WO2016017863A1 - Pharmaceutical composition for preventing or treating autoimmune diseases, containing human bone marrow-derived mesenchymal stem cells as active ingredient - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases, containing human bone marrow-derived mesenchymal stem cells as an active ingredient. The human bone marrow-derived mesenchymal stem cells of the present invention exhibit prophylactic or therapeutic efficacy on autoimmune diseases by having no toxicity, suppressing the generation of anti-dsDNA antibodies, proteinuria, and total IgG, inhibiting the proliferation of T cells and B cells, and inhibiting cytokine expression of T cells. Lupus causes inflammation in the kidney by autoantibodies and immune complexes, and increases the infiltration of immune cells, and the administration of the human bone marrow-derived mesenchymal stem cells of the present invention reduces the infiltration of immune cells into the kidney, thereby exhibiting a prophylactic or therapeutic effect on the lupus.

Description

 [Specification】

 [Name of invention]

 Pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising human bone marrow-derived mesenchymal stem cells as an active ingredient

Technical Field

 This patent application claims priority to Korean Patent Application No. 10-2014-0097129 filed with the Korean Intellectual Property Office on July 30, 2014, the disclosure of which is incorporated herein by reference.

 The present invention relates to a pharmaceutical composition for preventing or treating autoimmune diseases comprising human bone marrow-derived mesenchymal stem cells as an active ingredient.

[Technique to become background of invention]

 Autoimmune disease is a disease caused by the body's immune function attacking itself, which is formed over a long period of time, the symptoms persist chronically, and usually cause permanent damage to organs. There is no reality. While knowledge of autoimmune diseases has evolved over the past 20-30 years, the exact mechanism of production, the identity of autoantigens, and regulatory genes are still unclear. Autoimmune diseases can be broadly divided into organ-specific diseases and systemic diseases.

 Organ-specific autoimmune diseases are caused by immune response to organ-specific antigens and can occur in almost every organ in our body. Systemic autoimmune diseases are caused by immune responses against antigens expressed throughout the body, rather than by an immune response against any particular cell. These systemic autoimmune diseases can also cause disease selectively in specific organs. _

Examples of autoimmune diseases include: i) ^' Rheumatoid Arthr itis, ^{' where the} immune system attacks tissues of various joints; ii) autoimmune of the central nervous system induced by T cells. In severe cases, multiple neuritis (MS), which can lead to blindness, paralysis, and premature death, iii) pancreatic insulin production The cells immune cells are ^destroyed, saenggimyeo MHC genes are important immune-mediated or type I diabetes (Immune-Mediated or Type 1 Diabetes Mel 1 i tus), iv) an immune system disease appears to attack the intestinal inflammatory bowel diseases (Inf lammatory Bowel Di seases), v) Scleroderma, which induces thickening of the skin or blood vessels (vi), vi) systemic autoimmunity, accompanied by symptoms such as deep fatigue, rash, joint pain, and in severe cases, the immune system And systemic lupus erythematosus (SLE), which can damage the brain, lungs, and the like. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained. [Content of invention]

 Problem to be solved

 The present inventors have made intensive studies to develop stem cells that can effectively treat autoimmune diseases and that can be safely applied to the human body. As a result, mesenchymal stem cells obtained from human bone marrow are very effective in preventing or treating autoimmune diseases. By clarifying that the present invention has been completed.

 Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of autoimmune diseases. Other objects and advantages of the present invention will become apparent from the following detailed description and claims.

[Measures of problem]

According to one aspect of the present invention, in the pharmaceutical composition for preventing or treating autoimmune diseases comprising human bone marrow-derived mesenchymal stem cells as an active ingredient, the human bone marrow-derived mesenchymal stem cells are (0 CD105, CD29, Positive immunological properties to at least one surface antigen selected from the group consisting of CD44, CD73 and CD90; And (ii) negative immunological properties to at least 1 ^' species of surface antigen selected from the group consisting of CD34, CD45 and HLA-DR. The present inventors have made diligent research to develop enjoyable cells that can effectively treat autoimmune diseases and safely apply them to the human body. As a result, the mesenchymal stem cells obtained from human bone marrow can be used to prevent or treat autoimmune diseases. It proved very effective.

 The human bone marrow-derived mesenchymal enjoyable cell of the present invention is at least one selected from the group consisting of CD105, CD29, CD44, CD73 and CD90 which are antigen-labeled antigens, preferably at least two, more preferably at least three More preferably at least 4 and most preferably 5 surface antigens. According to one embodiment of the present invention, the human bone marrow-derived mesenchymal stem cells of the present invention express 100% of the stem cell marker surface antigens CD105, CD29, CD44, CD73 and CD90, and more preferably 80-100 % Expression, even more preferably 90-99¾>.

 Meanwhile, the human bone marrow-derived mesenchymal stem cells of the present invention exhibit negative immunological characteristics in CD34, CD45 and HLA-DR which are surface antigens of hematopoietic stem cells. According to one embodiment of the present invention, the human bone marrow-derived mesenchymal stem cells of the present invention express 0-10% of CD34, CD45 and HLA-DR, which are surface hematopoietic stem cell-labeled surface antigens, and more preferably 0.1. -7%, most preferably 0.1-5%.

 The unit used while referring to the expression rate of the surface antigen refers to the ratio of cells expressing the surface antigen in the cells to be analyzed. For example, the 95% expression rate of CD29 surface antigen means that 95% of the cells express the CD29 surface antigen in the cell sample.

According to one embodiment of the invention, the autoimmune diseases which can be prevented or treated by the composition of the present invention are lupus (systemic lupus erythematosus), rheumatoid arthritis (rheumatoi d arthr itis), systemic scleroderma (Progressive systemi c sclerosi s) , Sc leroderma), atopic dermatitis, alopeci alopecia, Type I or immune-mediated diabetes, psoriasis, pemphigus, asthma, aphthous stomatitis, chronic thyroiditis, inflammatory enteritis, Behcet's disease, Crohn's disease, dermatomyositis, polymyositis, multiple sclerosis sclerosis, Autoimmune hemolytic anemia, Autoimmune encephalomyelitis, Myasthenia gravis, Grave's disease, Polyarteritis nodosa, Ankylosing spondylitis , Fibromyalgia syndrome or Temporal arteritis.

 According to another embodiment of the present invention, the autoimmune disease which can be prevented or treated by the composition of the present invention is lupus, rheumatoid arthritis, multiple sclerosis or type I or immune-mediated diabetes.

 According to a particular embodiment of the present invention, the autoimmune disease that can be prevented or treated by the composition of the present invention is lupus.

 According to one embodiment of the invention, human bone marrow-derived mesenchymal enjoyment cells of the present invention express / secrete TGF | 3 (transforming growth factor beta) -l in cells.

 TGF- is known to have three subtypes of TGF-βΙ, 2 and 3 in humans, and is known to be a potent regulator that lowers the intensity of immune responses. Human bone marrow-derived mesenchymal stem cells of the present invention reduce autoimmune reactions by expressing / secreting TGF-βΙ, which lowers the intensity of immune response.

 Human bone marrow-derived mesenchymal stem cells of the present invention exhibit increased expression levels of COX-2, HO-1, IFN-γ, IL-4 and IL-10.

 As used herein, the term "high expression" or "increased expression" means that the degree of expression of the nucleotide sequence of interest in a sample to be investigated (e.g., human bone marrow-derived mesenchymal stem cells) is a general sample (e.g., normal cells). ), Which is high (preferably 1.2 times or more).

According to one embodiment of the invention, the human bone marrow-derived mesenchymal stem cells of the invention inhibit the production of anti—dsDNA antibodies. According to one embodiment of the present invention, the human bone marrow-derived mesenchymal stem cells of the present invention inhibit the production of proteinuria.

 According to one embodiment of the present invention, the human bone marrow-derived mesenchymal stem cells of the present invention inhibit the production of IgG.

 According to the present invention, the human bone marrow-derived mesenchymal stem cells of the present invention reduce immune cell infiltration. Immune cells are infiltrated at the site or organ where autoimmune disease occurs (e.g., in the case of lupus, immune cells are infiltrated by the kidney). The human bone marrow-derived mesenchymal stem cells of the present invention reduce autoimmune reactions by reducing such immune cell infiltration. Suppress it.

 According to one embodiment of the present invention, the human bone marrow-derived mesenchymal stem cells of the present invention inhibit the proliferation of T cells or B cells involved in immune response. According to the present invention, the proliferation inhibition of the T cells is by soluble mediator or cell-cell contact.

 According to one embodiment of the present invention, the human bone marrow-derived mesenchymal enjoyment cells of the present invention inhibit cytokine expression of T cells. Cytokines of T cells whose expression is inhibited by human bone marrow-derived mesenchymal stem cells of the present invention are

IL-2, IFN-g, IL-4, IL-5.

 According to one embodiment of the present invention, the pharmaceutical composition for preventing or treating autoimmune diseases of the present invention is a composition for parenteral administration, in particular, for endovascular administration, and is administered to the human bone marrow-derived intermediate when the composition is administered intravascularly. Lobe stem cells inhibit anti-dsDNA antibodies, proteinuria and total IgG production without inhibiting toxicity, inhibit the proliferation of T cells and B cells, and inhibit cytokine expression of T cells to prevent or prevent autoimmune diseases. Therapeutic efficacy.

 The composition of the present invention comprises (a) a pharmaceutically effective amount of the human bone marrow-derived mesenchymal enjoyment cells described above; And (b) a pharmaceutically acceptable carrier. The term "pharmaceutically effective amount" as used herein means an amount sufficient to achieve the therapeutic efficacy or activity of the human bone marrow-derived mesenchymal stem cells described above.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Of the present invention Pharmaceutically acceptable carriers included in pharmaceutical compositions are those commonly used in the preparation of lactose, dextrose, sucrose, sorbbi, manny, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid. Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, PBS ( phosphate buf fered sal ine) or a medium, but is not limited thereto.

 Human bone marrow-derived mesenchymal stem cells of the present invention are used by suspending the cells in a pharmaceutically acceptable carrier and filling them into vials, plastic bags, or syringes.

 In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

 The pharmaceutical composition of the present invention may be administered orally, topically (buccal, sublingual, skin or ocular), transdermal or parenteral (subcutaneous, intradermal, intramuscular, intravascular or intraarticular), preferably Parenteral mode of administration, more preferably intravascular administration.

 Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and reaction in response to the patient. It may be prescribed. Typical dosages of the pharmaceutical compositions of the invention are 102-1010 cells per day on an adult basis.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of an excipient powder, powder, granule, tablet or capsule, and may further include a dispersing or stabilizing agent. Provided is a method for preventing or treating autoimmune diseases comprising administering to a subject a composition comprising human bone marrow-derived mesenchymal stem cells as an active ingredient: (i) Positive immunological properties to at least one surface antigen selected from the group consisting of CD105, CD29, CD44, CD73 and CD90; And (ii) negative immunological properties to at least one surface antigen selected from the group consisting of CD34, CD45 and HLA-DR.

 The method for preventing or treating chamoimmune diseases of the present invention is carried out using the above-described composition, and the common contents between the two are omitted in order to avoid excessive complexity of the present specification.

【Effects of the Invention】

 The features and advantages of the present invention are summarized as follows:

 (i) The present invention provides a pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising human bone marrow-derived mesenchymal stem cells as an active ingredient.

 (ii) Human bone marrow-derived mesenchymal stem cells of the present invention inhibit anti-dsDNA antibody, proteinuria and total IgG production without inhibiting toxicity, inhibit the proliferation of T and B cells, and cytokine expression of T cells By suppressing? It is effective in preventing or treating autoimmune diseases.

 (iii) Lupus causes inflammation in the kidney by autoantibodies and immunocomplexes, and increases the infiltration of immune cells. When administration of the human bone marrow-derived mesenchymal stem cells of the present invention, infiltration of immune cells into the kidney By reducing it has a prophylactic or therapeutic effect against lupus. [Brief Description of Drawings]

La-c is an image showing that the shape of the cells does not change during the second to ninth passage of human bone marrow-derived mesenchymal stem cells in the cell culture flask, and la is a bone marrow-derived mesenchymal stem of healthy donor 1 Cells are images, FIG. Lb is donor 2, and FIG. Lc is image of donor 3. FIG. Figure 2a-c is a graph showing the growth rate according to the culture period of healthy human bone marrow-derived mesenchymal stem cells. FIG. 2A is a graph showing donor 1, FIG. 2B is donor 2, and FIG. 2C is a growth rate of bone marrow-derived mesenchymal stem cells of donor 3 according to the culture period.

 3-5 are flow cytometer (FACs) using the third, fifth, seventh,

This is a graph analyzing surface antigens of mesenchymal stem cells during the 9th passage. Figure 3 shows the results of surface antigen analysis during the culture period of the mesenchymal stem cells of donor 1, Figure 3A is a graph of the surface antigen analysis at the third passage, B is the cell at the fifth passage, C Is a graph of the surface antigen analysis of cells recovered at the 7th passage, and D recovered at the 9th passage. 4 is an analysis result of mesenchymal stem cells of the donor 2 and FIG. 5 is an analysis result of the donor 3.

 Figure 6 is an image confirming the stability of cell culture through karyotyping during passage of bone marrow-derived mesenchymal stem cells of healthy donors. Figure 6a is an image showing the stability of the bone marrow-derived mesenchymal stem cells of the donor 1 during the subculture without change in karyotype, Figure 6b is the result of the donor 2, Figure 6c is an image showing the result of the donor 3. In each figure, A is the third passaged cell, B is the fifth, C is the seventh, and D is the ninth passage and the image confirmed the karyotyping from the recovered cells.

 Figure 7 is the result of measuring the gene expression of TGF-β, COX-2, H0-1 gene in human bone marrow-derived mesenchymal stem cells by RT-PCR.

 Figure 8 is the result of measuring the gene expression of CCL2, IFN-g, IL-10, IL-4, TGF-β in human bone marrow-derived mesenchymal stem cells by RT-PCR.

 9 is a graph confirming the expression level of TGF-β in secreted proteins secreted by the cell culture medium during the fourth passage of bone marrow-derived mesenchymal enjoyment cells of three donors.

 10 is a result of measuring the differentiation capacity of bone marrow-derived mesenchymal stem cells into adipocytes.

U is a result of measuring the immunogenicity of bone marrow-derived mesenchymal stem cells. 12 is a result of measuring the immune response inhibitory effect of bone marrow-derived mesenchymal stem cells.

Figure 13 shows the results of measuring cell population changes in MRL / lpr mouse organs. Phenotypic expression of mouse organ cells was determined by flow cytometry. Organ cells are B220-APC, CD4-APC, CDllc-APC, ^" CD3-PE, CD8-PE, CDllb-PE, CD138- APC + IgG-PE, CD4-FITC + Foxp3-PE and B220-APC + CD3- Staining with monoclonal antibodies such as PE.

 14 shows the results of measuring cytokine expression in MRL / lpr mouse spleen cells. Total RNA was isolated from immune cells of mouse spleen. Gene expression levels of IL-2, IFN-γ, IL-4, IL-10, TNF-α, IL-Ιβ, IL-12 and IL-6 were analyzed by RT-PCR. PCR products were electrophoresed on agarose gel and stained with ethidium bromide.

 15 is a result of measuring the survival rate (a), weight (b), anti-dsDNA amount (c), IgG amount (d) and proteinuria amount (e) following repeated administration of human bone marrow-derived mesenchymal stem cells. Mice divide into 3 counties. From 10 weeks to 20 weeks

Once every two weeks, 200 μl of a vehicle was intravenously injected into each mouse of the control group, and 1 × 10 6 to each mouse of the human bone marrow-derived mesenchymal stem cell treatment group once every two weeks from 10 to 20 weeks of age. Human bone marrow-derived mesenchymal stem cells were injected intravenously, once every two weeks from 10 to 20 weeks of age, 50 mg / kg of cyclophosphamide was intravenously injected into each mouse of the CPM-treated group. (A) Survival until 28 weeks of age. (B) Toxicity was measured by weighing mice up to 28 weeks of age. (C) Anti-dsDNA concentrations were measured every 2 weeks using ELISA. (2) IgG concentration is measured every 2 weeks using ELISA. (E) Proteinuria concentrations are measured every two weeks using ELISA. Significance was determined using the Student's t test on the control group (Op <0.05, ** p <0.01, *** p <0.001).

 16 shows the results of measuring anti-dsDNA amount (a), IgG amount (b) and proteinuria amount (c) following a single administration of human bone marrow-derived mesenchymal stem cells. Mouse

Divided into three counties. 200 μΐ vehicle was injected intravenously in each of the 9-week-old control mice, and 1 × 10 6 human bone marrow-derived mesenchymal stem cells were intravenously injected into each mouse of the _9- week-old human bone marrow-derived mesenchymal stem cell treatment group. 50 mg / kg CPM in each 9-week-old cyclophosphamide-treated group Intravenous injection. (A) Anti-dsDNA concentrations were measured every 2 weeks using ELISA.

(B) IgG concentration is measured every 2 weeks using ELISA. (C) Proteinuria concentration is measured every 2 weeks using ELISA. Significance was determined using the Student's t test on the control group (* p <0.05, ** ρ <0.01, *** ρ <0.001).

 Figure 17 shows the results of measuring the anti-dsDNA amount (a), IgG amount (b) and proteinuria amount (c) following a single administration of human bone marrow-derived mesenchymal stem cells. Mouse

Divided into three counties. A 200 μl vehicle was intravenously injected into each 12-week-old control mouse, and 1 × 10 6 human bone marrow-derived mesenchymal stem cells were intravenously injected into each mouse of the 12-week-old human bone marrow-derived mesenchymal stem cell treatment group. , 12-week-old cyclophosphamide treated group mice were injected intravenously with 50 mg / kg of CPM. (A) Anti-dsDNA concentrations were measured every two weeks using ELISA. (B) IgG concentration is measured every 2 weeks using ELISA. (C) Proteinuria concentration is measured every two weeks using ELISA. Significance was determined using the Student's t test on the control group (* p <0.05, ** p <0.01, *** p <0.001).

 Figure 18 is the result of measuring the anti-dsDNA amount (a), IgG amount (b) and proteinuria amount (c) following repeated administration of human bone marrow-derived mesenchymal stem cells. Mice divide into 3 counties. Once every three weeks from 12 to 20 weeks of age, 200 μl of a vehicle was intravenously injected into each mouse in the control group, and once every three weeks from 12 to 20 weeks of age, 1 x 106 human bone marrow-derived mesenchymal stem cells were intravenously injected into each mouse, and 50 mg / kg of cyc lophosphamide was intravenously injected into each mouse of the CPM-treated group once every two weeks from 12 to 20 weeks of age. (A) Anti-dsDNA concentrations were measured every 2 weeks using ELISA. (B) IgG concentration is measured every 2 weeks using ELISA. (C) Proteinuria concentration is measured every 2 weeks using ELISA. Significance was determined using the Student's t test for the control (* p <0.05, ** ρ <0.01, *** p <0.001).

19 shows the results of measuring the cell population change of MRL / lpr mouse organs after the treatment of human bone marrow-derived mesenchymal mesenchymal cells. Phenotype expression in mouse organ cells was measured by flow cytometry. Organ cells are B220-APC, CD4-APC, CDllc-APC, CD3-PE, CD8-PE, CDllb-PE, CD138-APC + IgG-PE, CD4-FITC + Foxp3-PE and B220-APC + CD3-PE Using mouse monoclonal antibodies such as Dyed. Significance was determined using the Student's t test against the control p <0.05).

 20 shows the results of measuring cytokine expression in MRL / lpr mouse spleen cells after human bone marrow-derived mesenchymal stem cell treatment. Total R A is isolated from immune cells of spleen cells. Gene expression levels of IL-2, IFN-γ, IL-4, IL-10, TNF-α, IL-Ιβ, IL-12 and IL-6 were analyzed using RT-PCR. PCR products were electrophoresed on agarose gel and stained with ethidium bromide. 21 shows the results of histological changes in MRL / lpr mouse kidney after human bone marrow-derived mesenchymal stem cells. MRL / lpr mouse kidneys were assessed with HE-stained sections. MRL / lpr mice 5 weeks old (a), vehicle-administered MRL / lpr mice 25 weeks old (b), human bone marrow-derived mesenchymal stem cell-administered 25 weeks old (c), CPM-administered MRL / lpr mice 25 (D) Age. x 400 times.

 22 is a result of measuring the survival rate (a), weight (b) according to the repeated administration of human bone marrow-derived mesenchymal stem cells. Mice divide into 3 counties. Once at 12 weeks of age, 200 μl of a vehicle was intravenously injected into each mouse of the control group, and once at 12 weeks of age, 4 X 104, 4 X 105, and 4 X 106 humans to each mouse of the human BM-MSC treated group. Bone marrow-derived mesenchymal stem cells were injected intravenously and once every 12 weeks of age, 50 mg / kg of cyclophosphamide was intravenously injected into each mouse in the CPM-treated group.

 (A) Anti-dsDNA concentrations are measured every two weeks using EUSA.

(B) IgG concentration is measured every 2 weeks using ELISA. (C) Proteinuria concentration is measured every 2 weeks using ELISA. Significance was determined using the Student's t test on the control group (* p <0.05, ** ρ <0.01, *** ρ <0.001).

 24 is a result of observing the histological changes in the kidney following the administration of human bone marrow-derived mesenchymal cell.

 25 is a schematic diagram showing the immune control of human bone marrow-derived mesenchymal stem cells.

Figure 26 shows the results of measuring the phenotype of human bone marrow-derived mesenchymal stem cells. Mouse bone marrow cells were stained using mouse monoclonal antibodies (CD34-PE, CD45-PE, CD103-PE, CD73-PE, CD90-PE, CD105-PE, CD44-FITC and Sca-1-FITC) . Mouse BM-MSCs cultured for 20 days were treated with mouse monoclonal antibody (CD34- (B) staining using PE, CD45-PE, CD103-PE, CD73-PE, CD90-PE, CD105-PE, CD44-FITC and Sca-1-FITC).

 27 shows the results of measuring the effect of human bone marrow-derived mesenchymal stem cells on B / T cell proliferation. Splenocytes are activated with LPS and ConA in the presence or absence of radioactive treated human bone marrow-derived mesenchymal stem cells in 96 well plates. Cell proliferation is assessed by [3H] -thymidine commonity. (M) BM-MSCs co-culture with Balb / c derived -B / T cells. (B) Co-culture with human bone marrow-derived mesenchymal stem cells ^ C57BL / 6 derived -B / T cells. Human bone marrow-derived mesenchymal stem cells are co-cultured with MRL / lpr-derived -B / T cells (c). Significance was determined using the Student's t test on the control group (* p <0.05, ** p <0.01, *** p <0.001).

 28 shows the results of measuring the effect of human bone marrow-derived mesenchymal stem cells on T cell cytokine production. Total RNA is isolated from Τ cells. Gene expression levels of IL-2, IFN-γ, IL-4, and IL-5 were analyzed using RT-PCR. (A) PCR products were electrophoresed on agarose gel and then stained with ethidium bromide. (B) IFN- Y and IL-2 levels in immune cell supernatants are measured using ELI SA.

 29 shows the results of measuring direct and indirect effects of human bone marrow-derived mesenchymal stem cells on B / T cell proliferation. B / T cells were co-cultured with human bone marrow-derived mesenchymal stem cells using a transwell system. Cell proliferation is assessed using [3H] -thymidine commonity. Splenocytes of MRL / lpr mice are activated with LPS in the presence or absence of irradiated MSCs in 96 well plates (a). Splenocytes of MRL / lpr mice are activated with ConA in the presence or absence of irradiated MSCs in 96 well plates (b).

Figure 30 is the result of measuring the expression of water-soluble elements in human bone marrow-derived mesenchymal stem cells. Human bone marrow-derived mesenchymal stem cells were harvested on day 20. (A) Gene expression levels of water soluble elements were analyzed using RT-PCR. (B) The level of water soluble urea in human bone marrow-derived mesenchymal stem cell supernatants was measured using ELISA. Significance was determined using the Student's t test for bone marrow cell populations (*** p <0.001). Figure 31 shows the result of measuring the expression of IDO in BM-MSC. (A) ID0 levels in human bone marrow-derived mesenchymal stem cell supernatants are measured by ELISA. Human bone marrow-derived mesenchymal stem cell-mediated T cell immune suppression mechanism (b).

Fig. 32 shows the results of measuring the motility behavior of T cells by human bone marrow-derived mesenchymal stem cells. Human bone marrow-derived mesenchymal stem cells were stained with CMTMR stain (red) for 15 min at 37 ^° C. T cells stained with CFSE dye (green) at 37 ° C for 15 minutes. Two-dimensional cell tracking was performed using Imar is software. Snapshot images of the interaction between human bone marrow-derived mesenchymal stem cells and T cells.

 33 shows the results of measuring the migration of T cells by human bone marrow-derived mesenchymal stem cells. Migration of T cells according to human bone marrow-derived mesenchymal stem cell concentration was measured by counting the number of cells located in the lower chamber after 1.5 hours of culture. Significance was determined using the Student's t test for the control (*** p <0.001). Figure 34 is the result of measuring the level of chemokine expression in human bone marrow-derived mesenchymal stem cells. Human bone marrow-derived mesenchymal stem cells were harvested on day 20. (A) Gene expression levels of chemokines were analyzed using RT-PCR. (B) CCL2, CCL5 and CXCL12 levels in human bone marrow-derived mesenchymal enjoyment cell supernatants were measured using ELISA. Significance was determined using the Student's t test for bone marrow cell populations ** p <0.001).

 Figure 35 shows the results of measuring the expression of chemokines after knocking down the CCL2 and CXCL12 genes, respectively, using s iRNA.

 36 shows the results of analyzing T cells involved in the movement of human bone marrow-derived mesenchymal stem cells.

 37 shows the results of analysis of contact patterns and contact time of human bone marrow-derived mesenchymal stem cells and T cells.

 38 shows the results of analyzing the role of CCR2 expressed in T cells in the contact of mouse MSCs and T cells.

[Specific contents to carry out invention] Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

 '

Example

Example 1 Isolation and In Vitro Proliferation of Bone Marrow-Derived Mesenchymal Stem Cells in Bone Marrow of Healthy Donors

About 30-50 mL of bone marrow was taken from the patient's posterior superior iliac spine (PSIS) using a Jamshidi needle and transferred to a heparin tube. The heparin tube containing bone marrow was transferred to a clean room and then the procedure was performed. Transfer the heparin tube containing bone marrow into a sterile tube and dilute it 1: 3 with the base medium (CSBM, CORESTEM Inc., South Korea), and add this dilution to the prepared FicolKFiocoll-PaqueTM PREMIUM, concentration 1.077 g / mL, GE Healthcare. The layers were dropped and centrifuged at 400Xg for 30 minutes. In the monuclear cell washing process, after the mononuclear cell layer separation, monocytes were added to the monocytes and centrifuged at 400 Xg for 10 minutes to harvest monocytes, and the monocytes were harvested. OOXg, centrifuged for 10 minutes. Washed monocyte cells were treated with 1% penicillin-streptomycin (Biochrmone, Germany), L-alanyl-L-glutamine (Biochrome, Germany), 10 (v / v) fetal bovine serum (FBS; Gibco, USA). After inoculation into a ΤΓ75 cell culture flask containing a basic medium (Nunc, USA) and incubated for 11 days at 37V, 7% C02 conditions. After incubation, the medium was exchanged twice every 3 or 4 days to remove cells that did not adhere to the bottom of the cell culture flask. At passage, adherent cells with at least 70-80% adhered to the bottom of the cell culture flask were trypsinized (0.125% trypsin-EDTA; Gibco, USA). Example 2: Cryopreservation and Thawing of Bone Marrow-derived Mesenchymal Stem Cells Cells were seeded in a ΊΤ75 cell culture flask (Nunc. USA) with 1.5 × 10 ⁵ −2 × 10 ⁵ cells per flask and incubated for 7 days at 37T 7% CO ₂ conditions. The medium was changed at 3 or 4 day intervals during cell culture.

 Cells proliferating at least 70-80% were harvested by trypsinization (0.125% trypsin-EDTA). The harvested cells were 20% fetal bovine serum and 10%

Cells were suspended in a cryopreservation medium 800 ^ consisting of CSBM with DMSOCdimethylsul foxide (Sigma, USA) at a density of 1 X 10 ⁶ cells / vial, followed by cryotube vial (Nunc). , USA). The cell suspension dispensed in the vial was placed in a cryopreservation container (Nalgene, USA) containing isopropyl alcohol. —801: Stored in a cryogenic freezer for 24 hours, and then transferred the vial containing cells to a liquid nitrogen storage tank the next day. Was

A vial containing 1 × 10 ⁶ cells / baral density cells was transferred from the liquid nitrogen storage tank to room temperature to remove liquid nitrogen and then quickly transferred to a 37 ^° C water bath. Cell suspensions thawed in vials were inoculated into cell culture flasks containing cell culture medium under sterile conditions. Density of the inoculated cells, 1.5 X 10 ⁵ per ΤΓ75 flask was such that 2 X 10 ⁵ cells present. Cell culture medium exchange was performed every 3 or 4 days, and the appearance and proliferation of the cells were observed under an optical microscope (Nikon, Japan). The passage of passage 2 (P2) to passage 9 (P9) was observed, as shown in Figure 1, it was confirmed that the uniform proliferation in the form of spindle like fibroblasts (Fig. La-c). Example 3: Analysis of Proliferation Rate of Bone Marrow-Derived Mesenchymal Stem Cells from Donors

Proliferation rate of stem cells was measured as the passage number of the isolated cells increased. Population ion doubling (PD) is an index indicating the proliferation rate of cells, and can be expressed as the expression of the cell growth rate (populat ion doubling, PD) = log (number of cells harvested / initial inoculation) / log 2 have. The initial inoculated cell number was determined as 1.5 X 10 ⁵ cells in a ΤΓ75 flask for each passage number, and the proliferation rate was determined by measuring the number of cells obtained after the culture. Recalling bone marrow-derived mesenchymal stem cells After the same separation method was obtained and subcultured. As a result of confirming the proliferation rate of the (P2-P11) cells for about 40 days through the growth curve graph, it was confirmed that the doubling time (populat ion doubling t ime. PDT) of the cell number was about 75.14 hours (FIG. ² ). Example 4 Surface Antigen Expression Analysis of Bone Marrow-Derived Mesenchymal Stem Cells from Donors

Fluorescence activated cell sorting (FACS) was used to identify stem cell labeled surface antigen expression characteristics of cells. Mesenchymal stem cells isolated from bone marrow were treated with 2% fetal bovine serum with 2 x 10 ⁶ cells at passage 3 (P3), passage 5 (P5), passage 7 (P7), and passage 9 (P9) during passage. Washed three times with the added DPBS (Gibco, USA), suspended at a concentration of 1 × 10 ⁵ cells / 100 ^, aliquoted in vitro and analyzed by FACS caliber (Cal ibur; Becton Dickinson, NJ, USA). Antibodies were composed of mesenchymal stem cell markers PE-CD29, PE-CD90, PE-CD44, PE-CD73 and PE-CD105 and hematopoietic stem cell markers PE-CD34 and PE-CD45. The factor PE-HLA-DR (MHC class Π) and the negative control antibody PE-immunoglobulin isotype IgGl were used, and all antibodies were produced using the product of Becton Dickinson, USA. It was. The used antibody was reacted with the cells in ice conditions for 30 minutes on ice, and the extra antibody was washed with DPBS (Gibco, USA), followed by flow cytometry.

More than 80% of the cells showed positive immunological properties for CD29, CD44, CD49C, CD73 and CD105, which are stem cell labeled surface antigens, and less than all negative immunological properties for hematopoietic stem cell labeled surface antigens CD34 and CD45. Indicated. As a result, the isolated cells were confirmed to have the characteristics of stem cells. In addition, since the HLA-DR (MHC class 11), which does not express histocompatibility antigen HLA-DR that causes rejection in tissue or organ transplantation, does not induce an immune response such as rejection that is a problem during transplantation of existing cells or tissues. Therefore, it could be confirmed that it can be used as a taga-derived cell (Fig. 3-5). Example 5: Karyotyping of Bone Marrow-Derived Mesenchymal Stem Cells from Donors Analysis was performed to confirm that the normal chromosome morphology and the number of chromosomes were maintained during cell isolation and culture. Inoculated with bone marrow-derived mesenchymal stem cells (P3, P5, P7, P9) by passage in a T75 flask with 1.75 X 10 ⁵ cells and then expanded to 75% density at 37 ^° C, 7% C02 Was harvested by trypsin treatment (0.125% trypsin-EDTA) and 25 GTG-banded metaphase of mitosis of harvested cells. As a result, the numerical and structural abnormality of the chromosome could not be found, and it was confirmed that the normal chromosome was maintained without changing the karyotype even in the continuous culture of the mesenchymal stem cells (FIGS. 6A-C). Example 6: Expression analysis of secretory factors of bone marrow-derived mesenchymal stem cells of donor

Factors secreted in human MSCs were confirmed by RT-PCR. To this end, first, human MSCs are recovered from the culture flasks, and then trizol 200! — 5 minutes at room temperature. 200 ml of chloroform was added to the same tube, followed by vortexing, reaction at room temperature for 5 minutes, and centrifugation at 4 ^° C and 12,000 rpm for 15 minutes. 500 ml of the supernatant was transferred to a new tube, and the same amount of isopropanol was added to react for 10 minutes at room temperature, followed by centrifugation at 4 ^° C and 12,000 rpm for 15 minutes. The supernatant was removed, washed with 1 ml of 7 EtOH in RNA pellet, and centrifuged at 4t ：, 12,000 rpm for 15 minutes to obtain RNA pellet. 50 ml of RNase free water was added to the obtained RAN, and the RNA pellet was dissolved at room temperature for 10 minutes, and then the concentration and purity of the RNA were measured using a nano-drop. CDMA was synthesized at a concentration of 1 mg of each RNA.

cDNA synthesis was performed according to the Maxime RT premix kit method. Reverse transcription reaction was carried out for 60 minutes at 45 ^° C in a nucleic acid amplifier (PCR machine), and 5 minutes at 95 ^° C for inactivation of reverse transcriptase. PCR was performed on the synthesized cDNA.

Real-time PCR machine used Rotor-Gene Q5 Plex. Proceeding with the gene-specific primer, the solution used in the test was Rotor-Gene SYBR green PCR kit 10 μ 1, 10 pmole F / R primer 0.6 μ 1, 1 yg template cDNA 2.0 μ 1, Rnase free water Put 7.4 μ 1 total Make a PCR lybe with a volume of 20 μ1 and PCR under the following conditions; Pre-denaturation: 95 ^° C 10 min, Denaturation: 95 ^° C 15 sec, Annealing & extension: 60 ^° C 1 min. Repeat this process 40 times.

 The gene expression of TGF-β, COX-2, H0-1 in human MSC was confirmed by RT-PCR method (FIG. 7A). TGF-β is an immunomodulatory cytokine and is known to induce differentiation of regulatory T cells, and is an important factor in controlling autoimmune diseases. C0X-2 is an enzyme that produces prostagl andin. Activation of C0X-2 in MSCs is known to produce prostaglandins and inhibit cellular responses proliferated by these substances. H0-1 is also an important factor in which MSCs are involved in regulating immune cells.

Treatment with IFN-g in human MSC was confirmed to increase the expression of ID0 along with TGF-β, COX-2, H0-1. ID0 is a protein that MSC plays an important role in suppressing the immune response and is a protein that is strongly involved in inhibiting the activity of macrophages, B cells, etc. (FIG. 7B). In addition to gene expression, human MSCs express proteins of TGF-β and prostaglandin E (PGE2). 8, it was confirmed that genes of CCL2, IFN-γ, IL-10, IL-4, and TGF-β are highly expressed in human MSCs. CCL2 secreted by MSC is a chemokine that acts critically in contact with immune cells, T cells. When the MSC expresses CCL2, the T cells move to where the MSC is located, thereby facilitating the MSC to inhibit T cells. 1_ increases the expression of B7-H1 in MSCs, stimulates contact with T cells, and induces an immunosuppressive response by MSCs. IL-4 is a cytokine that differentiates T cells into Th2 cells and activates M2 macrophages (anti-inflammatory) to inhibit the activity of Ml macrophages (inflammatory) and regulate inflammatory reactions. IL-10 secreted by MSC is a representative anti-inflammatory cytokine that regulates abnormally activated immune response. These results suggest that MSC may effectively suppress immune responses by regulating various chemokine and cytokine expressions. Example 7: Expression analysis of secretory factor of bone marrow-derived mesenchymal stem cells of donor Lupus was one of autoimmune diseases caused by disruption of the normal immune control system. To evaluate the expression level of the immunomodulatory factors secreted by mesenchymal stem cells themselves.

 Among the factors expressing bone marrow mesenchymal stem cells themselves, TGFP (transforming growth factor beta) -l expression affects inexperienced T cells and directly or indirectly differentiates into regulatory T cells. When the cells are administered to lupus patients, they may induce anti-inflammatory and excessively regulated immunity-regulating immunity, so at passage 4 (P4) of bone marrow-derived mesenchymal stem cells isolated from the donor. 50 μl of the cell culture solution was tested using the TGF-β ELISA kit (Quant ikine, R & D Systems) according to the kit's manual. As a result, when the passage 4 (P4) of the three donor bone marrow-derived mesenchymal stem cells, cells that secrete an average of 36 pg TGFP-1 per 10,000 cells, it was confirmed that the secretion of at least 20 pg (Fig. 9) . Example 8: Analysis of differentiation and immunomodulatory ability of bone marrow-derived mesenchymal stem cells of donor

Differentiation Capacity of Bone Marrow-derived Mesenchymal Stem Cells

 MSCs have multipotents that can differentiate into mature cells of various mesenchymal tissues such as chondrocytes, myocytes, adipocytes, and osteoblasts. Therefore, it was confirmed whether human MSCs can be differentiated into various cell systems in vitro.

 Cells were cultured in adipocyte differentiation medium for 14 days to analyze adipocyte differentiation capacity. Morphological changes of differentiated adipocytes were confirmed by oil red 0 staining. Differentiated adipocytes accumulated droplets, which appeared red when oil red 0 stained. This confirmed the differentiation into adipocytes (Fig. 10B).

Cells were cultured in osteoblast differentiation media for 21 days to analyze osteoblast differentiation. Alizarin red staining was used as a method of confirming differentiation, which is attached to calcium to give a red color. No staining with alizarin red was observed in the negative control of FIG. 10C. On the other hand, differentiated cells Stained red with Alizarin Red, confirming that a thorax was formed (FIG. 10D).

 Pellets were cultured for 28 days in chondrocyte differentiation medium to analyze chondrocyte differentiation capacity. Characterization of differentiated chondrocytes was performed immunological staining (Aggrecan staining method). Aggrecan used in the analysis is a protein polysaccharide present in cartilage. As can be seen in Figure 10, aggrecan protein was expressed in differentiated chondrocytes and chondrogenic formation was observed (Fig. 10E-G). Immune Regulation of Bone Marrow-derived Mesenchymal Stem Cells

 The immunogenicity and immunomodulation capacity of human MSCs against allogeneic immune cells were evaluated. PBMCs were isolated from blood of HLA type normal subjects and immunogenicity was measured by co-culture with three other human MSCs. Human MSC did not induce proliferation and IFN-Y production of allogeneic PBMCs, indicating no immunogenicity (FIG. 11).

 PBMC and humans proliferated by PHA, a lymphocyte proliferation inducer

The co-culture of MSCs confirmed the immune response inhibitory effects of human MSCs.

Proliferation of PBMC was induced by PHA, and proliferation was inhibited and production was inhibited by human MSC (FIG. 12). Example 9 Experimental Method for Determining Lupus Efficacy of MSC

 Human MSC

 Human systems were provided for use in animal experiments in the core. Lupus animal experiment

MRL / lpr mice, widely known as spontaneous lupus animal models, were used. Human MSCs were injected intravenously into mice at a concentration of lxlO ⁶ cells / mouse. As a positive control group, cyclophosphamide (CPM), an immunosuppressive agent, was used, and mice were injected intravenously at a concentration of 50 mg / kg. Mouse MSC Culture Balb / c mice were regenerated by the cervical spinal bone method, and single cells were obtained by separating the bone marrow in the femur and tibia using a 10 m syringe. The impurities were filtered out using a 0.7 μιιι filter and the cells were centrifuged at 1,200 rpm for 3 minutes. After centrifugation, the supernatant was removed and treated with ACK lysis buffer for 1 minute to remove red blood cells. The final isolated cells were diluted in a—MEM medium with a cell number of 1 × 10 ⁷ cells and incubated in a 37 ° C. incubator fed with 5% C02. New medium 1 111 £ was added daily for 5 days of incubation. On day 6 of culture, ci-MEM medium was removed, washed with PBS, and fresh medium 1 was added. Cells 20 days old were used for the experiment. Cell Phenotyping Analysis

Cell surface analysis was performed using a flow cytometer. The recovered cells (1 × 10 ⁶ cells /) were washed with 0.5% BSA / PBS. The supernatant was removed and 50% 0.5% BSA / PBS containing monoclonal antibody was added and stained at 4 ^° C. for 20 minutes. After washing again with 500 ^ PBS / BSA, 500 ^ of PBS / BSA was added and suspended. Monoclonal antibodies include PE-binding CD3, CD8, CD34, CD45, CD73, CD90, CD103, CD105 CDllc, CDllb, IgG ᅳ Foxp3, FITO binding CD44, Sac-1, CDllc, CD4, APC-binding B220, CD138 and CD4 Was used. Data analysis was analyzed using WinMDI software. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Was by using a reagent TRIZ0L separating total RNA from tissues and cells, the cDNA using 0.3 μ _§ total RNA was prepared 60 minutes at 42 ^° C, 5 minutes at 94 ^° C. PCR (polymerase chain react ion) was performed using the prepared cDNA 3 ^ and 10 M primer. The basic conditions of PCR are the pre-denaturation step ， 94 ^° C., starting at 5 min, the denaturation step ， 94 ° C. 30 sec 30 cycle ， annealing step, 72 ^° C 1 min and after-extension step at 72 ^° C. 5 minutes conditions were performed. The annealing temperature per primer was performed differently between 54-56 ^° C. The resulting PCR product was electrophoresed at 50 V after loading 8 ^ by 1% agarose gel. HE staining

 Kidneys extracted from MRL / lpr mice were fixed in 10 3/4 paraformaldehyde for 18 hours and then 4 paraffin sections were prepared. Paraffin was removed from the tissue slide using xylene, hydrated with ethanol and washed with running water for 10 minutes. After staining with hematoxylin solution for 1 minute, the solution was placed in running water for 1 minute to remove residual dye solution. Thereafter, staining was performed using an eosin solution for 1 minute. The stained slides were dehydrated using alcohol and xylene and observed and photographed with an optical microscope. Cell migration assay

Mobility of T cells relative to MSC was measured using a transwell. 1 X 10 ⁵ T cells were placed in the upper chamber, 0.3 X 10 ⁴ , 1 X 10 ⁴ , 3 X 10 ⁴ MSCs were added to the lower chamber and reacted for 1.5 hours in a 37 ^° C. incubator. The cell number then moved to the lower chamber was measured using a flow cytometer. Cell proliferation assay

Thymidine uptake is performed using cells isolated from the spleen of MRL / lpr mice. Splenocytes are diluted in a RPMI complete medium composition with a cell count of 1 × 10 ⁶ cells. Splenocytes were treated with 1 concentration of LPS and ConA, and treated with MSC by cell concentration, and then cultured in a 37 ^° C incubator. After 54 hours, thymidine was treated at a concentration of 1 μ / well, and after 18 hours of incubation, the cells were collected in a glass fiber filter using a cell harvester and then dried for 2 hours. The dried glass fiber filter was placed in a sample bag, and then the filter was thoroughly wetted and sealed with a scintillation cocktail. Finally, the glass fiber filter was measured using a beta counter to measure the amount of radiation introduced into the DNA of the cells. Enzyme Immunoassay (ELISA)

Sera and urine of mice were obtained and anti-dsDNA ELISA kit, IgG ELISA kit, proteinuria ELISA kit was used. In addition, TGF-β was obtained by obtaining a supernatant of MSC. EL ISA kits, PGE2 ELISA kits, and IDO EL ISA kits were used, and the procedure was described in each kit. Cell Imaging

 Cell images were measured using a cell culture microscope. MSc 5 μΜ

CMTMR dyes were treated and stained at 37 ^° C. for 15 minutes, and T cells were treated with 5 μM CFSE dye and stained at 371: for 15 minutes. 35誦then gave the MSC into the cells Τ of 5 X 10 ⁴ cells in culture dishes with 5 X 10 ³ cells was measured for cell imaging. Cell images were measured for 6 hours at 2 minute intervals and data were analyzed using Imaris software. Histopathology

Kidney tissues isolated from the mouse were fixed in 10% formaldehyde solution for 3 days, paraffin blocks were made, cut to 4 mm, and slides were prepared. H & E (hematoxylin-eosin) or PAS (periodicacid-Schiff) staining was used to observe histological changes. Immunohistochemical staining (Immuno-histochemistry) first put the slides in 0.01 M citrate solution _: heat to express the antigen. The slide was placed in a solution containing 3% ¾0 ₂ in methanol, and the endogenous peroxidase activity was blocked. Slides were reacted overnight at 4 ^° C by diluting CD3, B220, F4 / 80, Foxp3, CD209b antibodies. The 3,3'-diaminobenzi dine (DAB) staining was performed using the Vectastain ABC kit. After immunohistostaining, hematoxylin was stained as a control. Example 10: Characterization of lupus treatment of human MSC

To verify the therapeutic effect of human MSC, we adopted MRL / lpr, which is widely known as a naturally occurring lupus animal model. MRL / lpr shows high levels of autoantibodies such as anti-ssDNA antibody, anti-dsDNA antibody, and rheumatoid factor and high concentration of immunoglobulin, resulting in an increase in the amount of immunocomplexes. This excessive phenotype is due to an autosomal recessive mutation called lymphoproliferation (lpr). The lpr mutation, located on chromosome 19, is responsible for the Fas receptor Transform transcription. As a result, a deficiency in Fas signaling inhibits apoptosis and results in lupus symptoms. The difference from the classical NZB / W F1 used in lupus animal models is the rapid death of mice and the presence of lupus symptoms in both females and males.

MRL / MpJ-Faslpr / J mice were purchased as one of the MRL / lpr mice and analyzed before and after onset. As described above, the mouse is mutated by the Fas receptor, and lymphatic proliferation produces an immune complex, at which time the female is about 12 weeks old, and on average, the female begins to die at about 22 weeks old. First, 6-week-old mice before lupus and 25-week-old mouse organs were extracted and analyzed. After the onset, the weight and cell number of the mouse organs were increased than before the onset (Table 1). The flow cytometry was analyzed to observe the phenotypic changes of the cells obtained in each organ. The most noticeable change after the onset of MRL / lpr mice was an increase in the proportion of B220 ⁺ CD3 +, a phenotype of lymphoma in almost all tissues. In addition, the proportion of CD4 + Foxp3 ⁺ , the phenotype of Treg cells responsible for immunosuppressive reactions, decreased after the onset (FIG. 13). RT-PCR was performed after RNA was isolated from splenocytes of mice to analyze cytokine expression as MRL / lpr mice progressed. As a result, it was confirmed that the expression of inflammatory cytokines in the advanced mice was increased than before the onset (Fig. 14).

Table 1

W IJLpr mouse body weights, organ weights, changes in organ cell numbers ^

MSCs have been reported to be easy to transplant because of the low expression of HLA-DR, which is the main cause of graft-versus-host reaction, and little phenotype of CD40, CD80, and CD86 (Ryan JM, Barry FP, Murphy JM, Mahon BP. , J Infla Lond). Jul 26; 2: 8 (2005)).

MRL / lpr mice were used to measure the lupus improvement effect of human MSC. MSC was injected intravenously into mice at a concentration of lxlO ⁶ cells / mouse, and the administration time was injected 6 times at 2 week intervals starting at 10 weeks of onset of the onset of mice. As a measure of animal experiments, the survival rate and weight of the mice were checked weekly, and anti-dsDNA, proteinuria, and IgG were measured at two week intervals. Cyclophosphamide (eye 1 ophosphami de) was used as a positive control. MRL / lpr mice began to die at 13 weeks of age and 90% of mice died at 22 weeks of age. Human MSCs increased the survival rate of mice, 90% of mice survived by 28 weeks of age (FIG. 15A). Human MSC did not affect the weight of MRL / lpr mice, from which it can be seen that human MSC is not toxic to the mouse (Fig. 15b). The concentration of anti-dsDNA Ab in the blood of MRL / lpr mice increased from 10 weeks of age and peaked at 20 weeks of age. Human MSC had the effect of reducing the concentration of anti-dsDNA Ab in the blood (FIG. 15C). The concentration of total IgG in blood of MRL / lpr mice increased from 8 weeks of age, peaked at 20 weeks of age, and human MSC inhibited the increase of total concentration of IgG in blood (FIG. 15D). Proteinuria levels in MRL / lpr mice steadily increased from 20 weeks of age, and human MSC decreased proteinuria concentrations (FIG. 15E).

 In summary, human MSC inhibited the anti-dsDNA Ab, proteinuria, and total IgG production of MRL / lpr and increased mouse survival. It was similar to the therapeutic effect of cyclophosphamide in current clinical use. However, weight loss cyclophosphamide was toxic while human MSC was not toxic (FIG. 15).

In the following animal experiment, MRL / lpr mice were injected intravenously with human MSC of 1 × 10 ⁶ cells / mouse only once at 9 weeks before lupus onset to confirm the effect of improving lupus. Human MSC inhibited anti-dsDNA and IgG production in MRL / lpr mouse blood and proteinuria production. However, the effect was strongly observed for 3 weeks after administration, but after 4 weeks the treatment effect tended to be weak (FIG. 16).

The effect of lupus improvement was confirmed by intravenously injecting human MSC of 1 × 10 ⁶ cells / mouse into MRL / lpr mice only once at 12 weeks of onset of lupus. Human MSC inhibited anti-dsDNA and IgG production in MRL / lpr mouse blood and proteinuria production. However, the inhibitory effect was observed for 3 weeks after administration, but after 4 weeks the therapeutic effect tended to be weak (FIG. 17). Through the animal experiments, it can be seen that when the human MSC is administered to MRL / lpr mice, the effects vary depending on the frequency of administration. The effect lasted for 3 weeks after a single administration, but weakened after 4-5 weeks. In the following animal experiment, MRL / lpr mice were injected intravenously with human MSC at a concentration of 1 × 10 ⁶ cells / mouse three times at 12 week intervals from week 12. Human MSC inhibited anti-dsDNA, IgG production in MRL / lpr mouse blood and also inhibited proteinuria production (FIG. 18).

Mice were autopsied and analyzed for organ weight, immune cell phenotype, cytokine expression, and cellular invasion. As a result of measuring the organ weight of the mouse, the group of human MSC did not show a significant difference in organ weight compared to the control group. Was reduced (Table 2).

Table 2

 Changes in Body Weight, Organ Weight and Organ Cell Number in ¾¾yLpr Mice

The flow rate of immune cell phenotypes in mouse organs was measured by flow cytometry. The proportion of CD138 ⁺ IgG and lymphoma phenotype B220 ⁺ CD3 ^{+ in} the human MSC group was decreased compared to the control group. The ratio of CD4 + Foxp3 ⁺ was increased (FIG. 19). As a result of analyzing cytokine expression by separating RNA from splenocytes through RT-PCR, the amount of inflammatory cytokine expression in the MSC-administered group was significantly decreased compared to the control group (FIG. 20).

 Lupus disease causes inflammation in the kidneys by autoantibodies and immunocomplexes, increasing immune cell infiltration. Mouse kidneys were isolated and cell infiltration was confirmed by HE staining. Human MSCs were found to reduce immune cell infiltration into the kidneys (FIG. 21).

In order to determine the minimum concentration, frequency and route of administration of human MSCs in MRL / lpr mice, human MSCs were determined as 4xl0 ⁴ , 4xl0 ⁵ , Single intravenous administration at 12 weeks of age with 4xl0 ⁶ cells / head. Survival and weight were measured weekly, and serum was separated every other week to measure the concentration of anti-dsDNA antibody and IgG, and urine was separated to measure protein concentration. At 21 weeks of age the observations were terminated and the kidneys were removed for histological analysis.

 Human MSC increases MRL / lpr mouse survival and lowest effective concentration

4xl0 ⁵ cells / 40 g and had a therapeutic effect with a single intravenous administration (FIG. 22). Human MSC significantly inhibited anti-dsDNA antibody, IgG, and proteinuria production in a concentration-dependent manner, but the higher the concentration, the better the administration effect (FIG. 23). Histopathologically, the kidneys of the control group increased immune cell infiltration of T cells, B cells, neutrophils, macrophages, and dendritic cells by inflammatory reaction, and all decreased by human MSC administration. In contrast, the percentage of regulatory T cells was increased by human MSC administration (FIG. 24). Example 11: Study of immunosuppressive mechanism of MSC

 MSCs have been known to inhibit innate i unity and apoptotic immunity, as well as to inhibit direct contact between cells and soluble factors. Soluble factors secreted by MSC include NO, O, TGF-β, IL-10, and PGE2. These soluble factors are reported to inhibit the activity and function of T cells, B cells, NK cells and dendritic cells (Fig. 25). Mouse bone marrow-derived MSCs were used for mechanism studies. Flow cytometry was used to confirm the phenotype of MSCs produced in mouse bone marrow. The cell phenotype of MSCs derived from the bone marrow of mice decreased expression of the hematopoietic stem cell phenotypes CD34, CD45, and CD 103 compared to the bone marrow cells of the mouse, and the expression of MSC phenotypes CD73, CD90, CD 105, CD44, and Sca-1. This increased (Figure 26).

Inhibition of Proliferation of Mouse T and B Cells by MSC

It was confirmed by mitogen analysis whether MSC inhibited the proliferation of mouse Τ and Β cells. MSCs were prepared using bone marrow cells from Balb / c mice, and isolated from splenocytes of Balb / c (syngenic), C57BL6 (al logenic), and MRL / lpr (al logenic) mice. MSC and T cells were mixed at a ratio of 0.001-0.1: 1, and treated with ConA to T Proliferation of the cells was induced. Experimental results confirmed that MSC inhibits ConA-induced T cell proliferation (FIG. 27). MSC and B cells were 0.001-0. 1: 1 ratio and LPS treatment to induce the proliferation of B cells. Experimental results confirmed that MSC inhibits LPS-induced B cell proliferation (FIG. 27).

Inhibition of Cytokine Expression in Mouse T Cells by MSC

 Whether MSC inhibited cytokine expression in mouse T cells was confirmed by RT-PCR and ELISA. MSCs were prepared using bone marrow cells from Balb / c mice, and T cells were isolated from splenocytes of MRL / lpr (al logeni c) mice. MSC and T cells were mixed at a ratio of 0.1: 1, and ConA was used to induce cytokine expression of T cells. Experimental results showed that MSC inhibits the expression of IL-2, IFN-g, IL-4, IL-5 in T cells (FIG. 28). MSC immunosuppressive effect according to cell contact

 Transwell assay was performed to determine whether the MSC immunosuppressive effect was due to cell contact or water soluble mediators. Balb / C-derived MSC and MRL / lpr mouse-derived B / T cells were tested at a ratio of 1:10.

 When MSC and B cells are mixed and added to the lower well, MSC can secrete contact or water-soluble mediators to inhibit the proliferation of B cells, MSC to the upper well and MSC to add B cells to the lower well. Can inhibit the proliferation of B cells through soluble mediators only.

 As can be seen in Figure 29a, the addition of MSC to the upper well and the lower well inhibited the proliferation of B cells, but the addition of MSC to the upper well reduced the inhibitory effect. This means that MSCs inhibit B cell function not only by water soluble mediators but also by cell-cell interactions. 29B, it can be seen that MSC inhibits the proliferation of T cells by soluble mediator and cell-cell contact.

Soluble Factors Secreted by MSC RT-PCR and ELISA were performed to analyze the solubility factor secreted by MSC. MSC expressed more NO, TGF-β, PGE2 than the control group (Fig. 30). MSC itself does not express the inhibitory substance ID0 (FIG. 30), but it was confirmed that the amount of ID0 expression of MSC increased when co-cultured with T cells (FIG. 31).

Observation of Interaction Between MSC and T Cells

 In order to directly observe the interaction between MSC and T cells, the movement of cells was measured using a real-time microscope. Image analysis confirmed that the movement of τ cells was classified into three types. The first type of T cells migrated freely without any contact with MSCs and at a rate of about 5 / min (FIG. 32A). The second type of T cells were in continuous contact with MSC and the rate was reduced to 1.5 / min (FIG. 32B). The third type of T cells were in contact with MSCs during migration, which showed a rate of about 5 / min at the beginning of migration, but decreased at a rate of about 1 / min after contact with MSCs (FIG. 32C). When co-culture of two cells, it was confirmed that T cells gathered around the MSC to form a herd (FIG. 32D). Next, we studied the mechanism by which T cells gather around the MSC to form a herd. MSC was added to the lower wells of the transwell and T cells were added to the upper wells. After 90 minutes, the number of T cells that migrated to the lower wells was measured, and the migration of T cells toward MSC was measured. As the concentration of MSC in lower wells increased, it was confirmed that T cell migration increased (FIG. 33). Next, we tried to identify a substance that induces T cell migration. The expression level of chemokine, a representative substance causing migration, was analyzed by RT-PCR and ELISA. As shown in Figure 34, MSC was confirmed to express higher CCL2, CXCL12 than the control group. Analysis of chemokines involved in contact between mouse MSCs and T cells

In order to identify chemokines involved in the contact between mouse MSCs and T cells, each gene was knocked down by treating siRNAs of CCL2 and CXCL12 which are highly expressed in MSC. MSCs whose expression is reduced by each gene siRNA are named CCL2-KD MSCs and CXCL12-KD MSCs. CCL2 siRNA on mouse MSC (FIG. 35A) and CXCL12 s iRNA (FIG. 35B), respectively, inhibited the chemokine gene expression and protein production.

 MSC was added to the lower wells of the transwells and T cells were joined to the upper wells. At this time, MSC was used as a control MSC not treated anything, CCL2-KD MSC and CXCL12-KD MSC. The number of T cells that migrated to the lower wells was measured to determine the migration of T cells toward the MSC. As a result, the migration of T cells to CCL2-D MSCs was reduced (FIG. 35C). CXCL12-KD MSCs were the same as control MSCs, and T cells in the upper wells migrated to the lower wells in a concentration-dependent manner of MSCs. In the observation on the image, T cells were not observed to move in the direction of CCL2-KD MSC (FIG. 35D).

Analysis of the movement of the MSC

 The contact between MSC and T cells was confirmed and analyzed for their movement. T cells that do not contact MSCs based on contact with MSCs are called wandering cells, and T cells that try to contact MSCs are called searching T cells, and T cells that contact MSCs are called contacting T cells. (FIG. 36A).

 CCL2-KD MSC and CXCL12-KD MSCs were analyzed for changes in contact with T cells. CXCL12-KD MSC showed no significant difference in T cell contact pattern with control MSC. However, CCL2-KD MSC was found to increase the proportion of wander ing T cells and decrease the ratio of contact ing T cells compared to the control MSC (Fig. 36B).

 Wander ing T cells and searching T cells were free to move without a specific direction and contact ing T cells in contact with MSC was stagnant in one place with little movement (Fig. 36C). This movement was the same regardless of the MSC condition. The rate of contact ing T cells was lower than that of Wander ing T and searching T cells.

There was no difference from MSC, CCL2-KD MSC, CXCL12-KD MSC (FIG. 36D).

Contact time analysis of MSCs and T cells MSC and T cells were co-cultured and analyzed for 6 hours. The contact time between the MSC and Τ cells was indicated by a red arrow. 37A, the contact time of MSC and T cells is not constant and variously observed. However, the contact time of CCL2-KD MSC was shorter than that of control MSC and CXCL12-KD MSC.

 The number of T cells contacted with control MSC, CXCL12-KD MSC, and CCL2-KD MSC after 6 hours was 11.4, 10.6 and 10.2, respectively (Fig. 37B). However, it was found that the contact time between CCL2-KD MSC and T cells was significantly low. Contact times of control MSC, CXCL12-KD MSC, CCL2-KD MSC and T cells were measured at 107, 97 and 34 minutes, respectively (FIG. 37C). As a result, it was found that the chemokine CCL2, which is expressed by MSC, plays an important role in the contact between MSC and T cells. Role of CCR2 Expressing T Cells in Contact with Mouse MSCs and T Cells

 In order to complement the result that CCL2 is important when contacting mouse MSC and T cells, a test was performed using CCR2 antagonists. CCR2 is a chemokine receptor expressed in T cells as a receptor for CCL2. Treatment of CCR2 antagonists with T cells by concentration showed that cytotoxicity was observed at 100 u g / ml or higher (FIG. 38A).

 T cells treated with CCR2 antagonists at concentrations of 3, 10 and 30 g / ml were seeded into upper wells of the transwells. MSCs were inoculated into the lower wells and the T cell migration was measured. As the concentration of CCR2 antagonist increased, the number of T cells migrated to MSC decreased.

CCR2 antagonists were treated with 30 ug / ml all T cells and seeded in the upper wells of the transwells. The lower wells were treated with MSC at different concentrations and observed for T cell migration. T cells not treated with CCR2 antagonists increased the concentration of MSCs to MSCs. However, T cells treated with CCR2 antagonists inhibited migration to MSCs (FIG. 38C). Therefore, the interaction between CCL2 of MSC and CCR2 of T cells proved that contact between cells occurred. Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

[Patent Claims]

 [Claim 1]

 In the pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising human bone marrow-derived mesenchymal stem cells as an active ingredient, the human bone marrow-derived mesenchymal stem cells are (i) CD 105, CD29, CD44, CD73 and CD90 Positive immunological properties to at least one surface antigen selected from the group consisting of; And (Π) having immunological properties negative for at least one surface antigen selected from the group consisting of CD34, CD45 and HLA-DR.

[Claim 2]

 The method of claim 1, wherein the autoimmune disease is lupus (systemic lupus erythematosus), rheumatoid arthritis, progressive systemic sclerosis (Scleroderma), atopic dermatitis, alopecia areata, psoriasis, pemphigus ， Asthma, Aphthous stomatitis, Chronic thyroiditis, Inflammatory enteritis, Behcet's disease, Crohn's disease, dermatomyositis, Multiple myositis, Multiple sclerosis, Autoimmune hemolytic anemia (Autoi 隱 une) hemolytic anemia, autoimmune encephalomyelitis, myasthenia gravis, Grave's disease, polyarteritis nodosa, ankylosing spondylitis, fibromyalgia syndrome and tempoarthritis Pharmaceutical composition, characterized in that it is selected from the group consisting of (Temporal arteritis).

[Claim 3]

 The method of claim 1, wherein the human bone marrow-derived mesenchymal stem cells are transforming growth factor beta (TGF-β) -1, COX-2, HO-1, IFN- γ, IL-4 and

Pharmaceutical composition, characterized in that the expression level of IL-10 is increased.

[Claim 4] The pharmaceutical composition of claim 1, wherein the human bone marrow-derived mesenchymal stem cells inhibit the production of anti-dsDNA antibodies.

[Claim 5]

 The pharmaceutical composition of claim 1, wherein the human bone marrow-derived mesenchymal stem cells inhibit the production of proteinuria.

[Claim 6]

 The pharmaceutical composition of claim 1, wherein the human bone marrow-derived mesenchymal stem cells inhibit the production of IgG.

[Claim 7]

 The pharmaceutical composition of claim 1, wherein the human bone marrow-derived mesenchymal stem cells reduce immune cell infiltration into the kidney.

[Claim 8]

 The pharmaceutical composition according to claim 1, wherein the human bone marrow-derived mesenchymal stem cells inhibit the proliferation of T cells or B cells.

[Claim 9]

 The pharmaceutical composition of claim 8, wherein the inhibition of T cell proliferation is by a soluble medi ator or cell-cell contact.

[Claim 10]

 The pharmaceutical composition of claim 1, wherein the human bone marrow-derived mesenchymal stem cells inhibit cytokine expression of T cells.

[Claim 11] The pharmaceutical composition according to claim 1, wherein the composition is for parenteral administration.

[Claim 12]

 The pharmaceutical composition of claim 1, wherein the parenteral administration is intravascular administration.

[Claim 13]

 A method for preventing or treating autoimmune diseases comprising administering to a subject a composition comprising human bone marrow-derived mesenchymal enjoyable cells as an active ingredient: The human bone marrow-derived mesenchymal stem cells are (0 CD105, CD29, Positive immunological properties for at least one surface antigen selected from the group consisting of CD44, CD73 and CD90; and (Π) for at least one surface antigen selected from the group consisting of CD34, CD45 and HLA-DR It is characterized by having negative immunological properties.