WO2022108165A1

WO2022108165A1 - Method for producing exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells, and use thereof

Info

Publication number: WO2022108165A1
Application number: PCT/KR2021/015286
Authority: WO
Inventors: 이미연; 나득채; 김경운
Original assignee: 주식회사 온코인사이트
Priority date: 2020-11-18
Filing date: 2021-10-28
Publication date: 2022-05-27
Also published as: KR20220068148A

Abstract

The present invention relates to a method for producing exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells, and a use thereof, and exosomes according to one embodiment noticeably increase the generation of IL-10, which is an anti-inflammatory cytokine, and noticeably suppress the generation of inflammatory cytokines IL-2, IL-17 and IFN-γ in Th0 and Th17 cells, and thus do not have an effect on the activity of regulatory T cells (Treg), and therefore exhibit excellent benefits for the treatment of autoimmune diseases.

Description

Method for preparing exosomes isolated from dedifferentiated stem cell-derived mesenchymal stem cells and use thereof

It relates to a method for producing an exosome isolated from dedifferentiated stem cell-derived mesenchymal stem cells and a use thereof.

Mesenchymal stromal cells can be obtained from adult tissues such as bone marrow, adipose tissue, umbilical cord blood, and umbilical cord, and have the form of fibroblasts. The stem cells can proliferate indefinitely in vitro, and unlike blood stem cells, they can be differentiated into various types of important cell lines such as fat, osteocytes, chondrocytes, cardiomyocytes, and nerve cells. Research is being done.

On the other hand, autoimmune disease is a disease in which immune cells that defend the body from external invaders such as bacteria, viruses, and foreign substances attack themselves. Since autoimmune diseases can appear in all organs and tissues of the human body, all cells in the body are attacked or only cells in specific organs are destroyed. In addition, autoimmune diseases selectively attack specific organs or the whole body, such as rheumatoid arthritis.

As the types and symptoms of autoimmune diseases are diverse, the treatment methods for autoimmune diseases are also diverse. The goal of treatment for autoimmune diseases is to relieve symptoms, preserve function, or block the pathogenesis of the disease. Drugs for this purpose include steroids, nonsteroidal anti-inflammatory drugs, immunosuppressants, and the like, but long-term administration of these drugs often causes serious side effects.

Under this technical background, research has been continuously conducted to solve the problems related to the prevention and treatment of autoimmune diseases (Korea Patent Publication No. 10-2111836 (May 15, 2020)), but it is still insufficient to solve this problem. .

One aspect comprises the steps of isolating cells from a biological sample; preparing dedifferentiated stem cells from the isolated cells; Inducing mesenchymal stem cells from the prepared dedifferentiated stem cells; And to provide a method for producing an exosome comprising the step of isolating the exosome from the induced mesenchymal stem cells.

Another aspect is to provide a pharmaceutical composition for preventing or treating an autoimmune disease comprising the exosome prepared by the method for producing the exosome.

Another aspect is to provide a method for preventing or treating an autoimmune disease comprising administering the exosome prepared by the method for producing the exosome.

One aspect comprises the steps of isolating cells from a biological sample; preparing dedifferentiated stem cells from the isolated cells; Inducing mesenchymal stem cells from the prepared dedifferentiated stem cells; And it provides a method for producing an exo-some comprising the step of isolating the exosome from the induced mesenchymal stem cells.

The "biological sample" refers to a sample derived from an organism. The organism may be a mammal including a human. The biological sample may be derived from a tissue or body fluid of the body. The body fluids include blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, intestinal fluid, genitourinary fluid, breast milk, lymphatic fluid, semen, cerebrospinal fluid, intratracheal fluid , ascites, cystic tumor body fluid, amniotic fluid, or a combination thereof. The blood may be derived from peripheral blood. The blood may include blood cells. The blood cells may include one or more of red blood cells, white blood cells, and platelets. The white blood cells may include eosinophils, basophils, neutrophils, lymphocytes, monocytes, macrophages, and the like. The cells isolated from the biological cells may be mononuclear cells.

The “induced pluripotent stem cell (iPSC)” is also referred to as an induced pluripotent stem cell, and refers to a cell having pluripotency obtained by dedifferentiation from a differentiated cell (eg, a somatic cell). do. The dedifferentiated stem cells can differentiate into various organ cells. The dedifferentiated stem cells can be obtained by reprogramming the cells differentiated by the dedifferentiation 　inducing factors.

The "mesenchymal stem cell (MSC)" refers to a stem cell present in cartilage, bone tissue, adipose tissue, bone marrow stroma, etc. differentiated from mesoderm generated by division of a fertilized egg. The mesenchymal stem cells may be differentiated into osteoblasts, adipocytes, chondrocytes, etc. in vitro, and may express CD73, CD90 and CD105. In addition, the mesenchymal stem cells may not express c-kit, CD11b, CD19, CD14, CD34, CD45, CD14, CD79 and HLA-DR.

The "exosomes" refers to endoplasmic reticulum having a size of several tens to hundreds of nanometers (about 30 to 200 nm) composed of a double phospholipid membrane identical to the structure of a cell membrane. The exosome may include a protein, a nucleic acid (mRNA, miRNA, etc.) called a 　exosome 　cargo. The exosome 　 cargo may include a wide range of signaling factors, and the signaling factors may be specific to a cell type and may be differently regulated according to the environment of the secretory cell. The exosomes are intercellular signaling mediators secreted by cells, and various cellular signals delivered through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. can The exosome may include both a nano-sized vesicle secreted from mesenchymal stem cells and released into the extracellular space or a vesicle having a composition similar to that of the exosome (eg, exosome-like vesicle). have.

In one embodiment, the "inducing step" may include the following steps:

(a) reacting the prepared retrodifferentiated stem cells with a collagenase solution;

(b) centrifuging the reacted dedifferentiated stem cells;

(c) suspending the centrifuged retrodifferentiated stem cells in a medium; and

(d) stimulating the suspended retrodifferentiated stem cells with b-FGF.

The "inducing step" may further include washing the dedifferentiated stem cells with PBS before step (a).

The collagenase (collagenase) refers to an enzyme capable of hydrolyzing collagen or gelatin, which is a kind of hard protein. The collagenase may be any one of

types

1, 2, 3 and 4. The collagenase solution may contain collagenase at a concentration of 0.5 to 2 mg/mL. For example, the collagenase solution may contain collagenase type 4 (type 　IV 　Collagenase) at a concentration of 1 mg/mL. Step (a) may be to react for 5 to 15 minutes. Preferably, step (a) may be to react the prepared retrodifferentiated stem cells with a collagenase type 4 solution for 10 minutes.

The step (b) may be centrifugation at 1000 to 1200 rpm, preferably centrifugation at 1100 rpm. Step (b) may be centrifuged for 2 to 4 minutes. For example, step (b) may be centrifuging the dedifferentiated stem cells for 3 minutes.

The b-FGF (basic fibroblast growth factor), also known as FGF2 or FGF-β, refers to a growth factor encoded by the FGF2 gene. In step (d), the b-FGF may be stimulated at 0.5 to 1.5 ng/mL. For example, step (d) may be to stimulate the suspended dedifferentiated stem cells with 1 ng/mL of b-FGF.

The "inducing step" may further include stimulation with recombinant human TGF-β after stimulation with b-FGF. The recombinant human TGF-β is also referred to as rh TGF-β, and may be rh TGF-β1, rh TGF-β2 or rh TGF-β3.

The "step of isolating exosomes" may include the following steps:

(a) culturing the induced mesenchymal stem cells in a medium for 36 to 60 hours;

(b) centrifuging the cultured mesenchymal stem cell culture medium at 200 to 400 g at 0 to 5° C. for 5 to 15 minutes;

(c) centrifuging the supernatant of the culture solution centrifuged in step (b) at 1500 to 2500 g at 0 to 5° C. for 15 to 30 minutes;

(d) centrifuging the supernatant of the culture medium centrifuged in step (c) at 8,000 to 12,000 g at 0 to 5° C. for 20 to 40 minutes;

(e) filtering the supernatant of the culture medium centrifuged in step (d) with a filter paper; and

(f) centrifuging the culture solution filtered in step (e) at 80,000 to 120,000 g for 100 to 140 minutes.

Step (a) may be culturing for 48 hours.

The "medium" may include essential components necessary for cell growth, survival, and proliferation in vitro . For example, the medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), Knockout DMEM, E8 (Essential 8 Medium), SF-DMEM (serum free-Dulbecco Modified Eagle Medium), such as commercially prepared medium or artificially synthesized medium may be used, but is not limited thereto.

The medium may be α-Minimum Eagle's Medium. The α-Minimum Eagle’s Medium is also referred to as α-MEM, and refers to a medium in which amino acids and vitamins are additionally added to the components of MEM. Specifically, the α-MEM may include non-essential amino acids, glutamine, and 2-mercaptoethanol. In addition, the α-MEM may include antibiotics such as penicillin and streptomycin. The non-essential amino acids may refer to one or more selected from the group consisting of isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine and arginine.

The "centrifugation" can be carried out for any length of time at any speed and temperature suitable for the purpose as is known in the art. For example, the centrifugation is 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g, 2000 g, 3000 g, 4000 g, 5000 g, 6000 g, 7000 g, 8000 g, 9000 g, 10000 g, 20000 g, 30000 g, 40000 g, 50000 g, 60000 g, 70000 g, 80000 g, 90000 g, 100000 g, 200 to 400 g, 250 to 350 g, 1500-2500 g, 1800-2200 g, 1900 g-2100 g, 8,000-12,000 g, 9,000-11,000 g, or 80,000-120,000 g, 90,000-110,000 g. For example, the centrifugation may be performed at 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, 1-5 minutes, 5-10 minutes, 1-10 minutes, 5-15 minutes, 10-20 minutes, 15-20 minutes, 15-30 minutes, 20-30 minutes minutes, 20 to 40 minutes, 25 to 35 minutes, 20 to 60 minutes, 20 to 80 minutes, 20 to 100 minutes, 80 to 160 minutes, 100 to 140 minutes, 110 to 130 minutes. The centrifugation is 0 ℃, 1 ℃, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 40 It may be carried out at ℃, 50 ℃, 0 to 5 ℃, 0 to 10 ℃, 0 to 20 ℃ or 0 to 30 ℃, but is not limited thereto.

The filter paper may have a pore size of 0.22 μm, but is not limited thereto.

Another aspect provides a pharmaceutical composition for preventing or treating autoimmune diseases comprising the exosomes prepared by the above manufacturing method.

The term exosome and the like are the same as described above.

The "prevention" means any action that suppresses or delays the onset of the obesity or autoimmune disease, and the "treatment" means any action that improves or beneficially changes the symptoms of the autoimmune disease.

The "autoimmune disease" refers to a disease in which immune function is abnormal and immune cells attack organs or tissues of the body. The autoimmune disease can be divided into organ-specific autoantibody-related diseases and organ non-specific (systemic) diseases. The autoimmune disease is Hemophagocytic lymphohistiocytosis, systemic lupus erythematosus, Kikuchi's disease, vasculitis, Adult onset Still's disease, rheumatoid arthritis (rheumatoid arthritis) arthritis), Inflammatory Myositis, Behcet disease, IgG4-associated disease, Sjogren syndrome, Giant cell arteritis, Temporal arteritis, Type 1 diabetes, It may be selected from the group consisting of atopic dermatitis, Crohn's disease, systemic sclerosis, psoriasis, multiple sclerosis, and Graves hyperthyroidism, but is not limited thereto.

The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier is used in the sense of including excipients, diluents or adjuvants. The carrier may be, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pi It may be selected from the group consisting of rolidone, water, physiological saline, buffers such as PBS, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil. The composition may include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, or a combination thereof.

The pharmaceutical composition may be provided to the subject in any formulation according to a conventional method, respectively. The formulation may be an oral or parenteral formulation. In the pharmaceutical composition, the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule. The solid formulation may further include an excipient. The excipient may be starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, the solid formulation may further include a lubricant such as magnesium stearate or talc. In the pharmaceutical composition, the oral liquid formulation may be a suspension, an oral solution, an emulsion, or a syrup. The liquid formulation may contain water or liquid paraffin. The liquid formulation may include a wetting agent, a sweetening agent, a flavoring agent, or a preservative. Formulations for parenteral administration may be sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilizations, or suppositories. The non-aqueous solvent or suspending agent may include vegetable oil or ester. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester is, for example, ethyl oleate. The base of the suppository may be witepsol, macrogol, tween 61, cacao butter, laurin, or glycerogelatin.

The pharmaceutical composition may include the exosome in a pharmaceutically effective amount. The term "effective amount" refers to an amount sufficient to exhibit the effect of prophylaxis or treatment when administered to a subject in need thereof. The effective amount can be appropriately selected by those skilled in the art depending on the individual. disease severity, patient's age, weight, health, sex, patient's sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, factors including drugs used in combination with or concurrently with the composition used, and other medical fields can be determined according to well-known factors in The effective amount of the pharmaceutical composition is that the pharmaceutical composition contains the exosomes at least about 1 μg, at least about 2 μg, at least about 5 μg, at least about 10 μg, at least about 20 μg, at least about 50 μg, at least about 100 μg. , about 200 μg or more, about 400 μg or more, about 1 mg or more, about 2 mg or more, about 4 mg or more, about 10 mg or more, about 20 mg or more, about 40 mg or more, about 100 mg or more, about 1 μg to 1 mg, about 10 μg to 1 mg, about 1 to 100 μg, about 10 to 100 μg, about 10 to 200 μg, or about 10 to 500 μg may be included, but is not limited thereto. The effective amount may mean the amount of the exosome contained per 0.01 mL, 0.1 mL, 1 mL, 10 mL, or 100 mL of the pharmaceutical composition.

The pharmaceutical composition may be administered in a conventional manner via oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, or intradermal routes. The pharmaceutical composition may be administered in the form of an injection, for example, subcutaneously, intravenously or intramuscularly. In addition, the pharmaceutical composition may be administered systemically or locally, alone or in combination with other pharmaceutically active compounds. The dosage of the pharmaceutical composition is, for example, about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 10 mg/kg, based on an adult. , about 0.1 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 5 mg/kg, or about 1 mg/kg to about 3 mg/kg may be within the range. The administration is once a day, twice a day, three times a day, four times a day, once a week, once in two weeks, once in three weeks, once in four weeks, once in a month. It may be administered once, once every three months or once a year.

Another aspect provides a method for preventing or treating an autoimmune disease comprising administering the exosome prepared by the method for producing the exosome.

A pharmaceutical composition comprising an exosome prepared according to a manufacturing method according to an aspect significantly inhibits the production of inflammatory cytokines IL-2, IL-17, IFN-γ in T _h 0 and T _h 17 cells, and , It significantly increases the production of IL-10, an anti-inflammatory cytokine, and does not affect the activity of immunoregulatory T cells (T _reg ), so it shows a very good effect in the treatment of autoimmune diseases.

1A to 1C are diagrams analyzing cell shape and markers of dedifferentiated stem cell-derived mesenchymal stem cells according to an embodiment of the present invention. 1a shows the expression of NANOG, SSEA-4, TRA-181, which are markers of iPSCs confirmed by immunofluorescence, and FIG. 1b shows iPSC-derived mesenchymal stems. It shows the results of observing the cell morphology of the cells (iPSC-iMSC) for each stage of differentiation, and FIG. 1c shows CD34, CD31, which are reported as MSC markers in iPSC-derived mesenchymal stem cells (hiPSC-MSCs). , shows the results of confirming the expression of CD19, CD11, HALDR (negative marker), CD44, CD73, CD105, and CD90 (positive marker).

2A to 2C are diagrams analyzing physical properties and markers of exosomes isolated from dedifferentiated stem cell-derived mesenchymal stem cells according to an embodiment of the present invention. Figure 2a shows the size distribution of the exosomes measured using the NANOSIGHT instrument after the separation of the dedifferentiated stem cell-derived mesenchymal stem cell exo (iPSC-iMSC-Exo), Figure 2b is a transmission electron microscopy (Transmission electron microscopy) , TEM) shows the morphology of dedifferentiated stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo), and FIG. -Exo) shows the expression of TGS101, CD9, CD63 (positive marker), which are reported as markers of exosomes confirmed by Western blotting.

Figures 3a to 3c show the results of confirming the immunomodulatory ability of the dedifferentiated stem cell-derived mesenchymal stem cell exosomes to T _h 0 cells according to an embodiment of the present invention. Figure 3a shows IL-17, IFN-g and immunity generated in Th0 cells after co-culture of mononuclear cells cultured in Th0 cell differentiation conditions with dedifferentiated stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo). It is a result of FACS analysis of the expression of CD25+Foxp3+, a marker generated in regulatory T cells (Treg), Figure 3b is a graph showing the FACS result, Figure 3c is IL-17, IFN-g in the culture medium confirmed by ELISA and IL-10.

Figures 4a to 4c show the results of confirming the immune modulating ability of the dedifferentiated stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo) to T _h 17 cells according to an embodiment of the present invention. Figure 4a shows IL-17, IFN-g, and immunity generated in Th17 cells after co-culture of mononuclear cells cultured in Th17 cell differentiation conditions with dedifferentiated stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo). It is a result of FACS analysis of expression of CD25+Foxp3+, a marker generated in regulatory T cells (Treg), FIG. 4b is a graph showing the FACS result, and FIG. 4c is IL-17, IL-10 in the culture medium confirmed by ELISA. , and IL-2.

5a to 5d show the results of comparing the disease control efficacy of the dedifferentiated stem cell-derived mesenchymal stem cell exosomes and the mesenchymal stem cell exosomes in an arthritis animal model according to an embodiment of the present invention. Figure 5a shows the arthritis evaluation index of the group administered with the dedifferentiated stem cell-derived mesenchymal stem cell exo (iPSC-iMSC-Exo) and the mesenchymal stem cell exo (MSC-Exo), respectively, in the induction of the rheumatoid arthritis animal model. Figure 5b shows changes in CD4+T intracellular cytokines (CD4+IL-17A+, CD4+IL-10, etc.) in splenocytes of an animal model of rheumatoid arthritis confirmed by FACS, and Figure 5c shows rheumatoid arthritis Shows the histological evaluation results by H & E, toluidine blue, and safranin O staining of the arthritis animal model, Figure 5d is the inflammation index (inflammation score) and cartilage erosion score (Cartilage damage score) calculated in each group through (* p<0.05, ** p<0.01, *** p<0.001 ).

6a to 6d show the results of confirming the disease control efficacy in the arthritis animal model according to the concentration of the dedifferentiated stem cell-derived mesenchymal stem cell exosomes according to an embodiment of the present invention. Figure 6a shows the arthritis evaluation index of the group administered with each concentration of immunized stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo) in the induction of an animal model of rheumatoid arthritis, Figure 6b is confirmed by FACS shows changes in cytokines (CD4+IL-17A+, CD4+IL-10, etc.) in splenocytes in an animal model of rheumatoid arthritis, FIG. 6c shows H&E, toluidine blue, and Shows the results of histological evaluation by safranin O staining, and FIG. 6d shows the inflammation index and cartilage erosion score calculated in each group (* p<0.05, ** p<0.01, *** p<0.001 ). ).

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of induced Pluripotent Stem Cells (iPSC) from normal human peripheral blood mononuclear cells

Blood was obtained from a normal donor without disease with the approval of the institutional IRB and used. To isolate mononuclear cells, blood was centrifuged at 2,000 rpm for 30 minutes using a Ficoll Paque-PLUS (GE Healthcare). The isolated mononuclear cells were cultured in StemSpan-ACF (Stem Cell Technologies) supplemented with StemSpan cc110 at 37° C. and 5% CO ₂ conditions for 4 days. Thereafter, the cultured mononuclear cells were inoculated in a 24-well plate coated with vitronectin (Life Technologies, Invitrogen), and Sendai of the CytoTune®-iPS 2.0 Reprogramming Kit (Life Technologies, Invitrogen) according to the manufacturer's instructions. IPSCs were prepared using a virus (Sendai virus). Specifically, from 3 to 21 days after transduction, cells were cultured at 37° C. and 5% CO ₂ conditions, and on day 12, new iPSC colonies were individually picked and expanded for characterization. The medium was changed daily until iPSC colonies were formed. After manually picking iPSC colonies, they were cultured in TeSR-E8 medium (Stem Cell Technologies) in vitronectin-coated plates. Cultured cells were analyzed by flow cytometry for the expression of NANOG, SSEA-4, and TRA-181, which are markers of iPSCs, and identified as iPSCs.

Example 2. Preparation and identification of mesenchymal stem cells (iPSC-iMSC) derived from dedifferentiated stem cells

2-1. Preparation of mesenchymal stem cells derived from dedifferentiated stem cells

In order to induce mesenchymal stem cells from the retrodifferentiated stem cells prepared in Example 1, the retrodifferentiated stem cells cultured on a plate to 90% confluency (90% confluency) were washed once with PBS (1 x Phosphate buffered saline). did After that, 3 mL of a 1 mg/mL type IV collagenase solution was added to the plate and reacted for 10 minutes. After adding 6 mL of TeSR-E8 medium (Stem Cell Technologies), it was centrifuged at 1100 rpm for 3 minutes. 2 wells 3 mL culture medium per dish in 6-well ultra low attachment dishes (100 mm diameter) 20% α-Minimum Eagle's Medium (0.1 mM non-essential amino acids (100x) gibco, 1 mM L-glutamine, 0.1 mM 2-mer After suspending the cells with captoethanol, 1% penicillin, 1% streptomycin), differentiation was induced by culturing at 37° C. and 5% CO ₂ conditions for 4 days in the presence of 1 ng/mL of b-FGF.

After 4 days of differentiation, embryoid body (EB) was stimulated with recombinant human (rh) TGF-β1 in 100 mm dishes coated with 1% gelatin for outgrowth. In the same manner, iPSC-MSCs were cultured until 90% confluency (90% confluency) in a gelatin-coated 100 mm dish while changing the culture medium every other day.

2-2. Identification of mesenchymal stem cell markers derived from dedifferentiated stem cells

In Example 2-1, it was confirmed through a marker whether dedifferentiated stem cell-derived mesenchymal stem cells (iPSC-MSC) were prepared.

Specifically, the expression of NANOG, SSEA-4 and TRA-181, which are markers of dedifferentiated stem cells, was confirmed by the immunofluorescence method, and the cell shape of dedifferentiated stem cell-derived mesenchymal stem cells was confirmed at each stage of differentiation. In addition, the expression of CD34, CD31, CD19, CD11 and HALDR (negative markers), and CD44, CD73, CD105 and CD90 (positive markers), known as MSC markers, were investigated for iPSC-MSCs. After culturing iPSC-MSCs for about 4 weeks, cells at passage 5 or higher were confirmed by flow cytometry.

As a result, as shown in Figure 1a, it was confirmed that the dedifferentiated stem cells used in Example 2-1 express NANOG, SSEA-4 and TRA-181, and as shown in Figure 1b, the dedifferentiated stem cells Differentiation into mesenchymal stem cells was confirmed through cell shape.

1C shows the expression of MSC markers by iPSC-MSCs confirmed by flow cytometry. The retrodifferentiated stem cell-derived mesenchymal stem cells prepared in Example 2-1 expressed CD34, CD31, CD19, CD11 and HALDR, which are negative markers of MSC, in similar amounts as the isotype control, but CD44, a positive marker , CD73, CD105 and CD90 all expressed significantly higher amounts compared to the isotype control, confirming that dedifferentiated stem cell-derived mesenchymal stem cells were formed in Example 2-1.

Example 3. Isolation and identification of mesenchymal stem cell exo-some (MSC-Exo) and dedifferentiated stem cell-derived mesenchymal stem cell exo-some (iPSC-MSC-Exo)

3-1. Isolation of exosomes from mesenchymal stem cell exosomes and dedifferentiated stem cell - derived mesenchymal stem cells

Cord blood-derived MSCs and iPSC-MSCs were each cultured in a 100 mm dish to 100% confluence, and 5 mL of SF-DMEM (serum free-Dulbecco Modified Eagle Medium) was added thereto at 37°C and 5% CO ₂ conditions for 48 hours. cultured. The culture medium was centrifuged for 10 minutes at 4 °C and 300 g conditions. The obtained supernatant was again centrifuged at 4°C and 2000 g conditions for 20 minutes. The obtained supernatant was centrifuged for 30 minutes at 10,000 g with a high-speed centrifuge (Hitachi KoKi #S306352A, Japan) to remove cell debris. The supernatant was filtered through a 0.22 μm filter and then centrifuged for 120 minutes at 100,000 g in an ultra-high speed centrifuge (Thermo Scientific Fiberlite F50L-8x39 Rotor, Thermo Fisher Scientific). After taking out the tube from the centrifuge, the pellet part, which is the location of the exosomes, was marked in advance, all the rest except the pellet was removed to the opposite side, and the remaining pellet was suspended in PBS and stored at -80°C until use.

3-2. Identification of characteristics and markers of dedifferentiated stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo)

In order to confirm the size and uniform particle distribution of the separated exosomes, the exosomes separated by Example 3-1 were diluted in PBS and measured six times consecutively with a NanoSight (Malvern instruments Ltd, Malvern, UK) instrument. . In addition, the isolated exosomes were photographed with a transmission electron microscope (TEM). Then, in order to confirm whether the isolated exosome is correct, TSG101 (Santa Cruz Biotechnology), CD9 (Santa Cruz Biotechnology) and CD63 (Santa Cruz Biotechnology), known as exosome markers, were identified using a Western blot method.

As a result, as shown in Figure 2a, it was confirmed that the size of the material isolated from the dedifferentiated stem cell-derived mesenchymal stem cells is 100 to 200 nm, which is known as the size of the exosome by NanoSight, and the material is the exosome (iPSC). -iMSC-Exo). Figure 2b shows the result of confirming the exosomes using a transmission electron microscope.

In addition, Figure 2c shows that the exosomes (hiPSC-MSCs-Exo) isolated from dedifferentiated stem cell-derived mesenchymal stem cells are a control (Control: SF-DMEM medium) and dedifferentiated stem cell-derived mesenchymal stem cells themselves (hiPSCs). -MSC), it was confirmed that TSG101, CD9 and CD63, which are known as markers of exosomes, are expressed significantly more. Through this, it was confirmed that the material isolated in Example 3-1 was an exosome (iPSC-iMSC-Exo).

Example 4. Immune regulation ability of exosomes (iPSC-MSC-Exo) isolated from dedifferentiated stem cell-derived mesenchymal stem cells from T _h 0 cells

The immunomodulatory ability of iPSC-MSC-Exo obtained in Example 3 was confirmed, respectively.

Mononuclear cells obtained from normal human peripheral blood were differentiated into T _h 0 cells as follows. Specifically, the mononuclear cells to be differentiated into T _h 0 cells were transformed into RPMI 1640 supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine. iPSC-iMSC-Exo prepared in Example 3 (0, 0.1, 1) inoculated into medium, treated with anti-CD3 (1 μg/mL; BD Biosciences) and anti-CD28 (1 μg/mL; BD Biosciences) , and 10 μg) and 37° C. and 5% CO ₂ conditions were co-cultured for 48 hours. Then, using FACS, IL-17 and IFN-γ, which are pro-inflammatory cytokines produced in T _h 0 cells, and CD25+Foxp3+ expression, a marker of immunoregulatory T cells (T _reg ), were investigated. In addition, the contents of the inflammation-inducing cytokine IL-17 and the anti-inflammatory cytokine IL-10 in each culture were analyzed. Specifically, cytokine content and marker expression were analyzed using FACS and sandwich ELISA.

Monoclonal antibodies anti-CD4-PE/Cy7 (RPA-T4, IgG1, BioLegend, San Diego, CA, USA) and anti-CD25-APC (M-A251, IgG1, κ isotype, BD Biosciences) were administered for FACS. cells were used, washed by intracellular staining, then permeabilized and monoclonal antibody anti-IL-17-PE (eBio64dec17, IgG1, κ; eBioscience, San Diego, CA, USA), anti-IFN-γ-FITC (4S.B3, IgG1, κ; eBioscience), anti-IL-10-APC (JES3-19F1, IgG2a, κ; Biolegend), and anti-Foxp3-FITC (PCH101, IgG2a, κ; eBioscience) antibodies in the dark. It was incubated for 30 minutes at 4°C. In addition, the control group was stained with the same type of fluorescence-labeled immunoglobulin of the same type without antigen specificity with an isotype antibody. Intracellular cytokines were analyzed by flow cytometry (FACS Calibur; Becton Dickinson, San Diego, CA).

In addition, for sandwich ELISA, monoclonal IL-17, IFN-γ, IL-10 and IL-2 antibodies (R&D systems, USA) were first added to a 96-well plate (NUNC, Denmark) for sandwich ELISA at 4 μg/mL, respectively. 50 μl per well of the furnace was added and reacted at 4° C. overnight, and then 200 μl of blocking solution (1% (w/v) BSA/PBS (Phosphate-Buffered Saline) and 0.05% (v/v) tween 20/PBS) was added per well. and reacted at room temperature for 2 hours. As a control group, recombinant IL-17, IFN-γ, IL-10 and IL-2 (R&D, USA) were used to 78 pg/mL, 156 pg/mL, 312 pg/mL, 625 pg/mL, and 1250 pg, respectively. /mL, 2500 pg/mL, 5000 pg/mL concentrations were measured.

50 μl of a T _h 0 cell culture solution obtained by co-culture with an exosome to be measured together with a standard sample per well was added and reacted at room temperature for 2 hours. The reaction vessel was washed 4 times with a washing solution (0.05% (v/v) Tween 20/PBS) and biotinylated IL-17, IFN-γ, IL-10 and IL-2 antibodies were added at 200 ng/ After dilution to mL, 50 μl per well was added, followed by reaction at room temperature for 2 hours, and washing with a washing solution 4 times.

Finally, 50 μl per well of an extravidin-alkaline phosphatase conjugate (SIGMA, USA, #E2636) was diluted 1:2,000, reacted at room temperature for 2 hours, washed, and phosphate phosphate disodium salt hexahydrate (PNPP, Fluka)/Diethanolamine solution (DEA, 97 mL, NaN ₃ 0.2 g, MgCl ₂ H ₂ O 0.1 g, primary distilled water 800 mL) was dissolved at a concentration of 1 mg/mL, 50 μl per well was added, and after 30 minutes, the reaction was stopped with 0.2 M NaOH, and absorbance was measured at a wavelength of 405 nm.

The results are shown in Figures 3a to 3c. Figures 3a and 3b show the results of FACS analysis of the amounts of IL-17 and IFN-γ produced in T _h 0 cells and CD25+Foxp3+ expression produced in immunoregulatory T cells (Tregs), respectively, and Figure 3c is in the culture medium. The results of measuring IL-17, IFN-γ and IL-10 by ELISA are shown. Immunomodulatory stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo) inhibit the production of inflammatory cytokines IL-17 and IFN-γ, while not affecting the activity of immunoregulatory T cells (T _reg ). confirmed that it is not. In addition, it was confirmed that iPSC-iMSC-Exo inhibited the production of IL-17 and IFN-γ, which are pro-inflammatory cytokines, while further increasing the production of IL-10, an anti-inflammatory cytokine, in cell culture medium.

Example 5. Confirmation of immune modulatory ability of exosomes (iPSC-iMSC-Exo) isolated from dedifferentiated stem cell-derived mesenchymal stem cells in T _h 17 cells

The immunomodulatory ability of iPSC-iMSC-Exo and MSC-Exo obtained in Example 3 was confirmed, respectively. Specifically, in order to differentiate mononuclear cells obtained from normal human peripheral blood into T _h 17 cells, anti-CD3 (1 μg/mL; BD Biosciences), anti-CD28 (1 μg/mL; BD Biosciences), IL- Neutralizing antibody against 1β (20 ng/mL; R&D Systems, Minneapolis, MN, USA), IL-6 (20 ng/mL; R&D Systems), IL-23 (20 ng/mL; R&D Systems), IFN-γ (IFN-γ; 2 μg/mL; R&D Systems) and a neutralizing antibody against IL-4 (2 μg/mL; R&D Systems), and Example 4 except that T _h 17 was used instead of T _h 0 cells The same experiment was performed.

The results are shown in Figures 4a to 4c. 4a and 4b show the results of FACS analysis of the expression of IL-17 generated in Th17 cells and CD25+Foxp3+ generated in immunoregulatory T cells (Tregs), respectively, and FIG. 4c shows IL-17 and IL-10 in the culture medium. , and IL-2 measured by ELISA are shown. Inversely differentiated stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo) inhibit the production of IL-17, an inflammation-inducing cytokine, but do not affect the activity of immunoregulatory T cells (T _reg ) was able to confirm In addition, it was found that iPSC-iMSC-Exo decreased the production of IL-17 and IL-2, which are pro-inflammatory cytokines, and further increased the production of IL-10, an anti-inflammatory cytokine, in cell culture medium.

Example 6. Comparison of disease control effects in rheumatoid arthritis animal model of exosomes isolated from retrodifferentiated stem cell-derived mesenchymal stem cells (iPSC-iMSC-Exo) and mesenchymal stem cell exosomes (MSC-Exo)

6-1. Confirmation of treatment effect for rheumatoid arthritis through mean joint index

In order to confirm whether the exosomes (iPSC-iMSC-Exo) isolated from the dedifferentiated stem cell-derived mesenchymal stem cells obtained in Example 3 have an effect on rheumatoid arthritis, an animal model experiment was performed.

Specifically, in order to prepare an animal model of rheumatoid arthritis (CIA), 100 μg of type II collagen (CII) was mixed with Complete Freund's Adjuvant (CFA) at a 1:1 ratio in 6 to 7-week-old male DBA/1J mice. was mixed and injected subcutaneously into the tail (1st boosting). After 2 weeks, 100 μg of CII was mixed with IFA (Incomplete Freund's Adjuvant) in a 1:1 ratio and injected subcutaneously into the tarsal bone of the hind leg (secondary boosting) to induce rheumatoid arthritis. Rheumatoid arthritis animal models were analyzed in 4 groups, CIA control (n=4), iPSC-iMSC-Exo (50 μg) (n=4), iPSC-iMSC-Exo (100 μg) (n=4) and MSC-Exo (100 μg) (n=4), iPSC-iMSC-Exo or MSC-Exo for each group was administered 1 day before the 1st boosting and on the 2nd boosting day, a total of 2 doses were administered, and the difference in disease development by group Confirmed. The degree of disease development was confirmed through the following rheumatoid arthritis evaluation.

The evaluation of rheumatoid arthritis was performed using the mean arthritic score by Rosoliniec et al. (Coligan JE, Kruisbeek AM, Matqulies DH, Shevach EM, Strober W: Collagen-induced arthritis. Current protocols in immunology vol 3;15.5.1-19, 1996 ) was based on Specifically, for each mouse, the average value divided by 3 is obtained by summing the scores (Arthritis score) according to the scale below, and the average value obtained by adding up the values obtained by three observers in each mouse is used.

0 points: No edema or swelling.

Score 1: Mild swelling and redness localized to the foot or ankle joint.

Score 2: Mild swelling and redness from the ankle joint to the metatarsal.

Score 3: Moderate swelling and redness from the ankle joint to the tarsal bone.

4 points: swelling and redness from the ankle to the entire leg

As shown in FIGS. 5A and 6A , in the control group (CIA control) and the iPSC-iMSC-Exo administration group not administered with exosomes, compared with the MSC-Exo administration group, all of the mouse arthritis evaluation indices were reduced, and iPSC-iMSC- Exo (50㎍) and iPSC-iMSC-Exo (100㎍) administration group were compared, and it was confirmed that the mean joint index significantly decreased in a concentration-dependent manner. That is, it was found that iPSC-MSC-Exo had an excellent effect in treating rheumatoid arthritis in an animal model of rheumatoid arthritis.

6-2. Confirmation of treatment effect for rheumatoid arthritis through immunological evaluation

Using the rheumatoid arthritis animal model of Example 6-1, cytokines (IL-17, TNF-a, IL-1b, IL-10, IL-4, etc.) in mouse splenocytes for further evaluation of disease Changes were confirmed using ELISA as described in Example 4.

5B and 6B show changes in CD4+T intracellular cytokines (CD4+IL-17A+, CD4+IL-10, etc.) in splenocytes of rheumatoid arthritis animal model mice measured by FACS. There was no significant difference between the CIA group and the iPSC-iMSC-Exo and MSC-Exo groups, but between the iPSC-iMSC-Exo 100㎍ group and the iPSC-iMSC-Exo 50㎍ group, the inflammatory T cells, CD4+IL- 17A+ (%) cells were significantly reduced in the iPSC-iMSC-Exo 100 μg administration group.

The results in the rheumatoid arthritis animal model showed that the iPSC-iMSC-Exo administration group suppressed the production of inflammation-inducing cytokines (CD4+IL-17+), and the anti-inflammatory cytokines (CD4) compared to the MSC-Exo administration group. +IL-10+), and even when comparing the two groups iPSC-iMSC-Exo 50㎍, iPSC-iMSC-Exo100㎍, it was statistically significantly It was confirmed that it inhibits the production of IL-17+) and maintains the production of anti-inflammatory cytokines (CD4+IL-10+).

6-3. Efficacy evaluation for rheumatoid arthritis animal model using joint tissue slides

Using the rheumatoid arthritis animal model of Example 6-1, mice were sacrificed 5 weeks after the second boosting for further evaluation of the disease, and the leg joints of each mouse were excised and fixed in 10% formalin for 6 days, Calci -Decalcification was performed in a clear rapid solution for 7 hours. After fixation in 10% formalin for 2 days or in 10% formalin for 6 days, demineralized tissue was processed in calci-clear rapid for 7 hours by dehydration, clearing and paraffin infiltration in the following order.

No.No.	용기(Jar)Jar	시간hour
1One	흐르는 물(tap water) tap water	1 시간1 hours
22	70% EtOH70% EtOH	1 시간1 hours
33	80% EtOH80% EtOH	1 시간1 hours
44	90% EtOH90% EtOH	1 시간1 hours
55	95% EtOH # 195% EtOH #1	1 시간1 hours
66	95% EtOH # 295% EtOH #2	1 시간1 hours
77	100% EtOH # 1100% EtOH #1	1 시간1 hours
88	100% EtOH # 2100% EtOH #2	1 시간1 hours
99	크실렌 # 1 Xylene #1	1 시간1 hours
1010	크실렌 # 2 Xylene #2	1 시간1 hours
1111	파라핀 # 1 Paraffin #1	2 시간2 hours
1212	파라핀 # 2 Paraffin #2	2 시간2 hours

After the above-described tissue treatment, the tissue was embedded in paraffin and manufactured as a block. In order to perform joint tissue staining, the tissue was sectioned into 4 μm using a microtome. Each slide was prepared so that there was one tissue per slide. For hematoxylin & eosin staining, paraffin-fixed tissue slides are placed in an oven at 65°C to melt paraffin for 1 hour, and then in NeoClear solution and different concentrations of ethanol for tissue deparaffinization and hydration. They were sequentially immersed for 5 minutes. After hematoxylin staining was performed for 10 seconds, the hematoxylin residue used for staining was removed by immersing the slide in tap water for 10 minutes to avoid direct contact with the slide. The slide was immersed in the Eosin channel and stained for 10 minutes. For dehydration, sequentially immersed in ethanol and NeoClear solution of different concentrations, mounting was performed, and after drying in a cool place for more than a day, the tissue was checked through an optical microscope.

For Safranin O staining After staining in Weigert's iron hematoxylin working solution for 10 minutes, the slide was immersed in running water for 10 minutes to avoid direct contact with the slide to remove the hematoxylin residue used for staining. The slide was immersed in a channel of 0.05% Fast green (FCF) solution and stained for 10 minutes. The slides were immersed in a 1% acetic acid solution channel for 10 seconds. A 1% safranin O solution was dispensed, and the slides were stained for 10 minutes. For dehydration, they were sequentially immersed in different concentrations of ethanol and NeoClear solution. After mounting and drying in a cool place for at least one day, the tissue was confirmed through an optical microscope.

For toluidine blue staining, 0.1% toluidine blue solution was dispensed on the slides and stained for 10 minutes. For dehydration, they were sequentially immersed in different concentrations of ethanol and NeoClear solution. After mounting and drying in a cool place for more than one day, the tissue was confirmed through an optical microscope.

Histological evaluation of the rheumatoid arthritis animal model was performed according to the following evaluation criteria.

0 points: no inflammation

1 point: Slight thickening of the inner layer or some infiltrating cells in the lower layer.

2 points: Slight thickening of the inner layer and some infiltrating cells in the lower layer.

3 points: thickening of the sublininig layer, the influx of cells into the lining layer and the presence of cells in the synovial cavity.

Score 4: Many inflammatory cells are present in the infiltrated synovial membrane

0 points: no destruction

1 point: Limited minimal erosion.

2 points: Slight to moderate erosion in a limited area.

Score 3: There is more extensive erosion.

4 points: Most cartilage destruction is seen.

5c and 5d show the histological evaluation results of CIA, iPSC-iMSC-Exo and MSC-Exo. In the joint tissue, the average joint index in the joint tissue was significantly lower in the iPSC-iMSC-Exo group compared to the MSC-Exo group or the CIA group through the inflammation score and the cartilage damage score. confirmed that.

6c and 6d show the histological evaluation results of CIA, iPSC-iMSC-Exo 50 μg and 100 μg administration groups. Even when iPSC-iMSC-Exo 50 μg and iPSC-iMSC-Exo 100 μg administration group were compared, it was confirmed that the iPSC-iMSC-Exo concentration-dependent mean inflammation index and cartilage erosion score were low. That is, it was found that iPSC-iMSC-Exo had an excellent effect in treating rheumatoid arthritis in an animal model.

Claims

isolating cells from the biological sample;

preparing dedifferentiated stem cells from the isolated cells;

Inducing mesenchymal stem cells by culturing the prepared dedifferentiated stem cells in the presence of b-FGF; and

A method for producing an exosome comprising the step of isolating the exosome from the induced mesenchymal stem cells.
The method according to claim 1, wherein the isolated cells are mononuclear cells.
The method according to claim 1, wherein the inducing step comprises the following:

(a) reacting the prepared retrodifferentiated stem cells with a collagenase solution;

(b) centrifuging the reacted dedifferentiated stem cells;

(c) suspending the centrifuged retrodifferentiated stem cells in a medium; and

(d) stimulating the suspended retrodifferentiated stem cells with b-FGF.
The method according to claim 3, wherein the collagenase in step (a) is collagenase type 4 (type 　IV 　Collagenase).
The method of claim 3, wherein the medium in step (c) is αMEM (α-Minimum Eagle's Medium).
The method according to claim 3, further comprising the step of stimulation with recombinant human TGF-β after step (d).
The method according to claim 1, wherein the step of separating the exosomes comprises the following steps:

(a) culturing the induced mesenchymal stem cells in a medium for 36 to 60 hours;

(b) centrifuging the cultured mesenchymal stem cell culture medium at 200 to 400 g at 0 to 5° C. for 5 to 15 minutes;

(c) centrifuging the supernatant of the culture medium centrifuged in step (b) at 1500 to 2500 g at 0 to 5° C. for 15 to 30 minutes;

(d) centrifuging the supernatant of the culture solution centrifuged in step (c) at 8,000 to 12,000 g at 0 to 5° C. for 20 to 40 minutes;

(e) filtering the supernatant of the culture medium centrifuged in step (d) with a filter paper; and

(f) centrifuging the culture solution filtered in step (e) at 80,000 to 120,000 g at 0 to 5° C. for 100 to 140 minutes.
The method of claim 7, wherein the medium in step (a) is SF-DMEM (serum free-Dulbecco Modified Eagle Medium).
A pharmaceutical composition for preventing or treating an autoimmune disease comprising the exosome prepared according to claim 1.
The pharmaceutical composition for preventing or treating an autoimmune disease according to claim 9, wherein the autoimmune disease is rheumatoid arthritis.