KR20150020112A - Mesenchymal stem cells treated metformin having immuno-modulating activity and cell therapeutic agent for preventing or treating immune disease - Google Patents

Abstract

The present invention relates to a method for manufacturing mesenchymal stem cells, which mass-produce human transforming growth factor-β (TGF-β) and have immuno-modulating activity, mesenchymal stem cells manufactured by the method, and a cell therapeutic agent composition containing the mesenchymal stem cells for preventing or treating immune diseases, wherein the method includes treating the mesenchymal stem cells, of which CD105, CD29 and CD44 are positive immunological features and CD34, CD45 and HLA-DR are negative immunological features, with metformin, and cultivating the metformin-treated mesenchymal stem cells at a temperature of 28-42°C for 14-21 days. The metformin-treated mesenchymal stem cells having immuno-modulating activity increase expression of IDO, TGF-beta or IL-10 that is a factor having immuno-modulating activity, and augment the self phagocytosis and mitochondrial activity, thereby being useful as a cell therapeutic agent for treating immune diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to mesenchymal stem cells, metformin-treated mesenchymal stem cells having mesenchymal stem cells, metformin-treated mesenchymal stem cells,

The present invention relates to a mesenchymal stem cell having metformin-treated immunomodulatory ability and a cell therapeutic composition for preventing or treating immunological disease comprising the same.

Immunity is one of the biological self-protection systems for all external polymeric substances (antigens) that enter or are injected into living tissue. The main component of the immune system is lymphocytes, which are white blood cells that are made in the bone marrow and circulate along the blood to the lymphatic tissues or organs, mainly the lymph node splenic tonsil. B cells proliferate rapidly when stimulated by the appropriate antigen, forming a clone that produces a special antibody (immunoglobulin) that neutralizes the antigen. The antibody produced by B cells circulates in body fluids and performs humoral immunity. T cells are made from the thymus and migrate to lymphoid tissue, which is responsible for cell-mediated immunity that directly attacks the antigen.

In addition, one of the most important characteristics of all normal individuals is that they do not react negatively to the antigenic substances that make up the self, while many non-self antigens recognize it and can react to remove it I have the ability. Such a non-response of a living body to a self-antigen is called immunologic unresponsiveness or tolerance.

When problems arise in inducing or maintaining such self-tolerance, an immune response to the self-antigen occurs, thereby attacking the self-organization, resulting in various autoimmune diseases.

On the other hand, drugs that suppress autoimmune function are mainly used as a main method for treating such autoimmune diseases. However, because of the large number of side effects, it is difficult to use continuously, and the treatment is limited because it does not prevent recurrence sufficiently.

Therefore, a new therapeutic agent for immune diseases is being developed more effectively. Recently, a cell therapy agent for the treatment of immune diseases has been developed, and studies for using stem cells as a cell therapy agent are increasing.

In particular, the mesenchymal stem cells of the stem cells have not yet revealed the precise mechanism of action against the immunoregulatory ability, and research on the production of stem cells having excellent immunological disease treatment has been rarely conducted.

Accordingly, the inventors of the present invention found that when mesenchymal stem cells prepared by treating metformin have an excellent immunological disease therapeutic effect while studying a cell therapy agent for immunotherapy effectively using stem cells, .

Korean Patent Application No. 2007-0017970

Accordingly, an object of the present invention is to provide a method for producing mesenchymal stem cells having mass-production of human transgranular growth factor-beta (TGF-beta) and immuno-controlling ability, which comprises culturing mesenchymal stem cells with metformin .

It is another object of the present invention to provide a mesenchymal stem cell capable of mass-producing human transgranular growth factor-beta (TGF-beta) produced by the method of the present invention and having immunomodulating ability.

It is another object of the present invention to provide a cell therapy composition for preventing or treating immune diseases comprising mesenchymal stem cells having immunomodulating ability according to the present invention as an active ingredient.

In order to accomplish the object of the present invention as described above, the present invention provides a method for producing human transgranular growth factor-beta (TGF-β), which comprises the step of treating metformin in mesenchymal stem cells, A method for producing mesenchymal stem cells is provided.

In one embodiment of the present invention, the mesenchymal stem cells may be a method for producing mesenchymal stem cells, wherein CD105, CD29 and CD44 are positive and CD34, CD45 and HLA-DR have immunological characteristics .

In one embodiment of the present invention, the method may further comprise culturing the metformin-treated mesenchymal stem cells at a temperature of 28 to 42 DEG C for 14 to 21 days .

In one embodiment of the present invention, the mesenchymal stem cells may be isolated from peripheral blood or adipose tissue.

In one embodiment of the present invention, the metformin may be a method for producing mesenchymal stem cells, wherein the metformin is treated at a concentration of 0.2 to 2 mM based on the number of mesenchymal stem cells of 2 × 10 ⁵ to 1 × 10 ⁶ cells .

In one embodiment of the present invention, the mesenchymal stem cells can be increased in expression of at least one factor selected from the group consisting of IDO, TGF-beta and IL-10 by treatment with metformin.

The present invention also provides a mesenchymal stem cell capable of mass-producing human transgenic growth factor-beta (TGF-beta) produced by the method of the present invention and having immunomodulating ability.

The present invention also provides a cell therapy composition for preventing or treating immune diseases comprising mesenchymal stem cells of the present invention as an active ingredient.

In one embodiment of the present invention, the composition may be administered at a dose of 2 × 10 ⁵ to 2.5 × 10 ⁷ cells / kg of body weight of an injectable mesenchymal stem cell having immunomodulating ability.

In one embodiment of the present invention, the immunological disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, It is known that post-transplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto thyroiditis, polymyositis, scleroderma inflammatory diseases such as scleroderma, Addison disease, vitiligo, pernicious anemia, glomerulonephritis and pulmonary fibrosis, inflammatory bowel disease, Crohns disease ), Autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty (post-angioplasty) stent restenosis, chronic obstructive pulmonary disease (COPD), Graves disease, gastrointestinal allergy, conjunctivitis, atherosclerosis, coronary artery disease Angina, cancer metastasis, small artery disease, and mitochondrial disease.

The present invention provides a method for mass production of human transforming growth factor-beta (TGF-beta) by treating metformin in mesenchymal stem cells having positive CD905, CD29 and CD44 and immunologic characteristics of CD34, CD45 and HLA-DR A method for producing mesenchymal stem cells having immunomodulating ability, mesenchymal stem cells prepared by the method, and a composition for treating or preventing an immune disease containing the mesenchymal stem cells. The metformin-treated mesenchymal stem cells having the immunomodulatory effect according to the present invention have increased expression of any one or more of the factors selected from the group consisting of IDO, TGF-beta and IL-10, It has an effect of being useful as a cell therapy agent for the treatment of immune diseases since it has an increased self-phagocytosis and mitochondrial activity.

FIG. 1 shows the result of microscopic observation of the cell shape of mesenchymal stem cells isolated from peripheral blood and the result of analysis of cell marker expression of mesenchymal stem cells.
FIG. 2 is a graph showing the results of analysis of the degree of expression of IDO and TGF-beta in mesenchymal stem cells prepared by treating mesenchymal stem cells and metformin isolated from peripheral blood.
FIG. 3 shows the expression of HMGB-1, IL-6 and IL-1beta, which are genes related to inflammatory and tumor factors, in mesenchymal stem cells prepared by treating mesenchymal stem cells and metformin isolated from peripheral blood. The results are shown graphically.
FIG. 4 is a photograph showing the results of observing the activity of autophagic vacuoles and mitochondria through electron microscopy in mesenchymal stem cells prepared by treating mesenchymal stem cells and metformin isolated from peripheral blood. FIG.
FIG. 5 is a graph showing the results of analysis of IL-10 and TGF-beta expression levels in mesenchymal stem cells prepared by treating mesenchymal stem cells and metformin isolated from adipose tissue.
FIG. 6 is a photograph showing the degree of expression of IL-10 and IDO in mesenchymal stem cells prepared by treating mesenchymal stem cells and metformin isolated from adipose tissue by DAPI staining method.
FIG. 7 is a graph showing the results of immunohistochemical staining of a mouse model of osteoarthritis-induced mouse metastasis (Met-MSC) injected with human adipose tissue-derived mesenchymal stem cells prepared by treatment with metformin, a group of MSCs injected with metformin- (A), and the degree of destruction of cartilage was observed by staining with Indian ink staining method (B).
FIG. 8 is a graph showing the results of immunohistochemical staining of mesenchymal stem cells (MSC + Metformin), mesenchymal stem cells alone (MSC), metformin non-treated group (A), IBD disease activity (B), and length and thickness change (C) of the large intestine (IBD; inflammatory bowel disease group).
FIG. 9 is a graph showing the results of immunohistochemical staining of mesenchymal stem cells (MSC + Metformin), mesenchymal stem cells alone (MSC), metformin untreated group ), Inflammatory bowel disease (IBD), non-treated group (WT), and intranasal inflammatory cell infiltration and tissue morphology changes (A) and immunochromism staining B).
FIG. 10 is a graph showing the results of immunohistochemical staining of the human adipose tissue-derived mesenchymal stem cells (Met-MSC), mesenchymal stem cells alone (MSC), metformin untreated group prepared by treating metformin with IL-1Ra knockout mouse ) And a group that was not treated with anything (i.e., an arthritis invention group; IL-1Ra Knockout mouse).
FIG. 11 is a graph showing the results of human mesenchymal stem cells (Met-MSC), mesenchymal stem cells (MSC) and metformin-free mesenchymal stem cells (MSCs) prepared by treating metformin in an animal model of rheumatoid arthritis, In the treatment of rheumatoid arthritis.
FIG. 12 is a graph showing the results of immunohistochemical staining of a mouse model of osteoarthritis-induced mouse metastasis (Met-MSC) injected with human adipose tissue-derived mesenchymal stem cells prepared by treatment with metformin, a group of MSCs injected with metformin- And osteoarthritis mouse group (MIA; nontreated group), H & E staining, Toluidine staining, and saffranin O staining were performed to analyze the extent of osteoarthritis and cartilage destruction.

The inventors of the present invention have been studying to develop a cell therapy agent capable of effectively treating immune diseases, wherein the mesenchymal stem cell prepared by treatment with metformin is significantly superior to the mesenchymal stem cell without metformin treatment And confirmed through experiments.

The present invention is characterized by treating metformin of the following formula 1 with mesenchymal stem cells, wherein metformin is N, N-dimethylimidodicarbonimidic diamide by IUPAC nomenclature, molecular formula is C ₄ H ₁₁ N ₅ , and molecular weight is 129.16364 .

Accordingly, the present invention provides a cell therapy composition for the prevention or treatment of immune diseases comprising metformin-treated mesenchymal stem cells having immunomodulatory ability and comprising mesenchymal stem cells having the immunomodulating ability as an active ingredient .

The mesenchymal stem cell (MSC) is a stem cell which is isolated from bone marrow, blood, dermis and periosteum, and is a pluripotent stem cell capable of differentiating into various cells such as adipocytes, chondrocytes and bone cells. ) Or multipotent cells.

In particular, the mesenchymal stem cell used in the present invention may be an mesenchymal stem cell of an animal, preferably a mammalian animal, more preferably a human mesenchymal stem cell. The mesenchymal stem cells of the present invention may be derived from bone marrow, adipose tissue, peripheral blood, liver, lung, amniotic fluid, placenta chorioamnion or cord blood, preferably derived from adipose tissue or peripheral blood.

For reference, mesenchymal stem cells can be obtained from various sources as described above. Specifically, the process for obtaining mesenchymal stem cells is as follows: (1) a mammal including a human or a mouse, preferably a human Isolating mesenchymal stem cells from an mesenchymal stem cell source of, for example, blood or bone marrow or adipose tissue; (2) culturing the isolated cells in a suitable medium; And (3) removing floating cells in the culture process and subculturing the cells attached to the culture plate, thereby obtaining finally formed mesenchymal stem cells.

As the medium used in the above process, any medium generally used for culturing stem cells can be used. Preferably, it is a medium containing serum (for example, fetal bovine serum, horse serum and human serum). Mediums which can be used in the present invention include, for example, RPMI series, Eagles ' s MEM (Eagle's minimum essential medium, Eagle, H. Science 130: Proc. Soc. Exp (1986)), 199 Iscove's MEM (Iscove, N. et al., J. Exp. Med. 1963), F12 (Ham, Proc. Natl. Acad < / RTI > Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1963)), F10 (Ham, RG Exp. Cell Res. 29: 515 (1959)), a mixture of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102: 255 (1980)), Way-mouth's MB752 / 1 (Waymouth, CJ Natl. Cancer Inst. 1959) and McCoy's 5A (McCoy, TA, et al., Proc. Soc. Exp. Biol. Med. )), But is not limited thereto. The medium may include other components such as antibiotics or antifungal agents (e.g., penicillin, streptomycin) and glutamine.

In addition, identification of isolated mesenchymal stem cells can be performed by flow cytometry. Such flow cytometry analysis is carried out using a specific surface marker of mesenchymal stem cells. Preferably, mesenchymal stem cells which can be used for the preparation of mesenchymal stem cells having immunomodulating ability in the present invention are cells which are positive for CD105, CD29 and CD44, and which have immunologic characteristics of CD34, CD45 and HLA-DR .

Meanwhile, in the present invention, mesenchymal stem cells are used to treat mesenchymal stem cells derived from various sources for the treatment of immune diseases. Thus, new mesenchymal stem cells having immunomodulating ability have been prepared.

Metformin is currently used in the treatment of non-insulin dependent diabetes mellitus (NIDDM) as an oral antidiabetic agent and as a blood sugar drug, by lowering both the basal plasma glucose and postprandial plasma glucose Improves glucose tolerance in NIDDM patients. Metformin has been widely used as a treatment for type 2 diabetes, and clinical approaches have been made for polycystic ovary syndrome, weight loss, and cancer treatment. However, there has been no report on a technique for treating immune diseases using metformin and a method for producing mesenchymal stem cells using metformin.

In order to develop a new therapeutic agent for immune diseases using metformin and mesenchymal stem cells, the present inventors have found that mesenchymal stem cells can be treated with metformin, and the mesenchymal stem cells can be cultured and obtained by treatment with metformin, The expression of TGF-beta, IDO and IL-10 genes was significantly increased in the peripheral blood stem cells of the mesenchymal stem cells. In particular, when metformin was administered to the mesenchymal stem cells derived from peripheral blood, TGF-beta (Fig. 2 and Fig. 4).

Among these TGF-beta, IDO and IL-10, indoleamine 2,3-dioxygenase (IDO) is known to be a factor for measuring the function and activity of the immune system. IL-10 TGF-beta (transforming growth factor beta) is a secreted protein with three isoforms called TGF1, TGF2, and TGF3, and TGF is encoded as a large protein precursor , TGF1 contains 390 amino acids, and TGF2 and TGF3 contain 412 amino acids, respectively. In particular, TGF has growth inhibitory activity against most cells, so that TGF also has a growth inhibitory activity against cancer cells expressing normal TGF receptor (Twardzik et al., J. Natl. Cancer Institute, 81: 1182- 1185, 1989). Therefore, tumor cells may be inhibited from growth inhibition by TGF by inhibiting their TGF receptor expression. For reference, the expression of Foxp3 in the presence of TGF-beta is derived from naive T cells and the Tregs thus induced have the same immunosuppressive function. In addition, the deficiency of TGF-beta gene can not control the activity of inflammatory immune cells and can cause severe autoimmune diseases. Thus, the stimulation of TGF-beta production is associated with the ability to treat immune disorders.

Furthermore, the inventors of the present invention confirmed that mesenchymal stem cells of the present invention prepared by treating metformin have excellent autophagy activity and mitochondrial activity. Autophagy is a process in which bacteria degrade their own organelles or cellular components, It differs from heterophages in that the polymer accepts by intracellular ingestion such as negative action or phagocytosis. The cell functions to decompose its own protein in response to a nutrient deficiency and to remove unnecessary cellular components in the process of rebuilding the cells. The cell component is surrounded by a membrane derived from an endoplasmic reticulum to form a child cell, It fuses with the cotton to form the child's parosomes and break it down.

In addition, since such autophagic action can control the beneficial or deleterious effects of immunity and inflammation and has an effect of preventing infectious diseases, autoimmune and inflammatory diseases, the metformin-treated mesenchymal stem cells of the present invention are self- It is predominantly useful for the treatment of immune and inflammatory diseases. In addition, when the mitochondrial activity is not regulated, more severe inflammation occurs, and thus it is known that the adverse reaction of mitochondria to the development of immunological inflammatory diseases is very important. When the mitochondrial activity is decreased, the control system of the diseased cells becomes unstable, which causes excessive cell activity or proliferation.

Therefore, in the present invention, when methamphetamine treatment of mesenchymal stem cells was carried out in the present invention, autopagophageal folliculi increased and autophagic activity increased, as compared with the case without metformin treatment, and the increase of mitochondrial activity was confirmed, The mesenchymal stem cells can be treated more effectively for immune diseases.

In addition, the present invention can provide a method for producing mesenchymal stem cells, which mass produce human transgranular growth factor-beta (TGF-?) And have immunoregulatory ability, wherein said method comprises the steps of culturing CD105, CD29 and CD44, , CD45 and HLA-DR may include treating metformin with mesenchymal stem cells having negative immunological characteristics and culturing for 14 to 21 days at a temperature of 28 to 42 占 폚.

In the present invention, the mesenchymal stem cells capable of treating metformin may be mesenchymal stem cells isolated from peripheral blood or adipose tissue. The metformin may be 0.2 to 0.25 cells per 2 × 10 ⁵ to 5 × 10 ⁵ cells of mesenchymal stem cells. To a concentration of 2 mM.

In addition, the present invention can provide a cell therapy composition for preventing or treating immune diseases, which comprises mesenchymal stem cells prepared by treating the immunomodulatory metformin as an active ingredient.

In the present invention, the cell therapeutic agent refers to a substance that alters the biological characteristics of a cell such as autologous, allogenic, and xenogenic cells in vitro, in order to restore the functions of cells and tissues , Which is used for the purpose of treatment, diagnosis and prevention through a series of actions. Since 1993, the United States has been administering cell therapy products as medicines since 2002. These cell treatments can be classified into two categories. The first is stem cell therapy for tissue regeneration or restoration of long-term function, the second is immunization for controlling immune response such as inhibition of in vivo immune response or exaggeration of immune response Cell therapy.

The administration route of the cell therapeutic composition of the present invention can be administered through any conventional route as long as it can reach the target tissue. But are not limited to, parenteral administration, for example, intraperitoneal administration, intravenous therapy (i.v), intramuscular administration, subcutaneous administration, intradermal administration.

The composition may be formulated in a suitable form together with a pharmaceutical carrier commonly used in cell therapy. &Quot; Pharmaceutically acceptable " refers to compositions which are physiologically acceptable and which, when administered to humans, do not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or the like. Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, aqueous carriers for parenteral administration such as aqueous glucose and glycols, etc., and may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The composition may also be administered by any device capable of transferring the cell therapy agent to the target cell.

The cell therapeutic composition of the present invention may comprise a therapeutically effective amount of a cell therapy agent for the treatment of diseases. The term therapeutically effective amount refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, This includes an amount that induces relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the cell therapy agent contained in the composition of the present invention will vary depending on the desired effect. The optimal cell therapy agent content can therefore be readily determined by those skilled in the art and will depend upon a variety of factors including the nature of the disease, the severity of the disease, the amount of other ingredients contained in the composition, the type of formulation, and the age, weight, , The time of administration, the route of administration and the fraction of the composition, the duration of the treatment, the co-administered drug, and the like. It is important to include the amount by which all of the above factors are taken into account so as to obtain the maximum effect in a minimal amount without side effects. For example, the composition of the present invention may include a cell therapy agent administered at 2.5 × 10 ⁵ to 2.5 × 10 ⁷ cells per kg body weight of the administration subject.

The invention also provides a method of preventing or treating an immune disorder comprising administering to a mammal a therapeutically effective amount of the cell therapy composition of the invention.

As used herein, the term mammal refers to a mammal that is the subject of treatment, observation, or experiment, preferably a human.

In the treatment method of the present invention, when the cell therapeutic composition of the present invention is administered to an adult, the cell therapeutic agent contained in the composition is administered at a dose of 1 × 10 ⁴ to 1 × 10 ⁸ It is preferable to include the amount of cell water.

In the therapeutic method of the present invention, the composition comprising the cell treatment agent of the present invention as an active ingredient can be administered by a conventional route through rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, ≪ / RTI >

In addition, in the present invention, the mesenchymal stem cell prepared by metformin treatment and the cell therapeutic composition for treating an immunological disease comprising the cell, the immunological diseases to be treated include, but are not limited to, osteoarthritis, rheumatoid arthritis, (CNS), asthma, dermatitis, psoriasis, cystic fibrosis, posttransplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Systemic lupus erythematosus, Sjogren's syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia, Glomerulonephritis and pulmonary fibrosis, Inflammatory Bowel Disease, Crohns disease, autoimmunity It is well known that chronic obstructive pulmonary disease such as autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, The present invention relates to a pharmaceutical composition for the treatment of pulmonary disease (COPD), Graves disease, Gastrointestinal allergy, Conjunctivitis, Atherosclerosis, Coronary artery disease, Angina, And mitochondrial diseases.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.

≪ Example 1 >

Preparation of Metformin-Treated Mesenchymal Stem Cells and Analysis of Expression Levels of Immunomodulatory Gene

<1-1> Isolation and culture of mesenchymal stem cells with peripheral blood

After collecting 50cc of blood from normal subjects, PBMCs were separated and cultured for 5 days in a-MEM medium containing 20% FBS. Subsequently, the medium was replaced at intervals of 3 days. The cells were removed from the culture plate according to the degree of cell proliferation and cultured. The cultivation was carried out three times (passage 3) to obtain mesenchymal stem cells from peripheral blood. As a result, it was confirmed that CD105 + CD29 + CD44 was positive and that CD34, CD45 and HLA-DR were negative (refer to FIG. IA, in FIG. 1A, Picture, and the right picture is a picture enlarged 40 times).

<1-2> Isolation and culture of mesenchymal stem cells from adipose tissue

The fat tissue obtained by liposuction surgery or the fat tissue obtained after the surgical operation was washed with PBS containing 10% penicillin-streptomycin more than 10 times to remove blood and foreign substances, and the tissue was finely cut to 0.2-0.3 g . (Roche, Sandhofer Strasse, Mannheim, Germany) in a 0.2% collagenase solution and reacted at 100 rpm for 1 hour in a water bath at 37 ° C. The solution layer decomposed by the collagenase was separated from the undegraded fragments using a 100 μm mesh, and then an equal volume of PBS was added to the separated collagenase solution. Then, the mixture was centrifuged at 4 ° C and 1200 rpm for 5 minutes to remove the lipids and fat layers, and the collagenase supernatant was removed. (Cambrex, Walkersville, MD, USA), mesenchymal cell growth aids (Cambrex, Walkersville, MD, USA), 4 mM &lt; RTI ID = 0.0 &gt; L-glutamine and penicillin (0.025 unit / 500 ml) / streptomycin (0.025 mg / 500 ml)] and centrifuged at 4 ° C and 1200 rpm for 5 minutes. MSCGM is a medium based on DMEM (Dulbecco's modified Eagle's medium) containing fetal bovine serum. The supernatant was then removed and MSCs obtained were inoculated on a culture dish and cultured in MSCGM at 37 占 폚 in a 5% CO ₂ incubator. The cultures were incubated while changing the culture medium once every two days.

<1-3> Preparation of metformin-treated mesenchymal stem cells with immunoregulatory ability and analysis of expression level of immunoregulatory related genes in mesenchymal stem cells prepared

The mesenchymal stem cells isolated and cultured in Examples <1-1> and <1-2> were treated with metformin, and the expression of IDO, TGF-beta, IL-10 The changes were analyzed.

More specifically, mesenchymal stem cells in peripheral blood-derived is cultured under 37 ℃ temperature conditions by treating the metformin of 1mM concentration, based on the 2 × 10 ⁵ cells were prepared the mesenchymal stem cells with metformin this process, the adipose tissue-derived mesenchymal stem The cells were treated with metformin at a concentration of 1 mM for 2 × 10 ⁵ cells and cultured under the temperature condition of 37 ° C. to prepare metformin-treated mesenchymal stem cells.

At this time, mesenchymal stem cells not treated with metformin were used as a control group, and the degree of immunoregulatory gene expression was analyzed in metformin treated mesenchymal stem cells.

As a result, in the case of mesenchymal stem cells isolated from peripheral blood and treated with metformin, the expression of IDO and TGF-beta in mesenchymal stem cells was increased by metformin treatment, (FIG. 2).

In addition, these results were similar in mesenchymal stem cells isolated from adipose tissue and treated with metformin, indicating that the expression levels of IDO, TGF-beta and IL-10 were increased compared with mesotermal stem cells not treated with metformin (See FIG. 5).

Further, the present inventors have also confirmed the above results by DAPI staining method. Namely, DAPI (4 ', 6-diamidino-2-phenylindole) was used for staining and metformin-treated mesenchymal stem cells Of IL-10 and IDO were analyzed.

As a result, as shown in Fig. 6, when metformin was treated with mesenchymal stem cells derived from adipose tissue, it was found that red-stained IL-10 and IDO were further increased compared to the case without metformin treatment.

<1-4> Preparation of metformin-treated mesenchymal stem cells with immunoregulatory ability and the expression level of genes related to inflammatory and tumor factors in mesenchymal stem cells prepared

The mesenchymal stem cells isolated and cultured in Examples <1-1> and <1-2> were treated with metformin and then treated with HMGB-1, IL-6, IL -1beta was analyzed.

Specifically, metformin-treated mesenchymal stem cells were prepared by culturing adipocyte-derived mesenchymal stem cells with methamphetamine at a concentration of 1 mM for 5 × 10 ⁵ cells at 37 ° C.

At this time, mesenchymal stem cells not treated with metformin were used as a control, and expression of inflammatory and tumor factor genes in metformin treated mesenchymal stem cells was analyzed using realtime PCR method.

As a result, the expression levels of HMGB-1, IL-6 and IL-1beta were significantly decreased in mesenchymal stem cells isolated from adipose tissue and treated with metformin (see FIG. 3).

Based on these results, the inventors of the present invention found that mesenchymal stem cells prepared by treatment with metformin significantly increased the expression of IDO, IL-10 and TGF-beta, which have immunomodulatory properties, compared to those without metformin treatment , And the expression of HMGB-1, IL-6 and IL-1beta, which are related to inflammatory and tumor factors, was significantly reduced.

&Lt; Example 2 >

Analysis of autophagy activity and mitochondrial activity in mesenchymal stem cells prepared by treatment with metformin

From the peripheral blood-derived mesenchymal stem cells obtained by treating the mesenchymal stem cells derived from peripheral blood and cultured in Example 1 with metformin, the mesenchymal stem cells with increased immunoregulatory ability and the metformin-treated mesenchymal stem cells were subjected to electron microscopy And the changes of mitochondrial activity were observed.

Since autophagic action can modulate the beneficial or deleterious effects of immunity and inflammation and has the effect of preventing infectious diseases, autoimmune and inflammatory diseases, the present inventors have found that the metformin-treated mesenchymal stem cells of the present invention are self- It has a phagocytosis function to check if it is possible to treat immune and inflammatory diseases.

In addition, it is known that autophagy is involved in the degradation of microorganisms (viruses, bacteria, parasites, etc.) invading into the cell, and it is known to suppress the inflammatory reaction as well as to participate in the actual immune response and its regulation. This shows that it plays an important role in various pathophysiological processes and can regulate the self defense (immune) system in particular. Recently, it has been emphasized that the control of autophagy balance is important in the treatment of inflammatory bowel disease and various autoimmune diseases.

As a result, as shown in Fig. 4, metformin-treated mesenchymal stem cells showed an increase in the number of autophagic vacuoles (yellow arrow) compared with the group not treated with metformin (yellow arrow), activity of mitochondria And increased by metformin (red arrows point to mitochondria).

These results suggest that Met-MSC is effective in the treatment of mitochondrial diseases.

&Lt; Example 3 >

Effect of metformin-treated mesenchymal stem cells on osteoarthritis

In order to confirm whether metformin-treated mesenchymal stem cells prepared according to the present invention are actually useful for the treatment of immune diseases, the therapeutic effect of the osteoarthritis-induced animal model was investigated.

First, 5-week-old male Wistar rats (central laboratory animals), 200-250 g, were raised at 21-22 ° C for 12 hours in light-dark cycle to produce osteoarthritic animal models. Feed was supplied and raised. After that, 3 mg of monosodium iodoacdtate (Sigma, St. Louis, Mo.) was injected intra-articularly into the osteoarthritis-induced mouse animal by intraperitoneal injection of 26.5 g syringes to induce osteoarthritis.

Then, osteoarthritis-induced mice were injected with 2 × 10 ⁶ adipose tissue-derived mesenchymal stem cells and metformin-treated adipose tissue-derived mesenchymal stem cells, once a week, twice in total, iv. After 7 days, the degree of pain, which is an index for evaluating the effect of osteoarthritis - induced animals, was measured and the degree of cartilage destruction was analyzed by india ink staining.

As a result of the analysis, as shown in Fig. 7, in the mouse group injected with metformin-treated mesenchymal stem cells compared to the group injected with mesvastatin-treated mesenchymal stem cells, And the degree of cartilage destruction was restored to that of the group treated with metformin - treated mesenchymal stem cells. 7B, the upper and lower views show that the joint of the mouse in which osteoarthritis is induced is observed at different angles.

Further, after 7 days, the present inventors analyzed the degree of joint inflammation and cartilage destruction in osteoarthritis-induced animals after staining using H & E staining, Toluidin blue staining, and saffranin O staining method.

As a result, as shown in FIG. 12, in comparison with the mouse group injected with mesenchymal stem cells, mesenchymal stem cells injected with mesenchymal stem cells significantly decreased inflammatory cell infiltration and cartilage destruction Respectively.

<Example 4>

Effect of metformin-treated mesenchymal stem cells on inflammatory bowel disease

To prepare a mouse model of inflammatory bowel disease, C57BL / 6 (H-2kb) mice were used as test animals, and 3.5% dextran sulfate sodium (DSS) Induced inflammatory bowel disease. Then, mesenchymal stem cells derived from adipose tissue, which had been treated with adipocyte-derived mesenchymal stem cells and metformin at a concentration of 1 mM, were injected into the animal of inflammatory bowel disease twice at a dose of 2 × 10 ⁶ cells a total of twice a week, (Body weight, length of bowel, and disease activity index (DAI)).

As a result, as shown in FIG. 8, compared to the group treated with adipose tissue-derived mesenchymal stem cells, the metformin-treated mesenchymal stem cells showed that the weight of the mice reduced to inflammatory bowel disease was more effectively restored , IBD disease activity was significantly decreased (disease activity was almost zero), and the thickness and length of the colon were recovered.

Further, after 7 days, the present inventors analyzed the degree of TNF-α expression by intestinal inflammatory cell infiltration, changes in cell morphology and immunochemistry staining using an H & E staining method in an animal model of inflammatory bowel disease.

As a result, as shown in Fig. 9, mesenchymal stem cells treated with metformin compared to the group treated with adipose tissue-derived mesenchymal stem cells were observed to exhibit intramuscular inflammatory cell infiltration and cell morphology similar to normal mice In addition, TNF-a, which is an inflammatory cytokine in the intestine, was observed by immunochemistry. Similarly, mesenchymal stem cells treated with metformin were similar to normal intestinal tissues compared to those treated with mesenchymal stem cells alone The expression level of TNF-a was decreased.

&Lt; Example 5 >

Effect of metformin-treated mesenchymal stem cells on spontaneous inflammatory autoimmune arthritis

Furthermore, the present inventors conducted the following experiment to determine whether metformin-treated mesenchymal stem cells have therapeutic effects on arthritis.

In order to prepare an autoimmune arthritis mouse model, a mouse (IL-1Ra knockout mouse) lacking the IL-1 receptor gene was prepared according to the method disclosed by Y. Iwakura, The IL-1 receptor antagonist (IL-1Ra) acts directly on the IL-1 receptor and prevents IL-1α and IL-1β from acting on the receptor, thereby naturally inducing autoimmune arthritis disease Method.

Metformin-treated mesenchymal stem cells prepared according to the present invention were injected into mice suffering from arthritis, and metformin-treated mesenchymal stem cells were transplanted into the mice at a density of 2 x 10 &lt; ^{6 &} . After one week, the arthritic size of each mouse was measured. As a control group, arthritis-induced mouse group and arthritic mouse group injected with adipose tissue-derived mesenchymal stem cells (mesenchymal stem cell group without metformin treatment) were used as a control group.

The results of the analysis showed that mice treated with metformin-treated mesenchymal stem cells improved the symptoms of arthritis compared with those treated with mesenchymal stem cells only (see FIG. 10).

&Lt; Example 6 >

Effect of metformin-treated mesenchymal stem cells on induction of autoantigen activity to treat rheumatoid arthritis

To prepare the arthritis mouse model, DBA / 1J mice were mixed with 1: 1 type II collagen (CII) and CFA (adjuvant), and 100 ul of CII per mouse was injected into the tail base at a dose of 50 ul. A mixture of CII and IFA 1: 1 was injected at a dose of 100 ug / 50ul.

After 1 week of induction of arthritis in mice, mesenchymal stem cells or metformin-treated mesenchymal stem cells, which had not been treated with anything three times per week, were administered 1 x 10 ⁵ (per kg body weight) And evaluated.

For the measurement of arthritis index, three observers who did not know the contents of the experiment were evaluated for perception of joint inflammation three times a week for up to 10 weeks. At this time, the evaluation of arthritis was based on the average arthritis index by Rossoliniec et al., And the average score obtained by dividing the score of the three scales according to the following scale by three, except for the leg applied with CII / CFA at the second inoculation, The average value obtained by dividing the values obtained by the three observers in the animal model was used. The scores and criteria for arthritis evaluation are as follows.

0 point: There is no shortage or swelling.

1 point: mild swelling and redness limited to the foot or ankle joint

2 points: Mild swelling and redness from ankle joint to metatarsal

3 points: Moderate edema and redness from ankle joint to foot

4 points: edema and redness from the ankle to the whole leg

The best arthritis indices are four in the marine so the highest disease index is 16 per mouse.

As a result, as shown in Fig. 11, arthritis was further inhibited in the mouse group injected with metformin-treated mesenchymal stem cells compared with the mouse group injected with mesenchymal stem cells (see Fig. 11).

Therefore, the inventors of the present invention have found that mesenchymal stem cells prepared by treating metformin have an activity of effectively controlling the immune response and thus can be usefully used as a new cell therapy agent for the treatment of immune diseases, particularly autoimmune diseases I could.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (10)

A method for producing mesenchymal stem cells which mass-produce human transgranular growth factor-beta (TGF-beta) and immunoregulatory ability, comprising the step of treating metformin in mesenchymal stem cells. The method according to claim 1,
Wherein the mesenchymal stem cells are positive for CD105, CD29 and CD44, and have immunological characteristics of CD34, CD45 and HLA-DR negative. The method according to claim 1,
Culturing mesenchymal stem cells treated with metformin at a temperature of 28 to 42 DEG C for 14 to 21 days. The method according to claim 1,
Wherein said mesenchymal stem cells are isolated from peripheral blood or adipose tissue. The method according to claim 1,
Wherein the metformin is treated at a concentration of 0.2 to 2 mM based on the number of mesenchymal stem cells of 2 x 10 ⁵ to 1 x 10 ⁶ cells. The method according to claim 1,
Wherein the mesenchymal stem cells have increased expression of one or more factors selected from the group consisting of IDO, TGF-beta and IL-10 by treatment with metformin. A mesenchymal stem cell which mass produced human transgranular growth factor-beta (TGF-beta) produced by the method of claim 1 and has immune-controlling ability. A cell therapy composition for preventing or treating immune diseases comprising the mesenchymal stem cell of claim 7 as an active ingredient. 9. The method of claim 8,
Wherein said composition is administered at 2.5 × 10 ⁵ to 2.5 × 10 ⁷ cells / kg of body weight of the mesenchymal stem cells having immunomodulating ability. 9. The method of claim 8,
The immunological diseases are selected from the group consisting of osteoarthritis, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, posttransplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease Glomerulonephritis and pulmonary fibrosis, Inflammatory Bowel Disease, Crohns disease, Autoimmune Diabetes, autoimmune diabetes, autoimmune diabetes, autoimmune diabetes, Diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive cardiomyopathy, Or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and / or prophylaxis of chronic obstructive pulmonary disease (COPD), Graves disease, Gastrointestinal allergy, Conjunctivitis, Atherosclerosis, Coronary artery disease, Metastasis, small artery disease, and mitochondrial disease. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;

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