Therapeutic composition for immune diseases by mitochondrial transplantation

Abstract

The present invention relates to a composition of an immunomodulatory therapeutic agent by inducing transplantation of a mitochondrial derived from an induced pluripotent stem cell (iPSC), and it is confirmed that the mitochondrial derived from the iPSC according to the present invention overexpresses UCP2, and the expression of the UCP2 is related to an immunomodulatory effect, and more particularly, to a composition of an immunomodulatory therapeutic agent by inducing transplantation of the mitochondrial derived from the iPSC. In addition, it has been confirmed that mitochondrial function is enhanced after administration of an iPSC-derived mitochondrial, thereby increasing ATP synthesis and mitochondrial membrane potential. Moreover, it has been confirmed that the expression of immune cells associated with autoimmune diseases is regulated, and when used in combination with metformin, it has been confirmed that a synergistic effect on the regulation effect of immune cells has been confirmed. In addition, it was confirmed that the disease activity of autoimmune diseases is reduced, and the expression of autoimmune-related cells is suppressed. Moreover, it has been confirmed that, when a peripheral blood mononuclear cell is treated with an iPSC-derived mitochondrion, the expression of Th17 is suppressed and the expression of Treg is increased, thereby improving the immune regulation and disease activity of various immune diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to a composition for treating an immune disease by mitochondrial transplantation. BACKGROUND

[0002] An immune disease refers to a disease in which a constituent element of an immune system causes, mediates, or otherwise contributes to a pathological state, and in particular, an inflammatory disease is one of the most important health problems worldwide. Inflammation generally refers to a local protective reaction of body tissue against invasion of a host by external substances or harmful stimuli. Causes of inflammation include infectious pathogens such as bacteria, viruses, and parasites; physical causes such as burns or radiation exposure; chemical drugs such as toxins, drugs, or commercial preparations; immune reactions such as allergies and autoimmune reactions; or states related to oxidative stress.

[0003] Also, autoimmune diseases, one of the immune diseases, are characterized by spontaneous reactions caused by an immune system attacking its own organs. These reactions are caused by T lymphocytes recognizing auto-antigens, thereby inducing immune reactions on the humoral side (auto-antigen production) and on the cellular side (increased cytotoxic activity of lymphocytes and macrophages). Autoimmune diseases include rheumatic diseases, psoriasis, systemic dermatomyositis, multiple sclerosis, lupus erythematosus, or exacerbation of immune reactions caused by antigens, i.e., asthma, allergies to drugs or food, inflammatory bowel disease, etc. These diseases are limited and chronic, and can be fatal depending on the situation, and there is currently no treatment that can effectively treat the diseases. Therefore, any drug, medicine, or medium that can alleviate or relieve symptoms during the development of these diseases can be considered an important solution to the health of patients.

[0004] On the other hand, mitochondria are organelles of eukaryotic cells involved in the synthesis and regulation of adenosine triphosphate (ATP) as an energy source in cells. Mitochondria are involved in various metabolic pathways in the body such as cell signaling, cell differentiation, apoptosis, and control of cell cycle and cell growth. Mitochondria are organelles that have their own genes and play a central role in energy metabolism in cells. Mitochondria produce energy through the electron transport and oxidative phosphorylation processes, and play an important role in aspects such as the apoptosis signaling pathway.

[0005] It has been reported that a decrease in energy production due to a decrease in the function of mitochondria can cause various diseases. When the function of the electron transport chain reaction is decreased due to mutations in the mitochondrial genome and proteome, phenomena such as a decrease in ATP production, excessive production of reactive oxygen species, and a decrease in calcium regulation function occur. In this case, the membrane permeability of mitochondria changes, leading to functional abnormalities in apoptosis, which can cause cancer and intractable diseases.

[0006] It is reported that human diseases caused by abnormal mitochondrial function as described above include mitochondrial-related genetic diseases, diabetes, heart disease, senile dementia such as Parkinson's disease or Alzheimer's disease, and the occurrence and metastasis of various cancers. Also, a common feature found in more than 200 different types of cancer is impaired apoptosis function, increased inflammatory response, and increased abnormal metabolic action. Also, research on the correlation between mitochondria and immune diseases is currently being conducted.

[0007] There are signal transduction mechanisms closely related to cell death inside mitochondria, and if we can interpret and control these signals, they can be applied to disease treatment. Mitochondrial dysfunction is not a single symptom or disease, but can manifest as multiple diseases in multiple organs in the human body, and thus the possibility of a specific treatment method for lesions and diseases associated with diseased mitochondria is proposed, but there is an urgent need to develop precise targets or suitable treatment techniques. In particular, recent studies have shown that increased oxidative stress due to increased reactive oxygen species in cells is a basic substrate of cytology / molecular biology in cardiovascular diseases, metabolic diseases, and neurodegenerative diseases, but the effects of metabolic therapeutic agents or antioxidant treatments currently used in the clinical and preclinical stages are still controversial. Therefore, transplantation of undamaged mitochondria or gene therapy as a micro-targeting therapy that can regulate the expression of membrane proteins and matrix proteins that can directly control mitochondrial function can be a promising permanent or semi-permanent treatment. SUMMARY

[0008] TECHNICAL PROBLEM

[0009] The present application aims to provide a mitochondrion derived from an induced pluripotent stem cell (iPSC) and overexpressing uncoupling protein 2 (UCP2).

[0010] Still another object of the present application is to provide a pharmaceutical composition for preventing or treating an autoimmune disease, comprising an induced pluripotent stem cell (iPSC)-derived uncoupling protein 2 (UCP2) overexpression mitochondrion as an active ingredient.

[0011] Another object of the present application is to provide a food composition for preventing or improving autoimmune diseases, which comprises mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSC) as an effective ingredient.

[0012] Still another object of the present application is to provide a pharmaceutical composition for immunomodulation, which comprises mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSC) as an effective ingredient.

[0013] Still another object of the present application is to provide a food composition for immunomodulation, which comprises mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSC) as an effective ingredient.

[0014] Still another object of the present application is to provide a method for treating autoimmune diseases, which comprises the step of administering a pharmaceutically effective amount of the mitochondria as described above to an individual.

[0015] Technical Solution

[0016] To achieve the object, the present application provides mitochondria derived from induced pluripotent stem cells (iPSC) and overexpressing uncoupling protein 2 (UCP2).

[0017] Also, the present application provides a pharmaceutical composition for preventing or treating autoimmune diseases, which comprises mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSC) as an effective ingredient.

[0018] Also, the present application provides a food composition for preventing or improving autoimmune diseases, which comprises mitochondria overexpressing uncoupling protein 2 (UCP2) derived from induced pluripotent stem cells (iPSC) as an effective ingredient.

[0019] Also, the present application provides a pharmaceutical composition for immune regulation comprising induced pluripotent stem cell (iPSC)-derived uncoupling protein 2 (UCP2)-overexpressing mitochondria as an effective ingredient.

[0020] Also, the present application provides a food composition for immune regulation comprising induced pluripotent stem cell (iPSC)-derived uncoupling protein 2 (UCP2)-overexpressing mitochondria as an effective ingredient.

[0021] Also, the present application provides a method for treating an autoimmune disease, the method comprising the step of administering a pharmaceutically effective amount of the mitochondria as described above to an individual.

[0022] Effects of the Invention

[0023] It was confirmed that the iPSC-derived mitochondria of the present application overexpress UCP2, and that the expression of UCP2 is associated with immune regulation. Also, it was confirmed that the function of mitochondria is enhanced after administration of the iPSC-derived mitochondria, thereby increasing ATP synthesis and mitochondrial membrane potential. Also, it was confirmed that the expression of immune cells associated with autoimmune diseases is regulated, and that a synergistic effect on the immune cell regulation effect is confirmed when used in combination with metformin. Also, it was confirmed that the disease activity of autoimmune diseases is reduced, and the expression of autoimmune-related cells is inhibited. Also, it was confirmed that when peripheral blood mononuclear cells are treated with iPSC-derived mitochondria, the expression of Th17 is inhibited and the expression of Treg is increased, improving immune regulation and disease activity of various immune diseases, and thus can be usefully used in the field of immunotherapeutic agents. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure for quantitative analysis of UCP2 protein expression in mitochondria according to cell origin by Western blotting.

[0025] Figure 2 Figure for analysis of UCP2 protein expression in iPSC-derived mitochondria treated with UCP2 siRNA by Western blotting (A part: Western blotting result, B part: quantification of analysis result)

[0026] Figure 3 Figure for analysis of immune cell activity according to treatment of iPSC-derived mitochondria treated with UCP2 siRNA (A part: flow cytometry result, B part: quantification of Th17 expression, C part: quantification of IL-17).

[0027] Figure 4 Figures for confirming increase in mitochondrial function in mouse splenocytes treated with mitochondria derived from iPSC according to the present application (A section: confirmation of ATP content in cells, B section: confirmation of ATP synthesis ability, C section: confirmation of membrane potential).

[0028] Figure 5 Figures for confirming immune cell expression in mouse splenocytes treated with mitochondria derived from iPSC according to the present application (A section: confirmation of IFN-γ positive cells, B section: confirmation of IL-17 positive cells, C section: confirmation of Foxp3 positive cells, D section: confirmation of Th17 / Treg ratio).

[0029] Figure 6 Figures for confirming amount of IFN-γ and IL-17 in mouse splenocyte culture solution treated with mitochondria derived from iPSC according to the present application by ELISA (A section: quantification of IFN-γ, B section: quantification of IL-17).

[0030] Figure 7 Figures for confirming immune cell expression in mouse splenocytes treated with mitochondria derived from iPSC according to the present application in combination with metformin (A section: confirmation of IFN-γ positive cells, B section: confirmation of IL-17 positive cells).

[0031] Figure 8 Figures for confirming disease activity in an animal model of inflammatory bowel disease treated with mitochondria derived from iPSC (A section: confirmation of body weight change, B section: quantification of intestinal length).

[0032] Figure 9 Figures for confirming intestinal tissue damage in an animal model of inflammatory bowel disease treated with mitochondria derived from iPSC by H&E staining (A section: staining result, B section: quantification of intestinal damage index).

[0033] Figure 10 Figures for confirming immune cell expression in splenocytes of an animal model of inflammatory bowel disease treated with mitochondria derived from iPSC (A section: quantification of Th1, B section: quantification of Th2, C section: quantification of Th17).

[0034] Figure 11 Figures for confirming Th17 and Treg cell expression in splenocytes of an animal model of inflammatory bowel disease treated with mitochondria derived from iPSC by confocal analysis (A section: Th17 analysis result, B section: quantification of Th17, C section: Treg analysis result, D section: quantification of Treg).

[0035] Figure 12 Graph to confirm the expression of Th17 in peripheral blood mononuclear cells according to mitochondria derived from iPSCs of the present application by flow cytometry (A part: flow cytometry results, B part: quantification of Th17).

[0036] Figure 13 Graph to confirm the expression of Th17 in peripheral blood mononuclear cells according to mitochondria derived from iPSCs of the present application and MTDR Treg condition by flow cytometry (A part: flow cytometry results, B part: quantification of Th17).

[0037] Figure 14 Graph to confirm the amount of IL-17 in cell culture medium according to treatment of cells with mitochondria derived from iPSCs of the present application by ELISA.

[0038] Figure 15 Graph to confirm the expression of Treg in peripheral blood mononuclear cells according to mitochondria derived from iPSCs of the present application by flow cytometry (A part: flow cytometry results, B part: quantification of Treg).

[0039] Figure 16 Graph to confirm the expression of Treg in peripheral blood mononuclear cells according to mitochondria derived from iPSCs of the present application and MTDR Treg condition by flow cytometry (A part: flow cytometry results, B part: quantification of Treg).

[0040] Figure 17 Graph to confirm the amount of IL-10 in cell culture medium according to treatment of cells with mitochondria derived from iPSCs of the present application by ELISA.

[0041] Figure 18 Graph to confirm the immunomodulatory effect in wild type mouse spleen cells according to treatment of cells with mitochondria derived from iPSCs of the present application (A part: quantification of immune cell expression, B part: quantification of autoantibodies).

[0042] Figure 19 Graph to analyze the expression of pathogenic B cells in mouse B lymphocytes according to treatment of cells with mitochondria derived from iPSCs of the present application by flow cytometry (A part: flow cytometry results, B part: quantification of analysis results).

[0043] Figure 20 Graph to analyze the expression of immunomodulatory B cells in mouse B lymphocytes according to treatment of cells with mitochondria derived from iPSCs of the present application by flow cytometry (A part: flow cytometry results, B part: quantification of analysis results).

[0044] Figure 21 A graph for confirming the amelioration effect of lupus nephritis of a lupus animal model accompanied by metabolic abnormality by administration of iPSC-derived mitochondria of the present application through H&E staining.

[0045] A part: Confirmation of increase according to nephritis accompanied by metabolic abnormality;

[0046] B part: Confirmation of improvement of nephritis according to treatment with iPSC mitochondria;

[0047] C part: Confirmation of decrease of nephritis index in urine.

[0048] Figure 22 A graph for analyzing the B cell regulation effect of a lupus animal model accompanied by metabolic abnormality by administration of iPSC-derived mitochondria of the present application through flow cytometry (A part: flow cytometry results, B part: quantification of analysis results).

[0049] Figure 23 A graph for analyzing the pathogenic T cell regulation effect of a lupus animal model accompanied by metabolic abnormality by administration of iPSC-derived mitochondria of the present application through flow cytometry.

[0050] Figure 24 A graph for analyzing the B cell regulation effect of spontaneous lupus splenocytes by administration of iPSC-derived mitochondria through flow cytometry (A part: flow cytometry results, B part: quantification of analysis results).

[0051] Figure 25 A graph for analyzing the B cell regulation effect of serum-treated splenocytes of spontaneous lupus and metabolic abnormality accompanied by administration of iPSC-derived mitochondria through flow cytometry (A part: flow cytometry results, B part: quantification of analysis results).

[0052] Figure 26 A graph for confirming the T cell proliferation inhibition effect in allogenic reaction according to treatment with iPSC-derived mitochondria of the present application.

[0053] Figure 27 A graph for confirming the IFN-r and IL-10 specific increase effect in allogenic reaction according to treatment with iPSC-derived mitochondria of the present application through ELISA.

[0054] Figure 28 A graph for quantifying the disease index level of body weight of a GVHD animal model according to treatment with iPSC-derived mitochondria of the present application (A part: disease index change over time, B part: body weight change over time).

[0055] Figure 29Figures for quantification of pathogenic immune cell modulation in blood and spleen of GVHD animal model according to treatment with iPSC-derived mitochondria of the present application.

[0056] Figure 30 Figures for analysis of suppressive effect of inflammatory IL-17 expressing T cells of CD4 and CD8 lineage according to treatment with iPSC-derived mitochondria of the present application by flow cytometry (A part: analysis result, B part: quantification of Th17 and Tc17 cells).

[0057] Figure 31 Figures for analysis of subtypes of CD4 and CD8 lineage tissue-resident memory T cells according to treatment with iPSC-derived mitochondria of the present application by flow cytometry (A part: flow cytometry result, B part: quantification of CD4 and CD8 TRM).

[0058] Figure 32 Figures for confirmation of disease activity of acute psoriasis animal model after administration of iPSC-derived mitochondria of the present application (A part: illustration of psoriasis animal model and administration schedule, B part: macroscopic confirmation of psoriasis activity, C part: quantification of psoriasis index).

[0059] Figure 33 Figures for confirmation of inflammatory immune cell expression in acute psoriasis animal model after administration of iPSC-derived mitochondria of the present application by flow cytometry (A part: quantification of CD4 lineage cells, B part: quantification of CD8 lineage cells, C part: confirmation of Treg expression).

[0060] Figure 34 Figures for analysis of tissue-resident memory cell expression in acute psoriasis animal model after administration of iPSC-derived mitochondria of the present application by flow cytometry (A part: quantification of CD4 TRM, B part: quantification of CD8 TRM).

[0061] Figure 35 Figures for confirmation of histological changes in acute psoriasis animal model after administration of iPSC-derived mitochondria of the present application by H&E staining (A part: staining result, B part: quantification of staining result).

[0062] Figure 36 Figures for quantification of immune cell expression in gastric cancer patient-derived PBMC according to treatment with iPSC-derived mitochondria of the present application.

[0063] Figure 37 Figures for quantification of TRM subtypes in gastric cancer patient-derived PBMC according to treatment with iPSC-derived mitochondria of the present application.

[0064] Figure 38 A schematic diagram of an experimental schedule for a gastric cancer patient representative animal model to which iPSC-derived mitochondria of the present application are administered.

[0065] Figure 39 A graph for confirming a decrease in tumor size after administration of iPSC-derived mitochondria of the present application.

[0066] Figure 40 A graph for quantifying a decrease in gastric cancer tumor size after administration of iPSC-derived mitochondria of the present application.

[0067] Figure 41 A graph for analyzing immune cell subtypes of a gastric cancer patient representative animal model after administration of iPSC-derived mitochondria of the present application.

[0068] Figure 42 A schematic diagram of an experimental schedule for a liver cancer patient representative animal model to which iPSC-derived mitochondria of the present application are administered.

[0069] Figure 43 A graph for confirming a decrease in liver cancer tumor size after administration of iPSC-derived mitochondria of the present application.

[0070] Figure 44 A graph for confirming the quality of purification of iPSC-derived mitochondria of the present application and the delivery efficiency into chondrocytes (A part: confirmation of mitochondrial quality, B part: delivery efficiency and quantification).

[0071] Figure 45 A graph for confirming the chondrocyte metabolic factor expression modulation effect of iPSC-derived mitochondria according to the present application (A part: quantification of q-PCR results, B part: quantification of ELISA results).

[0072] Figure 46 A graph for analyzing the expression of inflammatory cell apoptosis factors of iPSC-derived mitochondria of the present application by Western blotting (A part: Western blotting results, B part: quantification of analysis results).

[0073] Figure 47 A graph for confirming the osteoarthritis improvement effect of iPSC-derived mitochondria of the present application by pain analysis (A part: visualization of the animal model construction process, B part: quantification of pain analysis results).

[0074] Figure 48 A graph for histological analysis of the cartilage damage protection effect of iPSC-derived mitochondria of the present application (A part: Safranin O staining and quantification, B part: immunohistochemical staining and quantification).

[0075] Figure 49FIG. 2 is a graph showing the analysis of immune cell subtypes in an osteoarthritis animal model administered with iPSC-derived mitochondria of the present application by flow cytometry. DETAILED DESCRIPTION

[0076] Hereinafter, embodiments of the present application will be explained in detail with reference to the accompanying drawings. In the following description, detailed explanation of technologies well-known to the one of ordinary skill in the art can be omitted. Also, in explaining the present application, if it is judged that detailed explanation of related known functions or configurations can unnecessarily obscure the gist of the present application, detailed explanation thereof can be omitted. Also, the terminology used in the present specification is the terminology that is used to appropriately describe a preferred embodiment of the present application, and can be changed according to the intention of one of ordinary skill in the art, or customs, etc.

[0077] Therefore, these terms should be defined based on the overall content of the present specification. Throughout the specification, when a certain part "comprises" a certain structural element, unless otherwise specifically noted, it means that other structural elements can also be included, rather than excluding other structural elements.

[0078] The present application provides a mitochondrion derived from an induced pluripotent stem cell (iPSC) and overexpressing uncoupling protein 2 (UCP2).

[0079] The "induced pluripotent stem cell (iPSC)" of the present application is a differentiated omnipotent stem cell that can be directly produced from an adult cell. The iPSC is transformed into an induced pluripotent stem cell by reprogramming through expression of specific transcription factor genes in an adult somatic cell. The key to iPSC preparation is the use of those reprogramming factors, and representative factors are "Yamanaka factors" used in the method published by the Yamanaka research team, i.e., transcription factors Oct4, Sox2, cMyc, Klf4. Through continued follow-up research on reprogramming factors, not only transcription factors but also new regulatory factors such as miRNA have been discovered. The iPSC preparation method is as follows: (1) after isolating and culturing the desired cell from an adult, (2) using a viral vector to isolate a gene capable of inducing a stem cell and introducing the isolated cell. (3) Then, the cell expressing the introduced gene is labeled with an embryonic stem cell marker Fbx15, and separated using an antibiotic. Then, the separated cell is cultured on a support cell layer according to a stem cell culture method. (4) A part of the transformed cell in the cultured cell is reprogrammed into an iPSC, and a colony similar to an embryonic stem cell is screened to prepare an iPSC.

[0080] The "mitochondria" of the present application is one of the organelles involved in cellular respiration, and the more active the cell's respiration, the more mitochondria the cell contains. The mitochondria are surrounded by a double membrane, and the inside is formed by a bumpy inner membrane called cristae. DNA and RNA exist inside the mitochondria, and the size is 0.2 to 3 μm, and it is involved in cellular respiration. The most important function of mitochondria is to synthesize ATP as an energy source from food that enters the body. There is a protein called "ATP synthase" in the inner membrane of the mitochondria, which functions to produce ATP. Hydrogen ions generated between the inner and outer membranes of the mitochondria by food enter the inner membrane of the mitochondria, and phosphate and ADP (a form in which 2 phosphates and adenosine are combined) are combined by ATP synthase to produce ATP (a form in which 3 phosphates and adenosine are combined). Also, mitochondria play a role in killing cells that have lost function, which is called apoptosis, and mitochondria cause apoptosis by absorbing DNA-damaged cells or cutting DNA. Also, it prevents cells that have lost function from being transformed into cancer cells or other cells. Mitochondria are structures that play a central role in governing respiration, and are also involved in the oxidation of pyruvate and acetyl CoA produced by the catabolism of sugars and lipids, and oxidative phosphorylation through the electron transport chain. As described above, mitochondria play a central role in the oxidation of sugars and fatty acids and the accompanying oxidative phosphorylation.

[0081] The "uncoupling protein 2 (UCP2)" of the present application is a mitochondrial uncoupling protein 2, which is a protein belonging to the mitochondrial anion transporter family. UCPs dissipate the mitochondrial membrane potential in the form of heat and uncouple oxidative phosphorylation in ATP synthesis, a process known as mitochondrial proton leakage. UCPs facilitate the delivery of protons from the inner to the outer mitochondrial membrane and reduce the mitochondrial membrane potential of mammalian cells, thereby reducing the production of reactive oxygen species. Unlike UCP1 and UCP3, which are mainly expressed in adipose tissue and smooth muscle, UCP2 is expressed in various tissues including the kidney, liver, gastrointestinal tract, brain, and skeletal muscle, although the exact mechanism of UCP2 is not yet known, it is known that its main function is related to the regulation of mitochondrial-derived reactive oxygen species.

[0082] According to an embodiment of the present application, the UCP2 can increase the function of iPSC-derived mitochondria.

[0083] According to an embodiment of the present application, the increase in the function of the mitochondria can be modulating immune cells, which can be cells selected from the group consisting of Th1, Th2, Th17, Treg, plasma B, Breg, Tfh, tissue-resident memory (TRM) T cells, Tc1, Tc2, and Tc17, the modulating immune cells can be reducing the expression of cells selected from the group consisting of Th1, Th2, Th17, plasma B, Tfh, tissue-resident memory (TRM) T cells, Tc1, Tc2, and Tc17, or can be increasing the expression of Treg or Breg.

[0084] According to an embodiment of the present application, the mitochondria can reduce the expression of IFN-γ or IL-17, or can increase the expression of IL-10.

[0085] Also, the present application provides a pharmaceutical composition for preventing or treating an autoimmune disease, comprising an induced pluripotent stem cell (iPSC)-derived uncoupling protein 2 (UCP2) overexpression mitochondria as an active ingredient.

[0086] The term "prevention" used in the present application means all acts of inhibiting the symptoms of a specific disease or delaying the progression of a specific disease by administering the composition of the present application.

[0087] The term "treatment" used in the present invention refers to all actions of improving or beneficially changing symptoms of a specific disease by administering the composition of the present invention.

[0088] The pharmaceutical composition of the present invention can further comprise an adjuvant in addition to the effective ingredient. The adjuvant can be used without limitation by any adjuvant known in the art, for example, the effect thereof can be increased by further comprising Freund's complete or incomplete adjuvant.

[0089] The pharmaceutical composition of the present invention can be prepared in a form in which the effective ingredient and a pharmaceutically acceptable carrier are mixed. Among them, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. The pharmaceutically acceptable carrier that can be used in the pharmaceutical composition of the present invention can include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, hydroxybenzoic ester, hydroxypropyl ester, talc, magnesium stearate, and mineral oil, but is not limited thereto.

[0090] The pharmaceutical composition of the present invention can be formulated into a powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, etc. oral dosage form, external agent, suppository, or sterile injection solution form, respectively, according to a conventional method.

[0091] When formulated, a diluent or excipient such as a common filler, extender, binder, wetting agent, disintegrating agent, surfactant, etc. can be used to prepare. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., which can be prepared by mixing at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. in the effective ingredient. And, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. can be used. Liquid preparations for oral administration include suspensions, internal liquids, emulsions, syrups, etc., which can contain excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to common diluents such as water, liquid paraffin, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used as non-aqueous solvents and suspensions. Semi-synthetic fatty acid esters (witepsol), tween 61, cocoa butter, glyceryl laurate, gelatin glycerin, etc. can be used as the base of the suppository.

[0092] The pharmaceutical composition of the present invention can be administered to an individual by various routes. It can be administered to an individual by various routes.

[0093] The amount of administration of the pharmaceutical composition of the present application is selected in consideration of the age, body weight, sex, physical condition, etc. of the individual. It is obvious that the concentration of the effective ingredient contained in the pharmaceutical composition can be variously selected depending on the subject, and preferably, is contained in the pharmaceutical composition at a concentration of 0.01 to 5000 μg / ml. When the concentration thereof is less than 0.01 μg / ml, the drug activity can not appear, and when the concentration thereof is more than 5000 μg / ml, a toxin can be generated to the human body.

[0094] According to an embodiment of the present application, the iPSC-derived mitochondria can increase the function of mitochondria in a cell, and the increase in the function of the mitochondria can be an increase in ATP synthesis or an increase in mitochondrial membrane potential.

[0095] According to an embodiment of the present application, the mitochondria can increase colon length.

[0096] According to an embodiment of the present application, the composition can inhibit intestinal tissue damage.

[0097] According to an embodiment of the present application, the mitochondria can reduce the amount of autoantibodies, which can be total IgG, IgG2a, or IgM.

[0098] According to an embodiment of the present application, the mitochondria can inhibit kidney damage, which can be a reduction in a factor selected from the group consisting of a vascular inflammation index, a glomerulitis index, urinary creatinine, and urinary albumin.

[0099] According to an embodiment of the present application, the mitochondria can reduce an allo response, which can increase the proliferation of T cells.

[0100] According to an embodiment of the present application, the mitochondria can reduce psoriasis disease activity, which can be an index selected from the group consisting of skin thickness, which can be epidermis thickness, scaling, or redness.

[0101] According to an embodiment of the present application, the mitochondria can inhibit the expression of a cartilage metabolism factor, which can be a factor selected from the group consisting of MMP3, MMP9, MMP13, and MCP1.

[0102] According to an embodiment of the present application, the autoimmune disease can be selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, Behcet's disease, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, asthma, primary biliary cirrhosis, dermatomyositis, Goodpasture syndrome, autoimmune meningoencephalitis, Sjogren syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, sarcoidosis, scleroderma, spondyloarthropathy, pernicious anemia, scleroderma, graft-versus-host disease, and a mitochondrial-related syndrome.

[0103] According to an embodiment of the present application, the autoimmune disease can be accompanied by a metabolic abnormality.

[0104] Also, the present application provides a food composition for preventing or ameliorating an autoimmune disease, which comprises induced pluripotent stem cell (iPSC)-derived uncoupling protein 2 (UCP2)-overexpressing mitochondria as an effective ingredient.

[0105] The term "ameliorating" used in the present application refers to all acts of at least reducing a parameter such as severity of symptoms associated with a state to be treated.

[0106] The food composition of the present application can comprise various flavoring agents or natural carbohydrates, etc. as additional ingredients, in addition to the effective ingredient of the present application, like a common food composition.

[0107] Examples of the natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; polysaccharides such as dextrin, cyclodextrin, etc.; and sugar alcohols such as xylitol, sorbitol, and erythritol, etc. Natural flavoring agents (thaumatin), stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used as the flavoring agents. The food composition of the present application can be formulated in the same manner as the pharmaceutical composition and used as a functional food, or can be added to various foods. Foods to which the composition of the present application can be added are, for example, beverages, meat, chocolates, foods, biscuits, pizza, ramen, other noodles, chewing gums, candies, ice creams, alcoholic beverages, vitamin complexes, and health supplement foods, etc.

[0108] Also, the food composition can contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjustors, stabilizers, preservatives, glycerin, alcohol, carbonating agents for carbonated beverages, and the like, in addition to the extract as an effective ingredient. In addition, the food composition of the present application can contain natural fruit juice and pulp for preparing a juice beverage machine vegetable beverage.

[0109] The functional food composition of the present application can be prepared and processed into the form of tablets, capsules, powders, granules, liquids, pills, etc. for preventing or improving autoimmune diseases. In the present application, the "health functional food composition" refers to a food prepared and processed using a material or ingredient having a beneficial functionality for the human body according to the Health Functional Food-related Law No. 6727, and refers to a nutrient or physiological action required for obtaining a beneficial effect for health use, such as the regulation of the structure and function of the human body, which is ingested. The health functional food of the present application can include general food additives, unless otherwise specified, and whether or not it is suitable as a food additive is determined according to the General Rules of the Food Additive Code approved by the Food and Drug Safety Department and the general test method, etc. according to the standards and specifications of the relevant items. The items listed in the "Food Additive Code" can include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, cinnamic acid, etc.; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sorghum pigment, guar gum, etc.; and mixed preparations such as L-glutamate sodium preparation, alkaline additive for flour, preservative, and tar pigment preparation, etc. For example, the health functional food in the form of tablets can be prepared by granulating the mixture of the effective ingredient of the present application and excipients, binders, disintegrants, and other additives according to the conventional method, and then adding a lubricant, etc. and compression molding, or directly compression molding the mixture. Also, the health functional food in the form of tablets can further include a flavoring agent, etc. as needed. In the health functional food in the form of capsules, hard capsules can be prepared by filling the mixture of the effective ingredient of the present application and excipients, etc. into ordinary hard capsules, and soft capsules can be prepared by filling the mixture of the effective ingredient of the present application and excipients, etc. into a capsule lipid such as gelatin. The soft capsules can include a plasticizer such as glycerol or sorbitol, a coloring agent, a preservative, etc. as needed. The health functional food in the form of pills can be prepared by molding the mixture of the effective ingredient of the present application and excipients, binders, disintegrants, etc. using the existing well-known method, and can be coated with sugar or other coating agents as needed, or can be coated on the surface with substances such as starch, talc, etc. The health functional food in the form of granules can be prepared by granulating the mixture of the effective ingredient of the present application and excipients, binders, disintegrants, etc. using the existing well-known method, and can include a flavoring agent, a flavoring agent, etc. as needed.

[0110] Also, the present application provides a pharmaceutical composition for immunomodulation, which comprises induced pluripotent stem cell (iPSC)-derived uncoupling protein 2 (UCP2)-overexpressing mitochondria as an effective ingredient.

[0111] Also, the present application provides a food composition for immunomodulation, which comprises iPSC-derived UCP2 overexpressing mitochondria as an effective ingredient.

[0112] Also, the present application provides a method for treating autoimmune diseases, which comprises the step of administering a pharmaceutically effective amount of the mitochondria as described above to a subject.

[0113] The treatment method of the present application comprises administering a therapeutically effective amount of the iPSC-derived UCP2 overexpressing mitochondria to a subject. The specific therapeutically effective amount for a particular subject is preferably applied differently according to the kind and degree of response to be achieved, the specific composition including whether other agents are used according to the situation, the age, body weight, general health status, sex, and diet of the subject, the time of administration, the route of administration, and the excretion rate of the composition, the period of treatment, various factors including drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field. The amount of the pharmaceutical composition of the present application is 0.0001 mg / kg to 100 mg / kg, preferably 0.01 mg / kg to 100 mg / kg, which can be administered 1 to 6 times a day. However, it is obvious to those skilled in the art that the amount of each effective ingredient to be taken or administered should be such that the content of each effective ingredient does not become high enough to cause side effects. Therefore, preferably, the effective amount of the composition suitable for the purpose of the present application should be confirmed in consideration of the above matters.

[0114] The subject can be applied to any mammal, which includes not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs, and cats.

[0115] The iPSC-derived UCP2 overexpressing mitochondria of the present application can be administered to mammals such as rats, mice, livestock, humans, etc. through various routes. All administration methods can be contemplated, for example, administration can be made through oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine, intradural, or intracerebroventricular injection.

[0116] Best Mode for Carrying Out the Invention

[0117] Hereinafter, the present application will be explained in more detail through examples. It is obvious to those skilled in the art that these examples are only for a more specific explanation of the present application, and the scope of the present application is not limited to these examples.

[0118] Example 1: Confirmation of the correlation between UCP2 expression and activity in iPSC-derived mitochondria

[0119] 1-1. Confirmation of UCP2 expression according to cell type

[0120] Confirmation of UCP2 expression according to cell type was performed to confirm the expression of uncoupling protein 2 (UCP2) in mitochondria derived from induced pluripotent stem cell (iPSC) of the present application and the correlation between the expression of UCP2 and the activity of mitochondrial transplantation. Specifically, mitochondria were isolated from muscle cells (L6) and iPSC (isolated using Mitochondria Isolation Kit, Thermo, Cat # 89874), protein was extracted from each of the mitochondria, and then the expression of UCP2 was confirmed by Western blotting. Also, in order to isolate mitochondria derived from iPSC, mitochondria were isolated from WTC11 cell line, which is a human-derived iPSC cell line, and 8 to 10 μg of mitochondria were obtained from 1 x 10 6 cells of WTC11 cell line.

[0121] As a result, as shown in Figure 1 , it was confirmed that the expression of UCP2 in mitochondria derived from iPSC of the present application was increased by 4 to 6 times compared to L6 cells, which are a muscle cell line, and thus the expression of UCP2 was significantly increased.

[0122] 1-2. Confirmation of correlation between expression of UCP2 in mitochondria derived from iPSC and immunomodulatory effect

[0123] It was confirmed that mitochondria derived from induced pluripotent stem cell (iPSC) of the present application overexpress UCP2, and thus whether the expression of UCP2 is related to the immunomodulatory ability of mitochondria derived from iPSC was confirmed. Specifically, iPSC was treated with UCP2 siRNA and mitochondria were obtained after culturing the iPSC. Mitochondria isolated from iPSC treated with Control siRNA were used as a control group. Then, the expression of UCP2 in the obtained mitochondria was analyzed by Western blotting.

[0124] Then, in order to confirm the immunomodulatory effect, splenocyes obtained from wild-type mice were treated with mitochondria derived from iPSC at 1 x 10 6at a concentration of 1 x 10 5 cells / well in a 24-well plate, and treated with 0.5 pg / ml of anti-CD3 and 100 ng / ml of LPS as a cell stimulation condition, and then treated with 5 pg of isolated mitochondria and reacted for 24 hours. After the reaction, the cultured spleen cells were analyzed using flow cytometry to confirm the phenotype of immune cells. The mitochondria and drug treatment conditions of each group are shown in Table 1.

[0125] Table 1

[0126]

[0127] As a result, as shown in Figure 2 , it was confirmed that the expression of UCP2 in iPSC-derived mitochondria was significantly reduced when treated with UCP2 siRNA.

[0128] Also, it was confirmed that in iPSC-derived mitochondria, when UCP2 was reduced, the expression of Th17 cells, which are autoimmune pathogenic immune cells, was increased, and the amount of IL-17, which is a cytokine associated with autoimmune diseases, was significantly increased Figure 3 .

[0129] Example 2: Confirmation of increased intracellular mitochondrial function of iPSC-derived mitochondria

[0130] It was confirmed whether the UCP2-overexpressing mitochondria derived from the induced pluripotent stem cell (iPSC) of the present application increased intracellular mitochondrial function after being transplanted into cells. Specifically, after obtaining mouse spleen cells, they were seeded at a cell concentration of 1 x 10 5 cells / well in a 96-well plate coated with anti-CD3 at a concentration of 0.5 pg / ml. 0.125 pg of mitochondria isolated from iPSC (isolated using a Mitochondria Isolation Kit, Thermo, Cat # 89874) was added and cultured for 24 hours (iPSC group). After the culture, the intracellular ATP concentration was measured using ATPlite (PerkinElmer, Cat # 6016943). Also, 2 mM of ADP was added to the cells and incubated at a temperature of 37°C for 45 minutes, and then the ATP synthesis ability was evaluated using an ATP assay kit, and the mitochondrial membrane potential stained with JC-1 was analyzed by flow cytometry. The non-treated control group was used as a control group.

[0131] As a result, as shown in Figure 4As shown, it was confirmed that compared with the control group, there was no significant difference in intracellular ATP content in the iPSC-treated group, but ATP synthesis was significantly increased. Figure 4 Part A and Figure 4 Part B). Furthermore, JC-1 staining confirmed mitochondrial membrane potential, showing a significantly increased red / green JC-1 ratio in the iPSC-treated group compared to the control group, thus indicating an increased membrane potential. Figure 4 (Part C of the iPSCs). Therefore, it was confirmed that iPSC-derived mitochondria enhance intracellular mitochondrial function.

[0132] Example 3: Confirmation of mitochondrial regulatory immune cells derived from iPSCs

[0133] 3-1. Confirming the regulation of immune cells in mouse spleen cells

[0134] The study aimed to confirm whether UCP2 overexpression of mitochondria derived from induced pluripotent stem cells (iPSCs) of this invention regulates immune cells. Specifically, after obtaining mouse spleen cells, 1×10⁻⁶ cells were used... 6 Cells were seeded at a concentration of 0.5 μg / ml in 24-well plates coated with anti-CD3 and cultured. Then, 0.5 μg of iPSC-derived mitochondria obtained in the same manner as in Example 1 were added and the cells were cultured for 72 hours. IFN-γ-positive and IL-17-positive cells were then confirmed by flow cytometry, and FoxP3-positive cells, a marker of Treg immune regulatory cells, were confirmed. The Th17 / Treg ratio was then analyzed, and the amounts of IFN-γ and IL-17 in the culture supernatant were determined by ELISA.

[0135] The results confirmed that, compared with the control group, the number of IFN-γ positive cells and IL-17 positive cells in the iPSC group was significantly reduced. Figure 5 Part A and Figure 5 Part B of the study showed a significant increase in the number of FoxP3-positive cells, a marker of Treg immune regulatory cells. Figure 5 Part C). Furthermore, due to the decreased Th17 / Treg ratio, increased expression of Tregs on immunomodulatory cells was confirmed (…). Figure 5 (Part D).

[0136] Furthermore, the results of ELISA measurements of IFN-γ and IL-17 expression in the culture supernatant confirmed that, compared with the control group, the levels of IFN-γ and IL-17 in the iPSC group culture medium were significantly reduced. Figure 6 Part A and Figure 6Part B of Example 1).

[0137] 3-2. Confirmation of the effect of metformin in combination with mitochondria in mouse splenocytes

[0138] Whether the immune cell regulation effect is increased when the iPSC-derived UCP2 overexpressing mitochondria of the present application is used in combination with metformin was confirmed. Specifically, cells obtained from the spleen of a mouse were seeded at a cell concentration of 1 x 10 6 in a 24-well plate coated with aCD at a concentration of 0.5 μg / ml. Then, 0.5 μg of mitochondria isolated from iPSCs cultured for 3 days with 0.5 mM of metformin was added and cultured for 72 hours (metformin group). After the end of the culture, IFN-γ positive cells and IL-17 positive cells were analyzed by flow cytometry. A group treated only with iPSC-derived mitochondria was used as a control group.

[0139] As a result, it was confirmed that IFN-γ positive cells were reduced in the metformin group treated in combination with metformin compared to the control group (p < 0.05) (FIG. 1A, Part A), and IL-17 positive cells were significantly reduced (p < 0.01) (FIG. 1A, Part B), thus confirming that the use of iPSC-derived mitochondria in combination with metformin increases the immune cell regulation effect. Figure 7 Figure 7 As a result, it was confirmed that IFN-γ positive cells were reduced in the metformin group treated in combination with metformin compared to the control group (p < 0.05) (FIG. 1A, Part A), and IL-17 positive cells were significantly reduced (p < 0.01) (FIG. 1A, Part B), thus confirming that the use of iPSC-derived mitochondria in combination with metformin increases the immune cell regulation effect.

[0140] Example 4: Confirmation of the disease regulation of iPSC-derived mitochondria in an animal model of inflammatory bowel disease

[0141] 4-1. Construction of an animal model of inflammatory bowel disease

[0142] The disease improvement effect of the iPSC-derived mitochondria of the present application in autoimmune diseases was confirmed. Specifically, an animal model of inflammatory bowel disease, which is an autoimmune disease, was constructed, and 3% dextran sodium sulfate (DDS) was fed to 7-week-old male B6 mice. On the 5th day of feeding, ordinary drinking water was changed, and 10 μg of iPSC-derived mitochondria was intraperitoneally injected on the 2nd, 6th, and 9th days of the experiment (iPSC mito group). Then, the body weight of the mouse was measured once a day until the end of the experiment, and the time point at which the experiment ended was humanely sacrificed, and the small intestine tissue was obtained and the intestinal length was measured. A normal (Nomal) group, which is a wild-type mouse, and a solvent control (Vehicle) group, which was injected with the same amount of physiological saline, were used as control groups.

[0143] As a result, as shown in FIG. 2, the iPSC mito group showed a significant increase in the intestinal length compared to the normal group (p < 0.05) and the vehicle group (p < 0.05), and the intestinal length was significantly increased compared to the normal group (p < 0.05) and the vehicle group (p < 0.05), thus confirming that the iPSC-derived mitochondria of the present application has a disease improvement effect in an animal model of inflammatory bowel disease. Figure 8 ​As shown in FIG. 4-1A, it was confirmed that the body weight of mice in the group injected with iPSC-derived mitochondria was significantly recovered at the end of the experiment compared to the solvent control (Vehicle) group. Also, it was confirmed that the intestinal length of mice was significantly increased compared to the solvent control (Vehicle) group (FIG. 4-1B), and thus it was confirmed that it improved inflammatory bowel disease. Figure 8

[0144] 4-2. Confirmation of improvement of intestinal tissue damage

[0145] It was confirmed whether the iPSC-derived mitochondria of the present application improved the intestinal tissue damage in inflammatory bowel disease. Specifically, the intestinal tissue was obtained from the sacrificed mice in the above-described Example 4-1 and paraffin blocks were prepared. Then, sections were cut using a microtome, and then stained using Hematoxylin & Eosin (H&E). After staining, the intestinal tissue was confirmed, and the loss of epithellum, crypt damage, depletion of goblet cells, infiltration of inflammatory cells, and histological score were quantified.

[0146] As a result, it was confirmed that the small intestinal tissue damage was improved in the iPSC mito group compared to the solvent control (Vehicle) group (FIG. 4-2A), and the loss of epithellum, crypt damage, depletion of goblet cells, infiltration of inflammatory cells, and histological score were significantly reduced, respectively (FIG. 4-2B), and thus it was confirmed that the iPSC-derived UCP2-overexpressing mitochondria improved inflammatory bowel disease. Figure 9 Figure 9

[0147] Example 5: Confirmation of immune cell regulation of iPSC-derived mitochondria in an animal model of inflammatory bowel disease

[0148] ​​​To confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present application regulate immune cells in inflammatory bowel disease. Specifically, splenocytes were obtained from the sacrificed mice in Example 4, and the obtained splenocytes were stimulated with phorbol myristate acetate (PMA) and Ionomycin for 4 hours. Then, the activities of Th1 (CD4+IFN-γ+), Th2 (CD4+IL-4+), and Th17 (CD4+IL-17+) were analyzed by flow cytometry. Also, the expression of Th17 (CD4+IL-17+) and Treg (CD4+, CD25+, and Foxp3+) cells was confirmed by confocal analysis.

[0149] As a result, as shown in Figure 10 Table 2, it was confirmed that the activities of Th1, Th2, and Th17 cells were significantly reduced in the iPSC mito group compared to the vehicle group, and thus it was confirmed that the iPSC-derived mitochondria regulate the expression of autoimmune cells.

[0150] Also, as shown in Figure 11 Table 3, the results of confocal analysis of the spleen sections at the time point at which the experiment was completed confirmed that the expression of Th17 cells was reduced and the expression of Treg was significantly increased in the iPSC mito group compared to the vehicle group, and thus it was confirmed that the iPSC-derived mitochondria regulate the expression of autoimmune cells, and thus it was confirmed that the iPSC-derived UCP2-overexpressing mitochondria improve inflammatory bowel disease.

[0151] Example 6: Confirmation of immune cell regulation by iPSC-derived mitochondria

[0152] 6-1. Confirmation of Th17 cell inhibitory effect

[0153] To confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present application also inhibit the expression of Th17 cells, which are pathogenic cells associated with autoimmune diseases, in human peripheral blood mononuclear cells (PBMCs). Specifically, human PBMCs were stimulated with 10 ng / ml of LPS and 5 x 10 5The cells were seeded at a concentration of 2 x 105cells / well in a 48-well plate coated with aCD at a concentration of 2 μg / ml. After 24 hours of culture under stimulation, 0.5, 1.5, and 2.5 μg of mitochondria subjected to post-separation of mitochondria by MTD R were added to the iPSC cells and cultured for 72 hours. Then, the expression of Thl7 cells was confirmed by flow cytometry. Also, the expression of MTDR-negative Thl7 cells and positive Thl7 cells was confirmed by flow cytometry, and the amount of IL-17 in the culture supernatant was measured using ELISA. A non-treated control group (Nil) and a solvent control (Vehicle) group as a negative control group were used as control groups.

[0154] As a result, as shown in FIG. 6A, it was confirmed that the expression of Thl7 cells was significantly increased in the solvent control (Vehicle) group compared to the Nil group, but the expression of Thl7 cells was decreased in a concentration-dependent manner in the group treated with the iPSC-derived mitochondria of the present application. Figure 12

[0155] Also, it was confirmed that there was no significant difference in the inhibitory effect on the expression of Thl7 cells under the condition in which the iPSC-derived mitochondria did not move intracellularly as an MTDR-negative condition, but the iPSC-derived mitochondria inhibited the expression of Thl7 cells under the condition in which the iPSC-derived mitochondria moved intracellularly as an MTDR-positive condition. Figure 13

[0156] Also, by confirming the amount of IL-17 secreted from Thl7 cells in the culture supernatant, it was confirmed that the amount of IL-17 in the culture supernatant of the solvent control (Vehicle) group was significantly increased compared to the Nil group, indicating that the stimulation effect was good, and in this condition, the amount of increased IL-17 was significantly decreased in the group treated with the iPSC-derived mitochondria. Figure 14

[0157] 6-2. Confirmation of Treg cell increasing effect

[0158] ​​​To confirm whether the iPSC-derived UCP2-overexpressing mitochondria regulate the expression of Treg cells, which are immunoregulatory cells in autoimmune diseases. After stimulating human peripheral blood mononuclear cells (PBMCs) with 0.5 μg / m of anti-CD3 and 10 ng / ml of LPS for 24 hours in the same manner as in Example 6-1, 0.5, 1.5, and 2.5 μg of mitochondria, which were post-isolated by staining with MitoTracker®, were added to the iPSC cells and cultured for 72 hours. Then, the expression of Treg cells was confirmed by flow cytometry, and the expression of Tregs according to the positive Treg condition was confirmed by flow cytometry, and the amount of IL-10 in the culture supernatant was measured using ELISA. A non-treatment control group (Nil) and a solvent control (Vehicle) group, which is a negative control group, were used as control groups.

[0159] In the condition in which the iPSC-derived mitochondria did not undergo intracellular movement as the MitoTracker® negative condition, an increase in Tregs was not observed, but in the condition in which the iPSC-derived mitochondria underwent intracellular movement as the MitoTracker® positive condition, it was confirmed that the iPSC-derived mitochondria increased Tregs, and as a result, as shown in Figure 15 Figure 16 ).

[0160] In addition, the results of confirming the amount of IL-10 secreted from Treg cells in the culture supernatant confirmed that the amount of IL-10 was significantly increased in the group treated with the iPSC-derived mitochondria compared to the Vehicle group Figure 17 ).

[0161] Example 7: Confirmation of the effect of iPSC-derived mitochondria on improving lupus accompanied by metabolic abnormalities

[0162] 7-1. Confirmation of the B cell regulatory effect of iPSC-derived mitochondria

[0163] ​To confirm whether the iPSC-derived UCP2-overexpressing mitochondria modulate B cells, which are immune cells associated with lupus disease, and autoantibodies, BALB / c mice, which are wild-type mice, were humanely sacrificed and spleen tissues were obtained. Then, splenocytes were isolated from the spleen tissues and treated with R848 (SML0196) at a concentration of 100 ng / ml as a lupus-inducing condition. Then, under B cell activation conditions, treatment was performed with anti-IgM at 10 μg / ml and soluble CD40 ligand at 1 μg / ml, and stimulation was performed at a temperature of 37℃ for 4 days. The iPSC-derived mitochondria were administered at a concentration of 1 μg / ml or 10 μg / ml during the treatment with the lupus-inducing and B cell activation conditions. After the completion of the reaction, each of the cells and culture solution was obtained, and the expression of IgM plasma B cells and IgG2a plasma B cells, which are lupus pathogenic immune cells, and Bregs, which are immune regulatory cells, was analyzed by flow cytometry. Also, the amount of total IgG, IgG2a, and IgM, which are lupus pathogenic autoantibodies, was measured by ELISA.

[0164] Also, to further confirm the B cell modulating effect of the iPSC-derived UCP2-overexpressing mitochondria, an A20 cell line, which is a mouse B lymphocyte cell line, was obtained from an ATCC cell line, and each of the cells was inoculated into a 24-well plate at a cell concentration of 1 × 105cells / well, and treated with the iPSC-derived mitochondria under the same lupus environment conditions and B cell stimulation conditions as described above and stimulated for 4 days. Then, the cells were collected, and the expression of IgG2a and Breg cells was analyzed by flow cytometry. 6

[0165] As a result, when the splenocytes derived from wild-type mice were treated with the iPSC-derived mitochondria under the lupus environment conditions, it was confirmed that the expression of IgM plasma B cells and IgG2a plasma B cells, which are lupus pathogenic B cells, was significantly reduced, and the expression of Breg cells was significantly increased (FIG. 1A). Figure 18 Also, it was confirmed that the amount of lupus pathogenic autoantibodies in the culture solution was significantly reduced due to the treatment with the iPSC-derived mitochondria (FIG. 1B). Figure 18

[0166] Also, when the A20 cell line, which is a mouse B lymphocyte cell line, was treated with the iPSC-derived mitochondria under the lupus environment conditions and B cell activation conditions, it was confirmed that the expression of IgG2a cells, which are lupus pathogenic B cells, was significantly reduced (FIG. 2A). Figure 19 ​​), as immunoregulatory cells of Bregs increased in a concentration-dependent manner ( Figure 20 ), it was confirmed that the iPSC-derived UCP2-overexpressing mitochondria modulate lupus-related cells.

[0167] 7-2. Confirmation of improvement of lupus nephritis by iPSC-derived mitochondria

[0168] The activity of lupus nephritis was confirmed to confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present application improve refractory metabolic abnormality-accompanied lupus disease. Specifically, 8-week-old Balb / c mice were treated with 10 μl of R848 solution prepared at a concentration of 5 mg / ml using acetone as a solvent on the back of the right ear 3 times a week for 6 weeks. Also, in order to induce metabolic abnormality, high fat diet (HFD) was fed (R848+HFD group). The mice were intravenously injected with 10 μg of iPSC-derived mitochondria once a week (R848+HFD+iPSC mito group). Wild type mice (WT group), general lupus mice (R848 group) were used as control groups. Then, at the end of the experiment, the mice were humanely sacrificed, kidney tissues were obtained from the mice and sectioned, the obtained sections were stained with H&E, the activity of lupus nephritis was quantified by vasculitis score and glomerulonephritis score (GN score), and the basement membrane structure of the kidney tissue was confirmed using electron microscopy. Also, the urine of the mice was collected at 1st week and 5th week of the experiment, and the amount of urine creatinine and albumin was measured and quantified using Mouse Creatinine Parameter Assay kit (KGE005) and Goat anti-Mouse Albumin antibody Affinity Purified (A90-134A).

[0169] As a result, as shown in Part A of FIG. 7, Figure 21 As shown in Part A of FIG. 7, it was confirmed that the vasculitis score and glomerulonephritis score of the R848 group were increased compared to the WT group, but the vasculitis score and glomerulonephritis score of the R848+HFD group were significantly increased compared to the R848 group, and thus the metabolic abnormality significantly increased the activity of lupus disease. However, when the iPSC-derived mitochondria were administered to the R848+HFD group, it was histologically confirmed that the nephritis was reduced, and it was confirmed that the abnormal structure of the basement membrane was restored (Part B of FIG. 7). Figure 21 As shown in Part A of FIG. 7, it was confirmed that the vasculitis score and glomerulonephritis score of the R848 group were increased compared to the WT group, but the vasculitis score and glomerulonephritis score of the R848+HFD group were significantly increased compared to the R848 group, and thus the metabolic abnormality significantly increased the activity of lupus disease. However, when the iPSC-derived mitochondria were administered to the R848+HFD group, it was histologically confirmed that the nephritis was reduced, and it was confirmed that the abnormal structure of the basement membrane was restored (Part B of FIG. 7). Figure 21 As shown in Part A of FIG. 7, it was confirmed that the vasculitis score and glomerulonephritis score of the R848 group were increased compared to the WT group, but the vasculitis score and glomerulonephritis score of the R848+HFD group were significantly increased compared to the R848 group, and thus the metabolic abnormality significantly increased the activity of lupus disease. However, when the iPSC-derived mitochondria were administered to the R848+HFD group, it was histologically confirmed that the nephritis was reduced, and it was confirmed that the abnormal structure of the basement membrane was restored (Part B of FIG. 7).

[0170] And, in the first week of the experiment, there was no significant difference in the amount of creatinine and albumin in the urine samples of the R848+HFD group and the R848+HFD+iPSC mito group, but in the fifth week of the experiment, the urine creatinine and albumin of the R848 group increased, but the increased amount of urine creatinine and albumin was significantly reduced in the group administered with iPSC-derived mitochondria Figure 21 (C part of Example 7-2), and thus it was confirmed that the kidney damage was inhibited.

[0171] 7-3. Confirmation of immune cell regulation by iPSC-derived mitochondria

[0172] Whether the iPSC-derived UCP2-overexpressing mitochondria of the present application regulate immune cells in refractory metabolic abnormality-companied lupus disease was confirmed. Specifically, the spleen tissue was obtained from the mice sacrificed in Example 7-2, and the expression of lupus-related B cells and lupus-related T cells was analyzed by flow cytometry.

[0173] As a result, as shown in Figure 22 , it was confirmed that the expression of plasma B cells and IgG2a plasma B cells, which are pathogenic immune cells of lupus, was significantly reduced in the group administered with iPSC-derived mitochondria compared to the R484+HFD group. Also, a tendency that the expression of IL-10 plasma B cells, which are immune regulatory cells, was increased was confirmed.

[0174] And, as a result of confirming the T cell regulation effect, it was confirmed that the expression of Tfh (IL-21+CD4+T cells), Th1 (IFNg+CD4+T cells), and Th17 (IL-17+CD4+T cells), which are pathogenic immune cells of lupus, was significantly reduced in the group administered with iPSC-derived mitochondria compared to the R484+HFD group Figure 23 , and thus it was confirmed that the iPSC-derived mitochondria can regulate the activity of immune cells.

[0175] 7-4. Confirmation of the effect of iPSC-derived mitochondria to improve spontaneous lupus

[0176] To confirm whether the iPSC-derived UCP2-overexpressing mitochondria modulate immune cells of SKG mice that spontaneously induce autoimmune diseases, specifically, SKG mice that are 15 weeks old and are a mouse model of spontaneous autoimmune induction were humanely sacrificed, and splenocytes were isolated from the spleen tissue. Then, the splenocytes were treated with lupus environmental conditions and B cell activation conditions in the same manner as in Example 7-1, and at the same time, treated with iPSC-derived mitochondria at a concentration of 2 μg / ml or 10 μg / ml, and incubated at a temperature of 37°C for 4 days. After the incubation, the cells were collected, and the expression of plasma B cells, which are lupus pathogenic immune cells, and Bregs, which are immune regulatory cells, was analyzed by flow cytometry.

[0177] Also, to confirm the cell regulatory effect according to metabolic abnormalities, when treated with the lupus environmental conditions and B cell activation conditions, the cells were treated with serum obtained from SKG mice that were fed with HFD for 6 weeks, and after the incubation, the cells were isolated under the same conditions as above, and the expression of plasma B cells and Bregs was analyzed by flow cytometry.

[0178] As a result, as shown in FIG. 8, it was confirmed that the expression of plasma B cells, which are lupus pathogenic cells, in SKG mouse-derived splenocytes was significantly increased under lupus stimulating conditions, but the increased plasma B cells were significantly reduced when treated with iPSC-derived mitochondria. Also, it was confirmed that the expression of Bregs was increased in a concentration-dependent manner when treated with iPSC-derived mitochondria under lupus stimulating conditions. Figure 24

[0179] Also, it was confirmed that the expression of plasma B cells, which are lupus pathogenic cells, in SKG mouse splenocytes was significantly increased when treated with serum of SKG mice that were fed with HFD under lupus stimulating conditions, but the increased plasma B cells were significantly reduced when treated with iPSC-derived mitochondria. Also, it was confirmed that the expression of Bregs was significantly increased when treated with iPSC-derived mitochondria under lupus stimulating conditions, Figure 25 thereby confirming that the iPSC-derived UCP2-overexpressing mitochondria improve lupus.

[0180] Example 8: Confirmation of the effect of iPSC-derived mitochondria in improving transplant rejection diseases

[0181] 8-1. Confirmation of the effect of iPSC-derived mitochondria in modulating allogenic reactions

[0182] ​To confirm whether the iPSC-derived UCP2 overexpression of mitochondria in this invention improves graft-versus-host disease (GVHD), a disease of transplant rejection, and to confirm whether it regulates cell proliferation in alloresponse, specifically, 2 × 10⁻⁶ cells were used. 5 CD4+ T cells of 1 normal receptor (Bclb / c, responder) and 2 × 10 5 Spleen cells from recipients (homogeneous) or donors (C57BL / 6, stimulators, allogeneic) that have been irradiated and have had their T cells removed were co-cultured in each well of a 96-well plate. During co-culture, mitochondria derived from iPSCs at a concentration of 0.5 μl / ml were administered to the allogeneic model. Homogeneous Nil groups and allo-response groups were used as control groups. T cell proliferation (allogeneic response) was then confirmed in the cultured cells.

[0183] As a result, Figure 26 As shown, compared with the Nil group, T cell proliferation was significantly increased in the allogeneic response group, but when treated with iPSC-derived mitochondria, the increased T cell proliferation was significantly reduced.

[0184] 8-2. Confirm that mitochondria derived from iPSCs increase IFN-γ and IL-10 levels.

[0185] The study aimed to confirm whether the overexpression of mitochondria by UCP2 derived from iPSCs of the present invention specifically increases the levels of IFN-γ and IL-10, which are immunomodulatory factors in graft-versus-host disease (GVHD), a disease of transplant rejection. Specifically, the levels of IFN-γ, IL-10, and IL-17, an autoimmune pathogenic interleukin, were confirmed by ELISA in the cell culture medium cultured in Examples 8-1.

[0186] As a result, Figure 27 As shown, compared with the Allo response group, the group treated with iPSC-derived mitochondria was found to have a specific increase in IFN-γ and IL-10, which are immunomodulatory factors.

[0187] 8-3. Confirm that iPSC-derived mitochondria improve GVHD

[0188] To confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present application reduce disease activity as graft-versus-host disease (GVHD) of a transplant rejection disease. Specifically, to construct a GVHD model, recipient mice Balb / c (H-2k / d) were irradiated with 690 cGy of total body irradiation (TBI), and hematopoietic stem cells and splenocytes were isolated from the femur and tibia of donor mice C57BL / 6 (H-2k / b), and 5 x 105 6 of hematopoietic stem cells (BM, bone marrow) and 5 x 105 6 of splenocytes were transplanted into the recipient mice Balb / c (H-2k / d). Then, after aGVHD occurred, the iPSC-derived mitochondria (iPSC mito group) were intravenously injected once a week at a concentration of 10 μg / ml. Then, the disease activity and body weight of each group of mice were measured once a week. A syngeneic group using wild-type mice and a vehicle control group in which the same amount of solvent was administered after GVHD induction were used as control groups.

[0189] Results, as shown in Part A of Figure 28 , it was confirmed that the disease activity of the vehicle control (Vehicle) group continued to increase until the time point of the end of the experiment compared to the syngeneic (Syngenic) group, but the increased disease activity was significantly reduced in the iPSC mito group. Also, until the time point of the end of the experiment, it was confirmed that no weight loss was observed in the syngeneic (Syngenic) group, but a tendency of weight loss was observed in the vehicle control (Vehicle) group, and a tendency of recovery of reduced weight was observed in the iPSC mito group (Part B of Figure 28 ).

[0190] 8-4. Confirmation of the regulation of GVHD immune cells by iPSC-derived mitochondria

[0191] To confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present application regulate immune cells in graft-versus-host disease (GVHD) as a transplant rejection disease. Specifically, the mice of Example 8-3 were humanely sacrificed at the end of the experiment, and cells were isolated from the blood and spleen tissue. Then, the expression of Th17 cells as autoimmune pathogenic cells in the blood and CD8 TRM+IL-17+ cells as tissue-resident memory (TRM) cells and CD8 TRM+IL-17+ cells in the spleen were analyzed by flow cytometry.

[0192] As a result, as shown in Figure 29 Table 2, it was confirmed that Th17 cells and CD8 TRM+IL-17+ cells in the serum and spleen of the vehicle group were significantly increased compared to the syngenic group, but the increased Th17 cells and CD8 TRM+IL-17+ cells were significantly reduced in the iPSC-derived mitochondria group, thus confirming that the iPSC-derived UCP2 overexpressing mitochondria improved GVHD.

[0193] Example 9: Confirmation of the psoriasis improvement effect of the iPSC-derived mitochondria

[0194] 9-1. Confirmation of the effect of the iPSC-derived mitochondria on regulating IL-17 expressing immune cells

[0195] To confirm whether the iPSC-derived UCP2 overexpressing mitochondria of the present application improve psoriasis, which is a skin immunological disease, whether the subtypes of IL-17 expressing immune cells are regulated was confirmed. Specifically, BALB / C mice were humanely sacrificed, and after obtaining the spleen tissue, single cells were isolated. Then, the cells were inoculated in a 24-well plate at a cell concentration of 1 × 10 6 cells / well, and the expression of inflammatory T cells expressing IL-17 was stimulated with 2 μg / ml of CD3 and 100 ng / ml of LPS, while being treated with 0.4, 2, or 10 μg of the iPSC-derived mitochondria. Then, Th17 and Tc17 cells, which are inflammatory T cells, and tissue-resident memory (TRM) inflammatory T cells were analyzed by flow cytometry. The Nil group, which is a non-treatment control group, and the negative (-) control group, which stimulates the expression of inflammatory T cells, were used as control groups.

[0196] As a result, as shown in Figure 30 Table 3, it was confirmed that Th17 and Tc17 cells, which are inflammatory T cells, were significantly increased in the negative control group compared to the Nil group, but when treated with the iPSC-derived mitochondria, the increased Th17 cells were significantly reduced, and after treatment with the iPSC-derived mitochondria, the Tc17 cells were reduced in a concentration-dependent manner.

[0197] Also, it was confirmed that the CD4-positive, CD8-positive, CD4-positive IL-17-positive, and CD8-positive IL-17-positive TRM subtypes in the TRM of the negative control group were significantly increased compared to the Nil group, but when treated with the iPSC-derived mitochondria, the increased CD4-positive, CD8-positive, CD4-positive IL-17-positive, and CD8-positive IL-17-positive TRM subtypes were significantly reduced (Table 4). Figure 31 ​

[0198] 9-2. Confirm the effect of iPSC-derived mitochondria on improving psoriasis disease activity.

[0199] This study aimed to confirm whether the iPSC-derived UCP2 overexpression of mitochondria had an effect on improving psoriasis in an animal model of psoriasis. Specifically, hair was removed from the back and buttocks of SKG mice using depilatory cream. One day later, 62.5 mg of 5% imiquimod (IMQ) cream was applied topically to the back skin once daily to induce acute psoriasis. Three days after dry induction, 10 μg of iPSC-derived mitochondria was injected intraperitoneally into the mice. On the 7th day after dry induction, the mice were sacrificed humanely. Figure 32 (Part A of the study). After inducing psoriasis, clinical indicators of skin thickness, scaling, and redness on the back were measured and indexed daily.

[0200] As a result, Figure 32 As shown in Part B, compared with psoriasis-induced mice (SKG+IMQ), the group injected with mitochondria (iPSC mito) showed reduced skin lesions due to psoriasis induction. Furthermore, significant reductions in skin thickness, scaling, and redness index were observed. Figure 32 Part C).

[0201] 9-3. Confirm the effect of iPSC-derived mitochondria on improving psoriasis-associated immune cells.

[0202] The study aimed to confirm whether the iPSC-derived UCP2 overexpression of mitochondria of the present invention regulates the subtypes of inflammatory immune cells and tissue-resident memory cells in a psoriasis animal model. Specifically, at the end of the experiment, mice in each group of Examples 9-2 were humanely sacrificed and spleen tissue was obtained. Then, single cells were isolated from the spleen, and the isolated cells were divided into 1×10⁻⁶ cells. 6 Cells were seeded at a concentration of [specific concentration not specified] in 24-well plates and stimulated with PMA-lonomycin for 2 hours to induce T cell activation and cytokine secretion. Then, a 2-hour Golgi apparatus stop was performed to prevent T cell activation and cytokine secretion. Cells were then obtained, and the expression of CD4 lineage Th1, Th2, and Th17 cells and CD8 lineage Tc1, Tc2, and Tc17 cells in inflammatory immune cells, as well as the expression of Treg cells (immunomodulatory cells), was analyzed by flow cytometry. The TRM (tumor retrieval mechanism) of CD4 and CD8 lineage T cells was also analyzed.

[0203] As a result,Figure 33 Part A of FIG. 26, the expression of Th1 and Th17 cell subtypes of CD4 lineage was significantly reduced in the mitochondria-injected (iPSC mito) group compared to the psoriasis-induced mouse (SKG+IMQ) group, and Th2 showed a tendency of reduction. Also, it was confirmed that Tc1, Tc2, and Tc17 of CD8 lineage were significantly reduced (Part B of FIG. 26), and the expression of Treg was significantly increased (Part C of FIG. 26). Figure 33 Figure 33

[0204] Also, as a result of analyzing the subtypes of TRM, it was confirmed that the expression of CD4 TRM1 and CD4 TRM17 of CD4 lineage and CD8 TRM1 and CD8 TRM17 of CD8 lineage were significantly reduced in the mitochondria-injected group (iPSC mito) compared to the psoriasis-induced mouse (SKG+IMQ) group (Part 9-4-2 of FIG. 27). Figure 34

[0205] 9-4. Confirmation of histological protective effect of iPSC-derived mitochondria

[0206] It was confirmed whether the iPSC-derived UCP2-overexpressing mitochondria of the present application protected the skin tissue changes induced by psoriasis. Specifically, the skin tissue was obtained from the mice of each group sacrificed in Example 9-3 and sectioned. Then, hematoxylin & eosin (H&E) staining was performed, and the epidermis thickness was quantified.

[0207] As a result, as shown in FIG. 28, it was confirmed that the epidermis layer thickness was reduced in the mitochondria-injected group (iPSC mito) compared to the psoriasis-induced mouse (SKG+IMQ) group, and thus it was confirmed that the iPSC-derived UCP2-overexpressing mitochondria improved psoriasis. Figure 35

[0208] Example 10: Confirmation of anticancer effect of iPSC-derived mitochondria

[0209] 10-1. Confirmation that iPSC-derived mitochondria modulate immune cells of a gastric cancer patient

[0210] It was confirmed whether the iPSC-derived UCP2-overexpressing mitochondria of the present application had an anticancer effect on solid cancer. Specifically, the PBMCs derived from a gastric cancer patient were injected with 1×10 6 ​​​​cells were analyzed by flow cytometry. A solvent control (Vehicle) group, which was a negative (-) control group, was used as a control group.

[0211] As a result, as shown in Figure 36 FIG. 6, it was confirmed that the expression of CD4-positive IFN-r-positive Th1 cells, CD8-positive IFN-r-positive Tc1 cells, and CD8-positive GZMB-positive cells, which are immunomodulatory cells, increased after treatment with iPSC-derived mitochondria, and the expression of CD8-positive IL-17-positive Tc17 cells, CD4-positive CTLA-4-positive cells, and CD8-positive CTLA-4-positive cells significantly decreased compared to the negative control group.

[0212] Also, as shown in Figure 37 FIG. 7, it was confirmed that the expression of CD8-positive IFN-r-positive TRM significantly increased after treatment with mitochondria compared to the control group.

[0213] 10-2. Confirmation of anticancer effect of iPSC-derived mitochondria on gastric cancer

[0214] To confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present application have an anticancer effect on solid tumors, the tumor size inhibition effect in a gastric cancer xenograft animal model was confirmed. Specifically, PBMCs derived from a gastric cancer patient were intraperitoneally injected at a cell concentration of 1 x 10 6 into 6-week-old male NSG mice, which are immunodeficient mice. One week later, AGS cell lines, which are gastric cancer cell lines, were subcutaneously injected at a cell concentration of 5 x 10 6 . From the 4th week of the experiment, 10 pg of iPSC-derived mitochondria was intraperitoneally injected once a week, and at the 7th week of the experiment, the mice were humanely sacrificed. The specific experimental schedule is shown in Figure 38 FIG. 8. The HC group in which healthy adult-derived PBMCs were injected, the group in which gastric cancer patient-derived PBMCs and AGS cell lines were injected (GC Veh), and the AGS group in which only AGS cell lines were injected were used as control groups. Tumor tissues were obtained from the sacrificed mice, and the tumor volume was measured, and the spleen cells were isolated to analyze the immune cell subtypes.

[0215] As a result, as shown in Figure 39 FIG. 9, it was confirmed that the tumor volume continuously increased in the GC Veh group, reflecting the immune status of the gastric cancer patient, but the tumor volume of the group in which iPSC-derived mitochondria were injected significantly decreased, and the tumor volume at the time point at which the experiment ended also significantly decreased (p < 0.05). Figure 40 ).

[0216] And, confirming the results of immune cell subtypes in the spleen cells isolated from each group of mice, it was confirmed that the total CD8-positive cells of the group injected with iPSC-derived mitochondria increased compared to the GCVeh group, the expression of Tregs significantly increased, and the expression of Th17 significantly decreased Figure 41 ).

[0217] 10-3. Confirmation of anticancer effect of iPSC-derived mitochondria on liver cancer

[0218] To confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present application have an anticancer effect on solid tumors, the tumor proliferation inhibition effect in a liver cancer animal model was confirmed. Specifically, an animal model was prepared in the same manner as in Example 9-2, and the transplanted cancer cell line was replaced with the HepG2 cell line, which is a hepatocellular carcinoma cell line, instead of the AGS cell line. The specific experimental procedure is shown in Figure 42 The volume of the transplanted tumor was confirmed up to the time point at which the experiment ended, and a solvent control (Vehicle) group treated with the PBMC of a liver cancer patient and the HepG2 cell line and administered with the same amount of solvent was used as a control group.

[0219] As a result, as shown in Figure 43 , it was confirmed that the tumor volume of the group treated with iPSC-derived mitochondria continuously decreased compared to the solvent control (Vehicle) group, and the tumor volume was significantly reduced up to the time point at which the experiment ended.

[0220] Example 11: Confirmation of the effect of iPSC-derived mitochondria in improving osteoarthritis

[0221] 11-1. Confirmation of the effect of iPSC-derived mitochondria in regulating cartilage metabolic factors

[0222] To confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present application improve osteoarthritis, the quality of the purified mitochondria and the cartilage cell transplantation effect were analyzed, and the effect of regulating cartilage metabolic factors was confirmed. Specifically, after isolating the iPSC-derived mitochondria, the protein expression of tubulin as a cytosol factor and COX4 as a mitochondrial factor was analyzed by Western blotting to measure the quality of the mitochondria. Then, the mitochondria isolated from the cartilage cells were stained with MTDR, and the delivery effect into the cartilage cells according to the amount of mitochondria (0.5 or 1 μg) was analyzed by flow cytometry. A Nil group as a non-treatment control group and a solvent control (Vehicle) group treated only with the same amount of solvent were used as control groups.

[0223] Then, the chondrocytes obtained from the mice were stimulated with IL-1β, and then treated with 1 ng of iPSC-derived mitochondria and co-cultured. The Nil group was used as a non-treatment control group, and the Vehicle group treated with the same amount of solvent instead of the mitochondria was used as a control group. After 1 day of culture, the cells were obtained, and the expression of MMP3, MMP9, and MMP13 as chondroctic metabolic factors was analyzed by quantitative PCR (q-PCR), and the expression of MCP1 as a chondroctic metabolic factor in the culture solution was analyzed by ELISA.

[0224] Also, after 2 days of culture, the cells were obtained, and the protein expression and phosphorylation of RIP3 and MLKL as inflammatory cell apoptosis factors were analyzed by Western blotting.

[0225] The results of confirming the quality of the mitochondria are shown in FIG. 1, and as shown in FIG. 1, the iPSC-derived mitochondria were well isolated, and thus the protein expression of tubulin and COX4 was confirmed, and 1 μg of iPSC was delivered per 1 x 10 5 Figure 44

[0226] The results are shown in FIG. 2, and as shown in FIG. 2, the expression of MMP3, MMP9, and MMP13 as chondroctic metabolic factors was significantly increased in the Vehicle group compared to the Nil group, but the increased expression of the chondroctic metabolic factors was significantly reduced in the group treated with the iPSC-derived mitochondria. Also, the expression of MCP1 as a chondroctic metabolic factor in the culture solution was significantly reduced. Figure 45

[0227] Also, on the second day of culture, the results of confirming the protein expression and phosphorylation of the inflammatory cell apoptosis factors are shown in FIG. 3, and as shown in FIG. 3, the expression of RIP3 as an inflammatory cell apoptosis factor was significantly increased in the Vehicle group compared to the Nil group, and the expression and phosphorylation of MLKL were increased, but when treated with the iPSC-derived mitochondria, the increased expression of RIP3, the expression of MLKL, and the phosphorylation were all significantly reduced. Figure 46

[0228] 11-2. Confirmation that iPSC-derived mitochondria improve osteoarthritis

[0229] ​​​​To confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present application improve osteoarthritis, the improvement effect in an MIA-induced osteoarthritis animal model was confirmed. Specifically, 6-week-old male Wistar rats were raised in an environment having a temperature of 21 to 22°C and a 12-hour light-dark cycle, and were fed with sterilized water and feed. Then, monosodium iodoacetate (MIA, Sigma, ST. Louis, MO) was administered to the right knee of the rats at a dose of 3 mg / 50 μl to induce osteoarthritis. The MIA was dissolved in normal saline and administered. Then, it was divided into two groups: an MIA group to which an excipient of the drug was administered to the rats without any drug; and an mIPSC group to which the iPSC-derived mitochondria of the present application were administered into the joint cavity. The specific drug administration and experimental schedule are shown in Part A of FIG. 1. Figure 47 Then, pain caused by osteoarthritis was measured using a dynamic plantar aesthesiometer (Ugo Basile, Comerio, Italy), which is a pain measurement instrument, as follows: a mesh plate was placed on the measurement instrument, the rats were fixed in an acrylic animal holder and placed on the mesh plate, and then the right paw to which the drug was injected was pierced with the measurement instrument. After the piercing, the instrument automatically measured the time (s, seconds) and pressure (g) required for the paw to be retracted to confirm the recorded time and pressure, and the degree of pain was measured according to the time and pressure through a pain evaluation (Paw Withdrawal Latency).

[0230] As shown in Part B of FIG. 1, it was confirmed that the pain of the group to which the iPSC-derived mitochondria were administered was reduced compared to the MIA group, and thus the pain evaluation (Paw Withdrawal Latency) index was increased. Figure 47

[0231] 11-3. Confirmation of the effect of iPSC-derived mitochondria to protect cartilage

[0232] To confirm whether the iPSC-derived UCP2-overexpressing mitochondria of the present application improve osteoarthritis, cartilage tissue damage was confirmed. Specifically, the animal models of each group in Example 11-2 were humanely sacrificed at the end of the experiment, and the cartilage tissue was obtained and sectioned. Then, the sectioned cartilage tissue was stained with Safranin O to confirm the Mankin score of the cartilage tissue damage, and the expression of MMP3 in the cartilage was confirmed by immunohistochemical staining.

[0233] ​Results, as shown in Figure 48 Mankin index was significantly reduced compared to the MIA group, and the expression of MMP13 in cartilage tissue was significantly reduced.

[0234] 11-4. Confirmation of iPSC-derived mitochondria regulating immune cells in osteoarthritis

[0235] It was confirmed whether the iPSC-derived UCP2-overexpressing mitochondria of the present application regulate immune cells in osteoarthritis. Specifically, spleen tissues were obtained from the mice sacrificed in the Example 11-4, and T cell subtypes in the cells isolated from the spleen tissues were analyzed by flow cytometry.

[0236] Results, as shown in Figure 49 It was confirmed that the expression of Th1, Th2, and Th17 cells, which are autoimmune pathogenic immune cells, was significantly increased in the MIA group, but the increased expression of Th1, Th2, and Th17 was significantly reduced in the group administered with iPSC-derived mitochondria, and thus the expression of immune regulatory cells Treg was significantly increased.

[0237] Therefore, it was confirmed that the iPSC-derived mitochondria of the present application overexpress UCP2, and the expression of UCP2 is associated with immune regulation. Also, it was confirmed that the administration of iPSC-derived mitochondria enhances mitochondrial function, thereby increasing ATP synthesis and mitochondrial membrane potential. Also, it was confirmed that the expression of autoimmune disease-related immune cells was regulated, and when used in combination with metformin, a synergistic effect on immune cell regulation was confirmed. Also, it was confirmed that the disease activity of autoimmune diseases was reduced, and the expression of autoimmune-related cells was inhibited. Also, it was confirmed that when peripheral blood mononuclear cells were treated with iPSC-derived mitochondria, the expression of Th17 was inhibited and the expression of Treg was increased, improving immune regulation and disease activity in various immune diseases.

Claims

1. A mitochondrion, characterized in that, Induced pluripotent stem cell-derived and overexpressing uncoupling protein 2.

2. The mitochondrion of claim 1, wherein, The uncoupling protein 2 increases the function of induced pluripotent stem cell-derived mitochondria.

3. The mitochondrion of claim 2, wherein, The increase in the function of the mitochondria is modulating immune cells.

4. The mitochondrion of claim 3, wherein, The immune cells are cells selected from the group consisting of Th1, Th2, Th17, Treg, plasma cells, Breg, Tfh, tissue-resident memory T cells, Tc1, Tc2, and Tc17.

5. The mitochondrion of claim 3, wherein, The modulating immune cells is decreasing the expression of cells selected from the group consisting of Th1, Th2, Th17, plasma cells, Tfh, tissue-resident memory T cells, Tc1, Tc2, and Tc17.

6. The mitochondrion of claim 3, wherein, The modulating immune cells is increasing the expression of Treg or Breg.

7. The mitochondrion of claim 1, wherein, The mitochondria decrease the expression of IFN-gamma or IL-17.

8. The mitochondrion of claim 1, wherein, The mitochondria increase the expression of IL-10.

9. A pharmaceutical composition for preventing or treating an autoimmune disease, characterized by, A composition comprising induced pluripotent stem cell-derived uncoupling protein 2 overexpressing mitochondria as an active ingredient.

10. The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 9, characterized by, The mitochondria increase the function of intracellular mitochondria. 11.The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 10, characterized in that, The increase in the function of the mitochondria is an increase in ATP synthesis or an increase in mitochondrial membrane potential. 12.The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 9, characterized in that, The mitochondria modulate immune cells. 13.The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 12, characterized in that, The immune cells are cells selected from the group consisting of Th1, Th2, Th17, Treg, plasma cells, Breg, Tfh, tissue-resident memory T cells, Tc1, Tc2, and Tc17. 14.The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 12, characterized in that, The modulating immune cells is decreasing the expression of cells selected from the group consisting of Th1, Th2, Th17, plasma cells, Tfh, tissue-resident memory T cells, Tc1, Tc2, and Tc17. 15.The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 12, characterized in that, The modulating immune cells is increasing the expression of Treg or Breg. 16.The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 9, characterized by, The mitochondria increase colon length. 17.The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 9, characterized in that, The composition inhibits intestinal tissue damage. 18.The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 9, characterized by, The mitochondria decrease the amount of autoantibodies.

19. The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 18, wherein The autoantibodies are total IgG, IgG2a, or IgM. 20.The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 9, characterized in that, The mitochondria inhibit kidney damage.

21. The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 20, wherein The inhibition of kidney damage is a decrease in factors selected from the group consisting of vasculitis index, glomerulitis index, urine creatinine, and urine albumin.

22. The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 9, characterized by, The mitochondria decrease allogenic reactions.

23. The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 22, wherein The allogenic reactions increase the proliferation of T cells.

24. The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 9, wherein The mitochondria decrease psoriasis disease activity.

25. The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 24, wherein The psoriasis disease activity is an index selected from the group consisting of skin thickness, keratin, or erythema.

26. The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 25, wherein The skin thickness is epidermal thickness. 27.The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 9, characterized in that, The mitochondria inhibit the expression of cartilage metabolic factors.

28. The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 27, wherein The cartilage metabolic factors are factors selected from the group consisting of MMP3, MMP9, MMP13, and MCP1. 29.The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 9, characterized in that, The autoimmune disease is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, Behcet's disease, autoimmune cytopenias, autoimmune myocarditis, atopic dermatitis, asthma, primary biliary cirrhosis, dermatomyositis, Goodpasture's syndrome, autoimmune meningoencephalitis, Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, sarcoidosis, scleroderma, spondyloarthropathies, pernicious anemia, scleroderma, graft-versus-host disease, cancer, and mitochondrial-related syndromes.

30. The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 29, wherein The autoimmune disease is accompanied by a metabolic abnormality.

31. A food composition for preventing or ameliorating an autoimmune disease, characterized by, The mitochondria comprising overexpression of uncoupling protein 2 derived from induced pluripotent stem cells are used as an effective ingredient.

32. A pharmaceutical composition for immunomodulation, characterized by, The mitochondria comprising overexpression of uncoupling protein 2 derived from induced pluripotent stem cells are used as an effective ingredient.

33. The pharmaceutical composition for immunomodulation according to claim 32, characterized by, The mitochondria modulate immune cells.

34. The pharmaceutical composition for immunomodulation according to claim 32, characterized by, The immune cells are selected from the group consisting of Th1, Th2, Th17, Treg, plasma cells, Breg, Tfh, tissue-resident memory T cells, Tc1, Tc2, and Tc17.

35. The pharmaceutical composition for immunomodulation according to claim 32, characterized by, The modulating immune cells is reducing expression of cells selected from the group consisting of Th1, Th2, Th17, plasma cells, Tfh, tissue-resident memory T cells, Tc1, Tc2, and Tc17.

36. The pharmaceutical composition for immunomodulation according to claim 32, characterized by, The modulating immune cells is increasing expression of Treg or Breg.

37. A food composition for immunomodulation, characterized by, The mitochondria comprising overexpression of uncoupling protein 2 derived from induced pluripotent stem cells are used as an effective ingredient.

38. A method of treating an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-37. The method comprises the step of administering a pharmaceutically effective amount of the mitochondria according to claim 1 to an individual.

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