, experiments in which the cells were co-cultured with MSC_A, MSC_C, or MSC_J In the 2 groups, the values ​​were 22.50±2.17%, 25.72±1.53%, and 31.8%, respectively. The expression level of AURKA mRNA decreased to 7±1.99% (p<0.001). In the experimental group co-cultured with MSC_A, which also has a high cellularity, the number of cells was significantly higher than in the experimental group co-cultured with MSC_J. The proportion of cells that had died was significantly reduced (p<0.001).

[0114] Therefore, mesenchymal stem cells with low levels of AURKA and DOCK2 mRNA expression are prone to cell death. When co-cultured with apoptosis-induced muscle cells, the efficacy of suppressing cell death was found to be inferior. I understand.

[0115] Example 7: Correlation between suppression of AURKA and DOCK2 gene expression and cell death [Confirmation of the person in charge] 7-1. Cell death-related proteins by suppression of AURKA and DOCK2 gene expression (Confirmation of protein expression) Knockdown of the AURKA and DOCK2 genes leads to cell death. To confirm whether the inhibitory effect was affected, muscle cells in which cell death was induced were examined. mesenchymal stem cells, siNC, and siAURKA or siNC selected in Example 3-1 above. After co-culture of mesenchymal stem cells treated with DOCK2, the same procedure as in Example 6-1 was repeated. One method confirmed protein expression.

[0116] FIG. 6A shows the results of knockdown of the AURKA and DOCK2 genes. The mesenchymal stem cells were co-cultured with muscle cells in which apoptosis had been induced, and the AURKA gene and The results confirmed the expression of cell death-related proteins by suppressing the expression of the DOCK2 gene. do.

[0117] As a result, as shown in Figure 6A, the experimental group co-cultured with mesenchymal stem cells showed significantly higher cell death rates than the control group. Compared with muscle cells in which cell death was induced, cleaved caspase 3 and cleaved PAR The protein expression of P was significantly reduced compared to the control group in which cell death was induced. , mesenchymal stem cells, siNC treatment group, siAURKA treatment group, and siDOCK2 treatment group. In cultured muscle cells, the expression level of cleaved caspase 3 was 0.34±0. 0.4 times, 0.30±0.02 times, 0.52±0.05 times and 0.40±0.04 times It was confirmed that the effect was slight (p<0.001). , mesenchymal stem cells, siNC treatment group, siAURKA treatment group, and siDOCK2 treatment group. In cultured muscle cells, the expression level of cleaved PARP was 0.39±0.07 times, 0.45±0.04 times, 0.72±0.043 times, and 0.54±0.045 times It was confirmed that there was little difference (MSC, siNC, siDOCK2, p<0.001; siA URKA, p<0.01). In particular, siAU co-cultured with muscle cells in which cell death was induced The RKA-treated group showed significantly higher expression of cleaved caspase 3 and cleaved PARP than the siNC-treated group. A statistically significant increase in the amount of serotonin was confirmed (p<0.01).

[0118] 7-2. Reduction of cell death by suppression of AURKA and DOCK2 gene expression (Confirmation) To confirm the degree of reduction in cell death by suppressing AURKA and DOCK2 gene expression To achieve this, mesenchymal stem cells, siNCs, and the aforementioned examples were added to muscle cells in which cell death was induced. The mesenchymal stem cells selected in 3-1 and treated with siAURKA or siDOCK2 were then After co-culturing, the results were confirmed in the same manner as in Example 6-2.

[0119] FIG. 6B shows the results of knockdown of the AURKA and DOCK2 genes. The mesenchymal stem cells were co-cultured with muscle cells in which apoptosis had been induced, and the AURKA gene and These results confirm the degree of reduction in cell death due to suppression of DOCK2 gene expression.

[0120] As a result, as shown in Figure 6B, the experimental group co-cultured with mesenchymal stem cells showed significantly higher cell death rates than the control group. It was confirmed that cell death was significantly suppressed compared to muscle cells in which cell death was induced. Specifically, in the control group where cell death was induced, the cell death rate was 41.73± 1.03%, while the mesenchymal stem cell, siNC-treated group, siAURKA-treated group, s In the case of muscle cells co-cultured with iDOCK2 treatment, 17.93 ± 1.40%, respectively. The results were 17.79±2.06%, 32.75±2.24%, and 25.62±2.29%. In particular, siAU co-cultured with muscle cells in which cell death was induced was significantly increased (p<0.001). It was confirmed that the cell death rate in the RKA-treated group was significantly higher than that in the siNC-treated group. (p<0.001).

[0121] Therefore, knocking down AURKA may enhance the inhibitory effect on cell death. Therefore, the effect of mesenchymal stem cells on muscle cell death is It appears to be more affected by AURKA than DOCK2.

[0122] (7-3. Confirmation of changes in XCL1 protein expression levels due to suppression of AURKA gene expression) The effect of mesenchymal stem cells on muscle cell apoptosis suppression is directly related to the expression level of the AURKA gene. Specifically, the siRNs selected in Example 3-1 above were used to confirm whether they were closely related to each other. A knockdown of the AURKA gene in mesenchymal stem cells The primary antibody was XCL1 (R&D, Minneapolis, MN, USA) and β-actin (Santa Cruz Biotechnology, USA) were used. Western blotting was performed in the same manner as in Example 3-2.

[0123] Figure 6C shows the X-ray analysis of mesenchymal stem cells in which the AURKA gene was knocked down. This shows the results of confirming the amount of CL1 protein expression.

[0124] As a result, as shown in Figure 6C, AUKRA gene expression was suppressed. This resulted in a decrease in the expression of XCL1, a protein involved in suppressing cell death. The siAURKA-treated group showed a 0.44±1 increase in XCL1 expression compared with the siNC-treated group. The AURKA gene was reduced by 0.07 times (p<0.01). By directly influencing expression, mesenchymal stem cells have the effect of inhibiting apoptosis of muscle cells It can be shown that

[0125] Example 8. Relationship between AURKA mRNA expression level in mesenchymal stem cells and cell death in muscle tissue Checking for correlation] 8-1. Suppression of muscle cell apoptosis by AURKA mRNA expression in mesenchymal stem cells (Confirmation) The effect of mesenchymal stem cells on muscle cell death suppression by AURKA mRNA expression level was investigated using annexin. Specifically, the expression of IL-1 in the tail vein of a mouse model of muscle dystrophy (Mdx) was confirmed by immunohistochemistry. The mesenchymal stem cells were administered in a single dose at 1×10 cells / 100 μl. Based on the results of 7, the mRNA expression of AURKA was determined to have a direct effect on the suppression of cell death. MCS_A shows the highest expression level, MCS_E shows the intermediate level of AURKA mRNA expression level, and MCS_J, which had the lowest AURKA mRNA expression level, was used. After one week, the mice were sacrificed. The animals were killed, and the calf muscles were isolated to obtain tissue samples. V (Abcam, Cambridge, MA, USA), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), The tamper-evident results were obtained in the same manner as in Example 3-2, except that a tamper-evident result was obtained using a tamper-evident result ... The expression of proteins was confirmed.

[0126] Figure 7A shows the expression of AURKA mRNA in mesenchymal stem cells in a mouse model of muscle dystrophies. This result confirmed the effectiveness of inhibiting cell death in muscle tissue due to differences in the amount of ingested.

[0127] As a result, as shown in Figure 7A, when MCS_A and MCS_E were administered, Compared with the Mdx control group, the expression levels of annexin V were 0.58±0.05-fold and 0. The decrease was 67±0.04 times, but there was no statistically significant difference between the control group and the MCS_J group. In other words, the lower the AURKA mRNA expression level, the greater the cell death suppression effect. It was observed that the concentration of α-glucan in the blood decreased.

[0128] 8-2. The degree of muscle cell death depending on the AURKA mRNA expression level in mesenchymal stem cells (Confirmation) The degree of muscle tissue cell death inhibition due to AURKA mRNA expression was measured using Annexin V staining. Specifically, the Mdx mouse model was confirmed by the same method as in Example 8-1. After separating the calf muscle, the tissue was washed and cut into paraffin tissue sections. The cells were then incubated with 5% blocking solution at room temperature. After that, the cells were incubated at 4°C for 18 hours in a 1 / 500 concentration solution. The cells were reacted with annexin antibody (Abcam, Cambridge, UK) diluted to 100%. After washing the tissue, Alexa Fluor™ 594 AffiniPure Goat Anti-rabbit IgG (H+L) was used. ) secondary antibody (Thermo Fisher Scientific, Rockford, IL) and incubated at room temperature for 1 hour. The cells were then counterstained with Hoechst 33342 (Thermo Fisher Scientific, Rockford, IL). er-stain) and fluorescent staining was performed on the tissue using a Carl Zeiss LSM 700 confocal microscopy system. Optical images were obtained. The captured images were analyzed for quantitative analysis using Java Image J software. Analysis was performed and values ​​relative to the Mdx control group were calculated.

[0129] Figure 7B shows that AURKA mRNA expression in mesenchymal stem cells was significantly increased in a mouse model of muscle dystrophies. This result confirmed the difference in inhibitory effect on muscle tissue cell death due to differences in the amount of supplementation.

[0130] As a result, as shown in Figure 7B, MCS_A, MCS_E, and MCS_J are applied. When administered, the levels were 0.41±0.05 times and 0.43±0.05 times higher than the Mdx control group, respectively. The relative intensity of Annexin V was reduced by 0.07-fold and 0.64±0.06-fold, respectively. When MCS_A, which has the highest AURKA expression, was administered, M, which has the lowest AURKA expression, was administered. Compared with the administration of CS_J, the relative intensity of Annexin V was significantly reduced. In other words, mesenchymal stem cells with high AURKA mRNA expression are the cells of damaged muscle cells. Since the efficacy of suppressing death is even greater, the mesenchymal stem cells can be used as a therapeutic agent for muscle diseases. It can be used.

[0131] Example 9. AURKA mRNA expression level in mesenchymal stem cells and the inhibitory effect on muscle tissue fibrosis Check the results 9-1. Determination of muscle tissue survival level based on AURKA mRNA expression level in mesenchymal stem cells approval) Muscle dystrophy mouse model (Mdx) induced by AURKA mRNA expression in mesenchymal stem cells The number of mesenchymal stem cells remaining in the leg muscles of the mice was confirmed. In one method, the thigh and calf muscles of the Mdx mouse model were separated and then treated with Gentra Pur DNA was extracted using the Qiagen Tissue kit (Qiagen Inc.), and human Alu primers were used. Real-time PCR was performed using this.

[0132] Figure 8A shows the expression of AURKA mRNA in mesenchymal stem cells in a mouse model of muscle dystrophies. 10 is a graph confirming the degree of cell survival depending on the amount of the test substance.

[0133] As a result, as shown in Figure 8A, MSC_A, MSC_E, and MSC_J are injected. The number of mesenchymal stem cells remaining in the leg muscles of a mouse model of muscle dystrophies treated with IFN-γ was 41. 23±5.60, 42.68±8.71 and 16.96±8.44. In particular, A When MSC_J, which has the lowest URKA mRNA expression level, was administered, muscle dystrophic mice It was confirmed that the number of mesenchymal stem cells remaining in Del's leg muscles was the lowest. Mesenchymal stem cells with high AURKA mRNA expression levels migrate into injured muscle cells and The large number of cells remaining at the site of injury may aid in muscle recovery.

[0134] 9-2. Suppression of muscle cell fibrosis by AURKA mRNA expression in mesenchymal stem cells confirmation) The effect of AURKA mRNA expression on muscle tissue fibrosis was investigated using fibronectin. Specifically, fibronectin (Abcam, C) was used as the primary antibody. Branbridge, MA, USA), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). Protein expression was confirmed in the same manner as in Example 8-2, except that Ta.

[0135] Figure 8B shows the expression of AURKA mRNA in mesenchymal stem cells in a mouse model of muscle dystrophies. The results confirmed the inhibitory effect of expression level on muscle tissue fibrosis.

[0136] As a result, as shown in Figure 8B, the mice administered MCS_A, MCS_E, and MCS_J showed a significant improvement in the pulmonary function. In the control group, the concentrations were 0.54±0.04 times, 0.60±0.03 times, and 0. The expression level of fibronectin was reduced by 71±0.05 times. Mesenchymal stem cells with high RNA expression reduced fibronectin expression in muscle tissue. This means that fibrosis of damaged muscle cells is effectively suppressed.

[0137] 9-3. Confirmation of muscle cell fibrosis by AURKA mRNA expression in mesenchymal stem cells ) The effect of AURKA mRNA expression on muscle tissue fibrosis was investigated by comparing collagen accumulation and Specifically, the Mdx mouse model was examined using the same method as in Example 8-1. After cleaning the calf muscle tissue, I used picro-sirius red (Solution A) The reaction was allowed to proceed at room temperature for 1 hour. After that, the plate was washed twice with acidified water (Solution B) and then After mounting, images were taken using a Scanscope. The images were quantitatively analyzed using Java Image J software, and the relative differences between the Mdx control group and the control group were analyzed. The relative values ​​were calculated.

[0138] Figure 8C shows the expression of AURKA mRNA in mesenchymal stem cells in a mouse model of muscle dystrophy. The results confirm the degree of suppression of muscle tissue fibrosis depending on the expression level.

[0139] As a result, as shown in FIG. 8C, MCS_A, MCS_E, and MCS_J are applied. When administered, the levels were 0.26±0.04 times, 0.27±0.03 times, and 0.26±0.04 times higher than the control group, respectively. The accumulation of collagen was reduced by 0.73±0.04 times. Mesenchymal stem cells with high RNA expression reduce collagen accumulation in muscle tissue, It can effectively inhibit the fibrosis of injured muscle cells.

[0140] The foregoing description of the invention is given by way of example only and is intended to be illustrative and not restrictive to those skilled in the art to which the invention pertains. If so, the present invention may be adapted to other specific forms without changing the technical idea or essential features of the present invention. It will be understood that modifications are possible. The described embodiments should be considered in all respects as illustrative and not restrictive. It must be.

Claims

1. A mesenchymal stem cell genetically engineered to secrete or overexpress DOCK2 or AURKA relative to parental cells, DOCK2 or AURKA is a gene involved in adhesion, differentiation, chemotaxis, or proliferation; The mesenchymal stem cells have improved migration ability, colony formation ability, and cell proliferation ability, or have a shorter cell replication time, compared to parent cells.

2. mesenchymal stem cells that have been genetically engineered to secrete or overexpress AURKA relative to parental cells; A functional health food composition for preventing or improving muscle diseases.

3. The functional health food composition for preventing or improving muscle diseases according to claim 2, wherein the muscle diseases are induced by cell death.

4. A cell therapy agent comprising, as an active ingredient, mesenchymal stem cells that have been genetically engineered to secrete or overexpress AURKA compared to parent cells.

5. The cell therapy agent according to claim 4, which is for treating a muscle disease.

6. The cell therapy agent according to claim 5 , wherein the muscle disease is induced by cell death.

7. The cell therapy agent according to claim 5 , wherein the muscle disease is fibrosis of muscle tissue.

8. A pharmaceutical composition for preventing or treating muscle diseases, comprising as an active ingredient mesenchymal stem cells that have been genetically engineered to secrete or overexpress AURKA compared to parent cells.

9. The pharmaceutical composition for preventing or treating a muscle disease according to claim 8, wherein the muscle disease is induced by cell death.

10. The pharmaceutical composition for preventing or treating a muscle disease according to claim 8, wherein the muscle disease is fibrosis of muscle tissue.

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