KR102084974B1 - Pharmaceutical Compositions for Preventing or Treating Autoimmune Diseases Comprising Mesenchymal Stem Cells Derived from Human Bone-Marrow - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases comprising human bone marrow-derived mesenchymal stem cells as an active ingredient. Human bone marrow-derived mesenchymal stem cells of the invention inhibit anti-dsDNA antibody, proteinuria and total IgG production, inhibit proliferation of T cells and B cells, and inhibit cytokine expression of T cells without toxicity. It is effective in preventing or treating autoimmune diseases. Lupus causes inflammation in the kidneys by autoantibodies and immunocomplexes, and increases the infiltration of immune cells. When the human bone marrow-derived mesenchymal stem cells of the present invention are administered, lupus is reduced by infiltrating immune cells into the kidneys. It has a prophylactic or therapeutic effect.

Description

Pharmaceutical Compositions for Preventing or Treating Autoimmune Diseases Comprising Mesenchymal Stem Cells Derived from Human Bone-Marrow}

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.

Autoimmune disease is a disease caused by the body's immune function attacking itself, which is formed over a long period of time, symptoms persist chronically, and usually cause permanent damage to organs. The reality is that it is not. While knowledge of autoimmune diseases has evolved over the past 20-30 years, the exact mechanisms of production, the identity of autoantigens, and regulatory genes are still unclear. Autoimmune diseases can be broadly classified into organ-specific diseases and systemic diseases.

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

Examples of autoimmune diseases include i) Rheumatoid Arthritis, in which the immune system attacks tissues of various joints, and ii) autoimmunity of the central nervous system induced by T cells. Multiple sclerosis (MS), which can lead to blindness, paralysis, and premature death, is caused by the destruction of immune cells in the insulin-producing cells of the pancreas, and the MHC gene is an important immune mediator or type I diabetes. Mediated or Type 1 Diabetes Mellitus, ⅳ) Inflammatory Bowel Diseases, a disease caused by the immune system attacking the intestine, ⅴ) Scleroderma, which leads to the thickening of the skin or blood vessels (ⅵ). Systemic autoimmunity is accompanied by symptoms such as deep fatigue, rashes, and joint pain.In severe cases, systemic erythema may cause damage to the kidneys, brain, and lungs. And the like sex lupus (Systemic Lupus Erythematosus, SLE).

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 described.

Republic of Korea Patent Publication No. 10-2012-0018724

The present inventors earnestly researched to develop stem cells that can effectively treat autoimmune diseases and safely apply 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.

According to an 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 (i) CD105, CD29 Positive immunological properties to at least one surface antigen selected from the group consisting of, CD44, CD73 and CD90; And (ii) at least one surface antigen selected from the group consisting of CD34, CD45 and HLA-DR.

The present inventors earnestly researched to develop stem cells that can effectively treat autoimmune diseases and safely apply to the human body. As a result, mesenchymal stem cells obtained from human bone marrow are very effective in preventing or treating autoimmune diseases. It was identified.

Human bone marrow-derived mesenchymal stem cells of the present invention is at least one selected from the group consisting of CD105, CD29, CD44, CD73 and CD90 which are stem cell marker expression antigens, preferably at least two, more preferably at least three Even more preferably at least four, most preferably five surface antigens. According to one embodiment of the present invention, the human bone marrow-derived mesenchymal stem cells of the present invention express 70-100% of stem cell marker surface antigens CD105, CD29, CD44, CD73 and CD90, and more preferably 80-100 % Expression, even more preferably 90-99%.

Meanwhile, the human bone marrow-derived mesenchymal stem cells of the present invention exhibit negative immunological properties on CD34, CD45 and HLA-DR, which are surface antigens of hematopoietic stem cell markers. According to an 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 antigens, more preferably 0.1-7 % Expression, most preferably 0.1-5%.

The unit “%” used while referring to the expression rate of the surface antigen refers to the percentage 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 present invention, the autoimmune disease that can be prevented or treated by the composition of the present invention is lupus (systemic lupus erythematosus), rheumatoid arthritis, rheumatoid arthritis, systemic sclerosis, Scleroderma, Atopic dermatitis, alopecia areata, type I or immune-mediated diabetes mellitus, psoriasis, pemphigus, asthma, aphthitis, chronic thyroiditis, inflammatory enteritis, Behcet's disease, Crohn's disease, dermatomyositis, multiple myositis (polymyositis), multiple sclerosis, autoimmune hemolytic anemia, autoimmune encephalomyelitis, myasthenia gravis, Grave's disease, nodular polyarteritis nosa, polyarteritis nosa Ankylosing spondylitis, Fibromyalgia syndrome or Temporal arteritis.

According to another embodiment of the present invention, the autoimmune disease that 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 capable of being prevented or treated by the composition of the present invention is lupus.

According to one embodiment of the invention, human bone marrow-derived mesenchymal stem cells of the present invention express / secrete TGFβ (transforming growth factor beta) -1 in cells.

TGF-β is known to have three subtypes (TGF-β1, 2, 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 responses by expressing / secreting TGF-β1, which lowers the intensity of immune responses.

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 expression level of the nucleotide sequence in the sample to be investigated (eg, human bone marrow-derived mesenchymal stem cells) is a general sample (eg, normal cells). ), Which is high (preferably 1.2 times or more).

According to one embodiment of the present invention, the human bone marrow-derived mesenchymal stem cells of the present 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 kidneys). 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 the immune response.

According to the present invention, the inhibition of proliferation 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 stem cells of the present invention inhibit cytokine expression of T cells. The 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 invention, the pharmaceutical composition for preventing or treating autoimmune diseases of the present invention is a composition for parenteral administration, in particular, for vascular administration, and when the composition is administered in a blood vessel, the human bone marrow-derived intermediate Mesenchymal stem cells, while not toxic, inhibit anti-dsDNA antibodies, proteinuria and total IgG production, inhibit T cell and B cell proliferation, 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 stem cells described above; And (b) a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically effective amount” 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. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, phosphate buffered saline) or a medium and the like, 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 compositions of the present invention may be administered orally, topically (buccal, sublingual, dermal 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 vary depending on factors such as formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Typical dosages of the pharmaceutical compositions of the invention are 10 ² -10 ¹⁰ cells per day on an adult basis.

The pharmaceutical compositions of the present invention may be prepared in unit dosage 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 it may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

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

(Iii) The present invention provides a pharmaceutical composition for preventing or treating 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, inhibit proliferation of T cells and B cells, and express cytokine expression of T cells without being toxic. By inhibiting the drug exhibits a prophylactic or therapeutic effect on autoimmune diseases.

(Iii) Lupus causes inflammation in the kidney by autoantibodies and immunocomplexes, and increases the infiltration of immune cells. When the human bone marrow-derived mesenchymal stem cells of the present invention are administered, immune cells invade the kidney. By reducing it has a prophylactic or therapeutic effect against lupus.

Figure 1a-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, Figure 1a is bone marrow-derived mesenchymal stem of healthy donor 1 FIG. 1B is an image of donor 2, FIG. 1C is an 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 according to the culture period of bone marrow-derived mesenchymal stem cells of donor 3.
3-5 are graphs of surface antigen analysis of mesenchymal stem cells during third, fifth, seventh, and ninth passages using flow cytometers (FACs). Figure 3 is the result of analyzing the surface antigen during the culture period of the mesenchymal stem cells of the 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 collected at the 7th passage, and D recovered at the 9th passage. 4 is an analysis result of mesenchymal stem cells of donor 2 and FIG. 5 is an analysis result of donor 3.
Figure 6 is an image confirming the stability of cell culture through karyotype analysis 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 donor 1 during passage period without change in karyotype, Figure 6b is the result of donor 2, Figure 6c is an image showing the result of donor 3. In each diagram, 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 in the recovered cells.
Figure 7 is the result of measuring the gene expression of TGF-β, COX-2, HO-1 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-β1 in the secreted protein secreted by the cell culture medium during the fourth passage of bone marrow-derived mesenchymal stem cells of three donors.
10 is a result of measuring the differentiation ability of bone marrow-derived mesenchymal stem cells into adipocytes.
11 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, CD11c-APC, CD3-PE, CD8-PE, CD11b-PE, CD138-APC + IgG-PE, CD4-FITC + Foxp3-PE and B220-APC + CD3-PE Stain using a monoclonal antibody such as.
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-1β, 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 are divided into three groups. Once every two weeks from 10 weeks to 20 weeks of age, 200 μl of vehicle was injected intravenously into each mouse of the control group, and each of the human bone marrow-derived mesenchymal stem cell treatment groups was treated once every two weeks from 10 weeks to 20 weeks of age. Mice were injected intravenously with 1 x 10 ⁶ human bone marrow-derived mesenchymal stem cells, and 50 mg / kg of cyclophosphamide was injected intravenously in each mouse of the CPM treated group once every two weeks from 10 to 20 weeks of age. Survival until 28 weeks of age (a). To evaluate toxicity by measuring the weight of the mouse by 28 weeks of age (b). Anti-dsDNA concentrations were measured every 2 weeks using ELISA (c). IgG concentration is measured every 2 weeks using ELISA (d). Proteinuria concentration is measured every 2 weeks using ELISA (e). Significance is determined using the Student's t test on the control group (* p <0.05, ** p <0.01, *** p <0.001).
Figure 16 is the result 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. Mice are divided into three groups. 200 μl vehicle was injected intravenously into each mouse at 9 weeks of age, 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. , Nine-week-old cyclophosphamide treated group mice were injected intravenously with 50 mg / kg of CPM. Anti-dsDNA concentrations were measured every 2 weeks using ELISA (a). IgG concentration is measured every 2 weeks using ELISA (b). Proteinuria concentration is measured every 2 weeks using ELISA (c). Significance is determined using the Student's t test on the control group (* p <0.05, ** p <0.01, *** p <0.001).
Figure 17 is the result 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. Mice are divided into three groups. 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. Intravenously injected 50 mg / kg of CPM into each 12-week-old cyclophosphamide treated group mouse. Anti-dsDNA concentrations were measured every 2 weeks using ELISA (a). IgG concentration is measured every 2 weeks using ELISA (b). Proteinuria concentration is measured every 2 weeks using ELISA (c). Significance is 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 are divided into three groups. Once every three weeks from 12 to 20 weeks of age, 200 μl of a vehicle was injected intravenously into each mouse of the control group, and each of the human bone marrow-derived mesenchymal stem cell treatment groups once every three weeks from 12 to 20 weeks of age. Mice were intravenously injected with 1 x 10 ⁶ human bone marrow-derived mesenchymal stem cells, and 50 mg / kg of cyclophosphamide was intravenously injected into each mouse in the CPM-treated group once every two weeks from 12 to 20 weeks of age. Anti-dsDNA concentrations were measured every 2 weeks using ELISA (a). IgG concentration is measured every 2 weeks using ELISA (b). Proteinuria concentration is measured every 2 weeks using ELISA (c). Significance is determined using the Student's t test on the control group (* p <0.05, ** p <0.01, *** p <0.001).
19 shows the results of measuring cell group changes in MRL / lpr mouse organs after treatment with human bone marrow-derived mesenchymal stem cells. Phenotype expression in mouse organ cells was measured by flow cytometry. Organ cells are B220-APC, CD4-APC, CD11c-APC, CD3-PE, CD8-PE, CD11b-PE, CD138-APC + IgG-PE, CD4-FITC + Foxp3-PE and B220-APC + CD3-PE Staining using a mouse monoclonal antibody such as. Significance was determined using the Student's t test for 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 RNA is isolated from immune cells of spleen cells. Gene expression levels of IL-2, IFN-γ, IL-4, IL-10, TNF-α, IL-1β, 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 Age (d). 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 are divided into three groups. Once at 12 weeks of age, 200 μl of vehicle was intravenously injected into each mouse of the control group, and once at 12 weeks of age, 4 x 10 ⁴ , 4 x 10 ⁵ , 4 x 10 to each mouse of the human BM-MSC treated group. ⁶ Human 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.
FIG. 23 measures anti-dsDNA concentration using ELISA every two weeks (a). IgG concentration is measured every 2 weeks using ELISA (b). Proteinuria concentration is measured every 2 weeks using ELISA (c). Significance is determined using the Student's t test on the control group (* p <0.05, ** p <0.01, *** p <0.001).
24 is a result of observing the histological changes in the kidney following the administration of human bone marrow-derived mesenchymal stem cells.
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 using mouse monoclonal antibodies (CD34-PE, CD45-PE, CD103-PE, CD73-PE, CD90-PE, CD105-PE, CD44-FITC and Sca-1-FITC) Dyeing (b).
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 [ ³ H] -thymidine hybridization. BM-MSCs are co-cultured with Balb / c derived-B / T cells (a). Human bone marrow-derived mesenchymal stem cells are co-cultured with C57BL / 6 derived-B / T cells (b). Human bone marrow-derived mesenchymal stem cells are co-cultured with MRL / lpr derived-B / T cells (c). Significance is 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 T cells. Gene expression levels of IL-2, IFN-γ, IL-4, and IL-5 were analyzed using RT-PCR. PCR products were electrophoresed on agarose gel and then stained with ethidium bromide (a). IFN-γ and IL-2 levels in immune cell supernatants were measured using ELISA (b).
29 is a result of measuring the 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 [ ³ H] -thymidine hybridization. Splenocytes of MRL / lpr mice are activated with LPS in the presence or absence of MSCs that have been irradiated in 96 well plates (a). Splenocytes of MRL / lpr mice were activated with ConA in the presence or absence of irradiated MSCs in 96 well plates (b).
30 is a result of measuring the expression level of water-soluble elements in human bone marrow-derived mesenchymal stem cells. Human bone marrow-derived mesenchymal stem cells were recovered on day 20. Gene expression levels of water soluble elements were analyzed using RT-PCR (a). Levels of water soluble urea in human bone marrow-derived mesenchymal stem cell supernatants were measured using ELISA (b). Significance is determined using the Student's t test for the bone marrow cell population (*** p <0.001).
Figure 31 shows the result of measuring the expression of IDO in BM-MSC. IDO levels in human bone marrow-derived mesenchymal stem cell supernatants were measured using ELISA (a). Human bone marrow-derived mesenchymal stem cell-mediated T cell immune suppression mechanism (b).
32 is a result of measuring the motor 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 minutes at 37 ° C. T cells were stained with CFSE dye (green) for 15 minutes at 37 ° C. Two-dimensional cell tracking was performed using Imaris software. Snapshot images of the interaction between human bone marrow-derived mesenchymal stem cells and T cells.
Figure 33 shows the results of measuring the migration of T cells by human bone marrow-derived mesenchymal stem cells. T cell migration 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 is determined using the Student's t test for the control group (*** 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. Gene expression levels of chemokines were analyzed using RT-PCR (a). CCL2, CCL5 and CXCL12 levels in human bone marrow-derived mesenchymal stem cell supernatants were measured by ELISA (b). Significance is determined using the Student's t test for the bone marrow cell population (*** p <0.001).
35 is a result of knocking down the CCL2 and CXCL12 genes by siRNA and measuring the expression of chemokines, respectively.
36 shows the results of analyzing T cells involved in the movement of human bone marrow-derived mesenchymal stem cells.
Figure 37 shows the results of analysis of the contact pattern 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 between mouse MSCs and T cells.

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 collected from the patient's posterior superior iliac spine (PSIS) using a Jamshidi needle, and then 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 to a sterile tube and dilute 1: 3 with the basic medium (CSBM, CORESTEM Inc., Korea) and dilute this dilution with Ficoll (Fiocoll-Paque ^TM PREMIUM, concentration 1.077 g / mL, GE Healthcare). Layer was deposited onto the layer and centrifuged at 400 × g for 30 minutes. In the monuclear cell washing process, mononuclear cells were separated, and then monocytes were added to the monocytes, followed by centrifugation at 400 × g for 10 minutes to harvest monocytes, and the monocytes were harvested. After centrifuged for 10 minutes at 100 × g. Washed monocyte cells contain 1% penicillin-streptomycin (Biochrmone, Germany), L-alanyl-L-glutamine (Biochrome, Germany), 10% (v / v) fetal bovine serum (FBS; Gibco, USA) After inoculation into a T175 cell culture flask (Nunc, USA) containing the basal medium was incubated for 11 days at 37 ℃, 7% CO ₂ conditions. After incubation, 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 more than 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 to T175 cell culture flasks (Nunc USA.) 1.5 x 10 ⁵ per flask on - 2 x 10 ⁵ cells were inoculated into 37 ℃, 7% CO ₂ condition were cultured for 7 days. The medium was changed at 3 or 4 day intervals during cell culture.

Cells proliferated at least 70-80% were harvested by trypsinization (0.125% trypsin-EDTA). The harvested cells were present at a density of 1 × 10 ⁶ cells / vial in 800 μl of cryopreservation medium consisting of CSBM with 20% fetal bovine serum and 10% DMSO (dimethylsulfoxide, Sigma, USA). Were suspended and dispensed into cryotube vials (Nunc, USA). The cell suspension dispensed in the vial is stored in a cryopreservation container (Nalgene, USA) containing isopropyl alcohol and stored in a -80 ° C cryogenic refrigerator for 24 hours, and then the vial containing the cell is transferred to a liquid nitrogen storage tank the next day. Was

A vial containing 1 × 10 ⁶ cells / vial 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 per T175 flask, 1.5 x 10 ⁵ - was such that 2 x 10 ⁵ cells present. Cell culture medium exchange was performed at 3 or 4 day intervals, and the appearance and proliferation of the cells were observed under an optical microscope (Nikon, Japan). The passages of passages 2 (P2) to 9 (P9) were observed, and as shown in FIG. 1, it was confirmed that the cells proliferated uniformly in the form of spindles such as fibroblasts (FIGS. 1A-C).

Example 3 Analysis of Proliferation Rate of Bone Marrow-Derived Mesenchymal Stem Cells from Donors

The proliferation rate of stem cells according to the passage number (passage number) of the isolated cells was measured. Population doubling (PD) is an index indicating the proliferation rate of cells, and can be expressed as a formula (population doubling, PD) = log (final harvested cell number / initial seeded cell number) / log 2. The initial inoculated cell number was determined as 1.5 x 10 ⁵ cells in a T175 flask for each passage number, and the proliferation rate was determined by measuring the number of cells obtained after the culture. Bone marrow-derived mesenchymal stem cells were obtained through the above separation method and then passaged. As a result of confirming the proliferation rate of the cells (P2-P11) for about 40 days through the growth curve graph, it was confirmed that the population doubling time (PDT) 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 this added DPBS (Gibco, USA), suspended at a concentration of 1 × 10 ⁵ cells / 100 μL and aliquoted into tubes for analysis by FACS caliber (Calibur; 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 II) and the negative control antibody PE-immunoglobulin isotype IgG1 were used, and all antibodies were manufactured by Becton Dickinson, USA. . 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) and then subjected to flow cytometry.

More than 80% of the cells showed positive immunological properties for the stem cell labeled surface antigens CD29, CD44, CD49C, CD73 and CD105, and less than 5% negative for hematopoietic stem cell labeled surface antigens CD34 and CD45. It showed the chemical properties. As a result, it was confirmed that the isolated cells have the characteristics of stem cells. In addition, since the HLA-DR (MHC class II) that does not express the tissue-compatible antigen HLA-DR, which causes rejection in tissues or organ transplantation, does not induce an immune response, such as rejection, which 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 are maintained during cell isolation and culture. Inoculated with isolated bone marrow-derived mesenchymal stem cells (P3, P5, P7, P9) in a T75 flask with 1.75 x 10 ⁵ cells and then expanded to 75% density at 37 ° C and 7% CO ₂ Was harvested by trypsin treatment (0.125% trypsin-EDTA), and 25 of the metaphase of mitosis of harvested cells were GTG-banded. As a result, no numerical and structural abnormalities of chromosomes could be detected. Even in continuous culture It was confirmed that the change in karyotype did not occur and the normal chromosome was maintained (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, the human MSC was recovered from the culture flask, and the recovered cells were reacted with 200 μl of trizol for 5 minutes at room temperature. 200 ml of chloroform was added to the same tube, followed by vortexing for 5 minutes at room temperature, followed by centrifugation at 4 ° C and 12,000 rpm for 15 minutes. 500 ml of the supernatant was transferred to a new tube, and an equal amount of isopropanol was added thereto, followed by reaction at room temperature for 10 minutes, followed by centrifugation at 4 ° C. and 12,000 rpm for 15 minutes. The supernatant was removed, washed with 1 ml of 75% EtOH in RNA pellet, and centrifuged at 4 ° C. and 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. CDNA was synthesized at a concentration of 1 mg of each RNA.

cDNA synthesis was performed according to the Maxime RT premix kit method. The reverse transcription reaction was carried out for 60 minutes at 45 ° C. in a nucleic acid amplifier (PCR machine) and for 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 for the test was Rotor-Gene SYBR green PCR kit 10 μl, 10 pmole F / R primer 0.6 μl, 1 μg template cDNA 2.0 μl, Rnase free water 7.4 μl Add a PCR tube with a total volume of 20 μl and perform PCR under the following conditions; Pre-modification: 95 ° C. 10 min, denaturation: 95 ° C. 15 sec, annealing & extension: 60 ° C. 1 min. Repeat this process 40 times.

Gene expression of TGF-β, COX-2, HO-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, which is an important factor in regulating autoimmune diseases. COX-2 is an enzyme that produces prostaglandin. Activation of COX-2 in MSCs is known to produce prostaglandins and inhibit cellular responses proliferated by these substances. HO-1 is also an important factor involved in the regulation of immune cells by MSCs.

Treatment with IFN-g in human MSC was confirmed to increase the expression of IDO with TGF-β, COX-2, HO-1. IDO is a protein that MSC plays an important role in inhibiting the immune response and is a protein that is strongly involved in inhibiting the activity of macrophages, B cells and the like (Fig. 7B). In addition to gene expression, human MSCs expressed proteins of TGF-β and prostaglandin E (PGE ₂ ).

8 confirms that the genes of CCL2, IFN-γ, IL-10, IL-4, TGF-β are highly expressed in human MSC. CCL2 secreted by MSCs 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. IFN- [gamma] increases the expression of B7-H1 in MSCs and stimulates contact with T cells to induce 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 M1 macrophages (inflammatory) and regulate inflammatory responses. IL-10 secreted by MSC is a representative anti-inflammatory cytokine that regulates abnormally activated immune responses. Based on the above results, MSC is expected to effectively suppress the immune response by regulating various chemokine and cytokine expression.

Example 7: Expression analysis of secretory factor of bone marrow-derived mesenchymal stem cells of donor

Lupus is one of autoimmune diseases caused by disruption of the normal immune control system. The purpose of this study was to determine the expression level of immune regulatory factors secreted by mesenchymal stem cells themselves.

Among the factors expressing bone marrow mesenchymal stem cells themselves, TGFβ-1 expression is one of the factors influencing naïve T cells that directly or indirectly differentiate into regulatory T cells. When the cells are administered to lupus patients, they can induce anti-inflammatory and excessively regulated immunity-regulating immunity, so when 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 (Quantikine, R & D Systems) according to the kit's manual. As a result, it was confirmed that passage 3 (P4) of bone marrow-derived mesenchymal stem cells of three donors secreted TGFβ-1 per 36 cells on average 36 pg and secreted 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 O staining. Differentiated adipocytes accumulated droplets, which appeared red when oil red O 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 Figure 10C. On the other hand, differentiated cells were stained red by alizarin red, confirming that calcium 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 chondrocyte formation was observed (Fig. 10E-G).

Immune Regulation of Bone Marrow-derived Mesenchymal Stem Cells

The immunogenicity of human MSCs against allogeneic immune cells and their ability to modulate immune response were evaluated. PBMCs were isolated from blood of normal humans with different HLA types, and immunogenicity was measured by co-culture with three other human MSCs. Human MSC did not induce proliferation and IFN- [gamma] production of allogeneic PBMCs, indicating no immunogenicity (FIG. 11).

Co-culture of PBMC and human MSC proliferated by PHA, a lymphocyte proliferation inducing substance, was confirmed to suppress the immune response of human MSC. PHA induced proliferation of PBMCs, inhibited proliferative responses and inhibited IFN-γ production by human MSCs (FIG. 12).

Example 9 Experimental Method for Determining Lupus Therapeutic Effect of MSC

Human MSC

The human MSC was used for animal experiments in the core system.

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 1 × 10 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 sacrificed by the cervical spinal bone method, and the single cells were obtained by separating the bone marrow in the femur and tibia using a 10 ml syringe. The impurities were filtered using a 0.7 μm 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 α-MEM medium with a cell number of 1 × 10 ⁷ cells / ml and cultured in a 37 ° C. incubator fed with 5% CO ₂ . 1 ml of fresh medium was added daily for 5 days of culture. On day 6 of culture, α-MEM medium was removed, washed with PBS, and 1 ml of fresh medium was added. Cells 20 days old were used for the experiment.

Cell phenotype analysis

Cell surface analysis was performed using a flow cytometer. The recovered cells (1 × 10 ⁶ cells / ml) were washed with 0.5% BSA / PBS. The supernatant was removed and 50 μl of 0.5% BSA / PBS containing monoclonal antibody was added and stained at 4 ° C. for 20 minutes. After washing again with 500 μl of PBS / BSA, 500 μl of PBS / BSA was added and suspended. Monoclonal antibodies include PE-binding CD3, CD8, CD34, CD45, CD73, CD90, CD103, CD105, CD11c, CD11b, IgG, Foxp3, FITC-binding CD44, Sac-1, CD11c, CD4, APC-binding B220, CD138 And CD4 were used. Data analysis was analyzed using WinMDI software.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from tissues and cells using TRIZOL reagent. CDNA was synthesized at 42 ° C. for 60 minutes and 94 ° C. for 5 minutes using total RNA 0.3. PCR (polymerase chain reaction) was performed using 3 μl of the prepared cDNA and a primer of 10 pM. The basic conditions of PCR were pre-denaturation step, 94 ° C., 5 minutes starting, denaturation step, 94 ° C. 30 sec 30 cycles, annealing step, 72 ° C. 1 minute, and post-extension step at 72 ° C. for 5 minutes. Was carried out. The annealing temperature was performed differently between 54-56 ° C. per primer. The prepared PCR product was electrophoresed at 50 V after loading 8 μl into 1% agarose gel.

HE staining

Kidneys extracted from MRL / lpr mice were fixed in 10% paraformaldehyde for 18 hours and paraffin sections of 4 μm were made. 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. Then, stained for 1 minute using Eosin solution. The dyed 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 and diluted in RPMI complete medium composition at a cell number of 1 × 10 ⁶ cells / ml. The splenocytes were treated with LPS and ConA at a concentration of 1 μg / ml and the MSCs were treated by cell concentration and then cultured in a 37 ° C. incubator. After 54 hours, thymidine was treated at a concentration of 1 μCi / well, and after 18 hours of incubation, the cells were collected in a glass fiber filter using a cell harvester and dried for 2 hours. After putting the dried glass fiber filter into the sample bag, the filter was sufficiently wetted and sealed with a scintillation cocktail. Finally, the glass fiber filter was used to measure the amount of radiation introduced into the DNA of the cell using a beta counter.

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, a supernatant of MSC was obtained, and a TGF-β ELISA kit, a PGE2 ELISA kit, and an IDO ELISA kit were used, and the procedure was described in each kit.

Cell Image Measurement

Cell images were measured using a cell culture microscope. MSCs were treated with 5 μM CMTMR dye for 15 minutes at 37 ° C., and T cells were treated with 5 μM CFSE dye for 15 minutes at 37 ° C. 5 x 10 ⁴ cells T cells and 5 x 10 ³ cells were placed in a 35 mm culture dish, and cell images were measured. 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 and cut to 4 mm to prepare slides. 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 and heat to express the antigen. The slide was placed in a solution in which H ₂ O ₂ was added at 3% in methanol to block endogenous peroxidase activity. The slides were reacted overnight at 4 ° C. by diluting the CD3, B220, F4 / 80, Foxp3, CD209b antibodies. The Vectastain ABC kit was used to ensure good 3,3'-diaminobenzidine (DAB) staining. After immunostaining, 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 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, alters the transcription of the Fas receptor. As a result, a deficiency of Fas signaling inhibits apoptosis and results in lupus symptoms. The difference from NZB / W F1, which is used classically in lupus animal models, is that mice die rapidly and appear in both lupus symptoms in females and males.

MRL / MpJ-Fas ^lpr / J mice were purchased as one of the MRL / lpr mice and analyzed before and after onset. As described earlier, the mouse is mutated by the Fas receptor, which leads to lymphatic proliferation and production of an immunocomplex, at which point the female is about 12 weeks old, and on average the female begins to die at about 17 weeks old and about 22 weeks old. First, 6-week-old mice before the onset of lupus and 25-week-old mouse organs were extracted and analyzed. After the onset, it was found that 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 from 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 ratio of CD4 ⁺ Foxp3 ⁺ , a 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).

MSC has been reported to be easy to transplant because of its 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 Inflamm (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 1 × 10 ⁶ cells / mouse, and 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 mice were checked every week, and anti-dsDNA, proteinuria, and IgG were measured every two weeks. Cyclophosphamide 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 MRL / lpr mice was 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 blood levels of total IgG (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, total IgG production of MRL / lpr and increased the survival rate of mice. It was similar to the therapeutic effect of cyclophosphamide in current clinical use. However, weight loss cyclophosphamide was toxic, whereas 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, IgG production in MRL / lpr mouse blood and also inhibited proteinuria production. However, the effect was observed strongly for 3 weeks after administration, after 4 weeks showed a tendency to weaken the therapeutic effect (Fig. 16).

The effect of lupus improvement was confirmed by intravenous injection of 1 × 10 ⁶ cells / mouse of human MSC into MRL / lpr mice only once at 12 weeks of onset of lupus. Human MSC inhibited anti-dsDNA, IgG production in MRL / lpr mouse blood and also inhibited 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 the effect was 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. 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).

The flow rate of immune cell phenotypes in mouse organs was measured by flow cytometry, and 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 ⁺ 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 kidney by autoantibodies and immunocomplexes, increasing immune cell infiltration. Mouse kidneys were isolated and cell invasion 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 MSC in MRL / lpr mice, human MSC was administered once at 12 weeks of age at ⁴ × ^{10 4} , ⁴ × 10 ⁵ , ⁴ × ^{10 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. The observation was terminated at 21 weeks of age and the kidneys were removed for histological analysis.

Human MSC increased MRL / lpr mouse survival and the lowest effective concentration was ⁴ × 10 ⁴ cells / 40 g, 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). As a result of histopathology, the kidneys of the control group increased immune cell infiltration of T cells, B cells, neutrophils, macrophages, and dendritic cells due to inflammatory responses, and all decreased by administration of human MSC. In contrast, the percentage of regulatory T cells was increased by human MSC administration (FIG. 24).

Example 11: Study on the mechanism of immunosuppressive action of MSC

MSCs are known to inhibit innate immunity and apoptotic immunity and to inhibit direct contact between cells and soluble factors. Soluble factors secreted by MSC include NO, IDO, TGF-β, IL-10, PGE ₂ and the like. 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 CD103 compared to the bone marrow cells of the mouse, and increased expression of the MSC phenotypes CD73, CD90, CD105, CD44, and Sca-1. (FIG. 26).

Inhibition of Proliferation of Mouse T and B Cells by MSCs

It was confirmed by mitogen analysis whether MSCs inhibit the proliferation of mouse T and B cells. MSCs were prepared using bone marrow cells from Balb / c mice, and isolated from splenocytes of Balb / c (syngenic), C57BL6 (allogenic), and MRL / lpr (allogenic) mice. MSC and T cells were mixed at a ratio of 0.001-0.1: 1, and ConA treatment induced T cell proliferation. Experimental results confirmed that MSC inhibits ConA-induced T cell proliferation (FIG. 27). MSC and B cells were mixed at a ratio of 0.001-0.1: 1, and LPS treatment was used to induce 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 MSCs inhibited cytokine expression in mouse T cells was confirmed using 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 (allogenic) mice. MSC and T cells were mixed at a ratio of 0.1: 1, and ConA treatment induced 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 MSCs and B cells are mixed and added to the lower wells, the MSCs can secrete contact or water soluble mediators to inhibit the proliferation of B cells. MSCs are added to the upper wells and MSCs when B cells are added to the lower wells. 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 mediators 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 IDO (FIG. 30), but it was confirmed that the IDO expression of MSC increased when co-cultured with T cells (FIG. 31).

Observing Interactions Between MSCs and T Cells

In order to directly observe the interaction between MSC and T cells, the movement of the cells was measured using a real-time microscope. Through image analysis, it was confirmed that the T cell movement 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 μm / minute (FIG. 32A). The second type of T cells were in continuous contact with MSCs and the rate was reduced to 1.5 μm / min (FIG. 32B). The third type of T cells were cells in contact with MSCs during migration, which showed a rate of about 5 μm / min at the beginning of migration but decreased to about 1 μm / min after contact with MSC (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. The number of T cells that migrated to the lower wells after 90 minutes was measured to determine the migration of T cells towards the MSCs. As the concentration of MSC in the lower well 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 using 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 of mouse MSC with 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 degraded by each gene siRNA are named CCL2-KD MSCs and CXCL12-KD MSCs. Treatment of mouse MSC with CCL2 siRNA (FIG. 35A) and CXCL12 siRNA (FIG. 35B), respectively, inhibited the expression of each chemokine gene and the amount of protein production.

MSC was added to the lower wells of the trans wells 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-KD MSCs was reduced (FIG. 35C). The CXCL12-KD MSCs migrated to the lower wells of T cells in the upper wells in a concentration-dependent manner of the MSCs, with the same results as the control MSCs. In the observation on the image, it was not observed that the T cells move in the direction of the CCL2-KD MSC (FIG. 35D).

Analysis of the movement of the MSC

Contact between MSC and T cells was confirmed and analyzed for their movement. Based on contact with MSCs, T cells that do not contact MSCs are called wandering T cells, T cells that attempt to contact MSCs are called searching T cells, and T cells that have contacted 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 wandering T cells and decrease the ratio of contacting T cells compared to the control MSC (Fig. 36B).

Wandering T cells and searching T cells were free to move without a specific direction, contacting T cells in contact with MSC was confirmed that stagnated in one place with little movement (Fig. 36C). This movement was the same regardless of the MSC condition. The rate of contacting T cells was lower than that of Wandering T cells and searching T cells, which was not different from control MSC, CCL2-KD MSC, and 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. Contact timing of contact between MSC and T cells is indicated by a red arrow. 37A, the contact time between MSCs 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 control MSC, CXCL12-KD MSC, CCL2-KD MSC and T cells contacted for 6 hours were 11.4, 10.6, and 10.2, respectively, which were not significantly different (FIG. 37B). However, the time of contact showed 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

Tests were conducted using CCR2 antagonists to supplement the results that CCL2 is important when contacting mouse MSCs and T cells. CCR2 is a chemokine receptor expressed in T cells as a receptor for CCL2. Treatment of CCR2 antagonists by T cell concentration showed that cytotoxicity was observed at 100 μg / ml or higher (FIG. 38A).

T cells treated with CCR2 antagonists at concentrations of 3, 10 and 30 μg / 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.

30 μg / ml of CCR2 antagonist was treated to T cells and seeded into 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 merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (11)

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) surface antigens CD105, CD29, CD44, CD73 and CD90 Positive immunological properties for; And (ii) negative immunological properties of surface antigens CD34, CD45 and HLA-DR, wherein the autoimmune diseases include lupus (systemic lupus erythematosus), rheumatoid arthritis Progressive systemic sclerosis (Scleroderma), atopic dermatitis, alopecia areata, psoriasis, swelling, asthma, aphthous stomatitis, chronic thyroiditis, inflammatory enteritis, Behcet's disease, Crohn's disease, dermatomyositis , Polymyositis, multiple sclerosis, autoimmune hemolytic anemia, autoimmune encephalomyelitis, Myasthenia gravis, Grave's disease, nodular polyartositis noosa arteritis ), Ankylosing spondylitis, Fibromyalgia syndrome and Temporal arteritis Pharmaceutical composition, characterized in that it is selected from the group consisting of.
The method of claim 1, wherein the human bone marrow-derived mesenchymal stem cells have a level of expression of transforming growth factor beta (TGF-β-1), COX-2, HO-1, IFN-γ, IL-4 and IL-10 Pharmaceutical composition, characterized in that increased.
The pharmaceutical composition of claim 1, wherein the human bone marrow-derived mesenchymal stem cells inhibit the production of anti-dsDNA antibodies.
The pharmaceutical composition of claim 1, wherein the human bone marrow-derived mesenchymal stem cells inhibit the production of proteinuria.
The pharmaceutical composition of claim 1, wherein the human bone marrow-derived mesenchymal stem cells inhibit the production of IgG.
2. The pharmaceutical composition of claim 1, wherein the human bone marrow-derived mesenchymal stem cells reduce immune cell infiltration into the kidneys.
The pharmaceutical composition of claim 1, wherein the human bone marrow-derived mesenchymal stem cells inhibit proliferation of T cells or B cells.
8. The pharmaceutical composition of claim 7, wherein the inhibition of T cell proliferation is by soluble mediator or cell-cell contact.
The pharmaceutical composition of claim 1, wherein the human bone marrow-derived mesenchymal stem cells inhibit cytokine expression of T cells.
The pharmaceutical composition of claim 1, wherein the composition is for intravascular administration.
The pharmaceutical composition of claim 10, wherein the vascular administration is intravenous injection.

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