Stem cell-derived extracellular endoplasmic reticulum and its uses

Abstract

The present invention relates to a stem cell-derived extracellular vesicle and its use. The stem cell-derived extracellular vesicle of the present invention significantly reduces the amount of inflammatory cytokines such as TNF-α and IL-6 by highly expressing one or more proteins selected from COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF-β1, and TGM2, and is therefore effective in preventing, alleviating, improving, or treating various inflammatory diseases. In particular, when the stem cell-derived extracellular vesicle of the present invention is administered to an animal model of interstitial cystitis / bladder pain syndrome (IC / BPS), the bladder inner wall damaged during the IC / BPS induction process is restored, the degree of inflammation is alleviated, the intravesical pressure is restored, and the micturition cycle is similar to that of the control group, so that it has excellent therapeutic efficacy against interstitial cystitis / bladder pain syndrome. Furthermore, the stem cell-derived extracellular vesicles of the present invention significantly increase cell migration and have excellent wound healing effects, and therefore can be usefully used for wound healing.

Description

Detailed Description of the Invention

[0001] [Technical Field] The present invention relates to stem cell-derived extracellular vesicles and uses thereof.

[0002] [Background technology] In general, the inflammatory response is a biological defense reaction process for repairing and regenerating damage caused by invasion, which causes matrix changes in living cells and tissues. This reaction process involves the involvement of local blood vessels, various tissue cells in body fluids, and immune cells. While the inflammatory response normally induced by foreign invading bacteria is a defense system for protecting the living body, an abnormally excessive inflammatory response can lead to various diseases, collectively referred to as inflammatory diseases. Inflammatory diseases are life-threatening diseases that are caused by various inflammatory mediators secreted from target cells activated by external stimuli, which amplify and sustain inflammation. These diseases include acute inflammation, intrabladder diseases such as cystitis, intra-articular diseases such as rheumatoid arthritis, skin diseases such as psoriasis, and allergic inflammatory diseases such as bronchial asthma.

[0003] In particular, interstitial cystitis (IC) is a chronic bladder disease of unknown etiology characterized by symptoms of pain, e.g., pelvic pain, and lower urinary tract symptoms (LUTS), e.g., increased urinary frequency / urgency. In recent years, the term has evolved to include IC together with painful bladder syndrome (PBS) ([MacDiarmid SA et al. Rev Urol 2007;9(1):9-16]) or painful bladder syndrome (BPS) ([(van der Merve et al. European Urology 53(2008)60-67]), i.e., IC / BPS, or IC / PBS / BPS, to collectively describe this complex symptomatology.

[0004] The prevalence of IC / PBS / BPS varies from 67 to 230 cases per 100,000 women with clinically confirmed disease, and this figure is likely even higher because it is often misdiagnosed or underdiagnosed as endometritis, recurrent urinary tract infections, irritable bladder, or vulvar pain (Forrest JB et al. Clinical Courier 2006;24(3):1-8). IC has a substantial impact on quality of life, affecting travel, family relationships, and activities (Slade D et al. Urol 1997;49(5A Suppl):10-3), and is associated with depressive syndrome (Rothrock NE et al. J Urol 2002;167:1763-1767).

[0005] The single etiology of IC / PBS / BPS has not yet been identified, and it mainly causes hyperalgesia, chronic bladder pain, and dysuria (Forrest JB et al. Clinical Courier 2006;24(3):1-8).

[0006] Extracellular endoplasmic reticulum (EXTR) are lipid bilayer membrane-structured vesicles of various sizes secreted by various eukaryotic cells, including humans, animals, insects, plants, and microorganisms. Among these, microscopic vesicles with nano-sized particles are called exosomes. Exosomes contain specific molecules contained in cells, such as proteins, nucleic acids, lipids, and carbohydrates, and stably protect these molecules with the lipid bilayer. After secretion, they act as a signal transmitter, transmitting these molecules to other cells.

[0007] Exosomes are extracellular vesicles measuring tens to hundreds of nanometers in size and composed of a double phospholipid membrane, similar to the cell membrane, that contain exosomal cargo, including proteins and nucleic acids (e.g., mRNA and miRNA). Exosome cargo contains a wide range of signaling factors, which are cell-type specific and are known to be differentially regulated depending on the environment of the secreting cell. Exosomes are intercellular signaling mediators secreted by cells, and the various cellular signals transmitted through them are known to regulate target cell behaviors, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis. Exosomes contain specific genetic material and bioactive factors depending on the nature and state of the cell of origin. Exosomes derived from proliferating stem cells regulate cell behaviors, such as migration, proliferation, and differentiation, reflecting stem cell properties related to tissue regeneration (Nature Review Immunology 2002(2) 569-579).

[0008] However, despite various studies suggesting the possibility of using exosomes to treat some diseases, more thorough clinical and non-clinical research is needed. In particular, there is a need to scientifically investigate the various targets on which exosomes act and to develop technologies that can apply exosomes to the treatment of various diseases.

[0009] Therefore, the inventors have conducted extensive research into new uses for exosomes derived from stem cells, and have confirmed that exosomes isolated from stem cell culture medium can highly express one or more proteins selected from COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF-β1, and TGM2, thereby solving the safety issues of the stem cells themselves and the stem cell culture medium, and that these exosomes are effective in preventing, alleviating, improving, or treating various inflammatory diseases, including interstitial cystitis, and have thus completed the present invention.

[0010] Summary of the Invention [Problem to be solved by the invention] An object of the present invention is to provide stem cell-derived extracellular vesicles that are effective in preventing, alleviating, improving, or treating various inflammatory diseases, including interstitial cystitis.

[0011] Another object of the present invention is to provide a composition for preventing or treating inflammatory diseases or autoimmune diseases, which comprises the stem cell-derived extracellular vesicles as an active ingredient.

[0012] A further object of the present invention is to provide a composition for wound healing containing the stem cell-derived extracellular vesicles as an active ingredient.

[0013] [Means for solving the problem] To achieve the above-mentioned objectives, the present invention provides stem cell-derived extracellular reticulum that highly expresses one or more proteins selected from COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF-β1, and TGM2.

[0014] According to a preferred embodiment of the present invention, the stem cell-derived extracellular vesicles are capable of expressing the protein at a higher level than extracellular vesicles derived from stem cells cultured in two dimensions or extracellular vesicles derived from stem cells cultured in three dimensions without adding TGF-β to the culture medium.

[0015] According to a preferred embodiment of the present invention, the stem cell-derived extracellular vesicles are capable of highly expressing TGF-β1.

[0016] According to a preferred embodiment of the present invention, the stem cell-derived extracellular vesicles contain 50 to 1,000 pg / 1×10 TGF-β1. 9 It can be expressed in the amount of particles.

[0017] According to a preferred embodiment of the present invention, the stem cell-derived extracellular vesicles can highly express COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF-β1, and TGM2 proteins.

[0018] According to a preferred embodiment of the present invention, the stem cell-derived extracellular vesicles may be produced by a method comprising: (a) culturing stem cells isolated from a subject to form cell aggregates; and (b) three-dimensionally culturing the cell aggregates in a culture medium containing TGF-β (Transforming growth factor beta).

[0019] According to a preferred embodiment of the present invention, the stem cells may be mesenchymal stem cells.

[0020] According to a preferred embodiment of the present invention, step (a) may be performed by suspension culture of stem cells in a multi-well culture vessel.

[0021] According to a preferred embodiment of the present invention, the TGF-β may be TGF-β3.

[0022] According to a preferred embodiment of the present invention, step (b) may be performed by subjecting the cell aggregates to orbital shaking culture in a suspended state.

[0023] According to a preferred embodiment of the present invention, the rotary shaking culture may be carried out at a rotation speed of 50 to 70 rpm.

[0024] According to a preferred embodiment of the present invention, the stem cell-derived extracellular vesicles may have an average diameter of 30 to 150 nm.

[0025] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or autoimmune diseases, which comprises the stem cell-derived extracellular vesicles as an active ingredient.

[0026] According to a preferred embodiment of the present invention, the inflammatory disease or autoimmune disease may be cystitis, rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic fibrosing alveolitis, polymyositis, dermatomyositis, localized scleroderma, systemic cutaneous sclerosis, colitis, inflammatory bowel disease, Sjögren's syndrome, Raynaud's phenomenon, Bechet's disease, Kawasaki's disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, graft-versus-host disease (GVHD), or Crohn's disease.

[0027] According to a preferred embodiment of the present invention, the cystitis may be one or more selected from interstitial cystitis, chronic cystitis, and ketamine-induced cystitis.

[0028] In order to achieve yet another object of the present invention, the present invention provides a pharmaceutical composition for wound healing, which contains the stem cell-derived extracellular vesicles as an active ingredient.

[0029] [Effects of the Invention] The stem cell-derived extracellular vesicles of the present invention significantly reduce the amount of inflammatory cytokines such as TNF-α and IL-6 by highly expressing one or more proteins selected from COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF-β1, and TGM2, and are therefore effective in preventing, alleviating, improving, or treating various inflammatory diseases.

[0030] In particular, when the stem cell-derived extracellular vesicles of the present invention are administered to an animal model of interstitial cystitis / bladder pain syndrome (IC / BPS), the bladder lining damaged during the IC / BPS induction process is restored, the degree of inflammation is alleviated, intravesical pressure is restored, and a micturition cycle similar to that of the control group is observed, thereby demonstrating excellent therapeutic efficacy for interstitial cystitis / bladder pain syndrome.

[0031] Furthermore, the stem cell-derived extracellular vesicles of the present invention significantly increase cell migration and have excellent wound healing effects, and therefore can be usefully used for wound healing.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS [FIG. 1] A diagram showing the process of 3D culture of mesenchymal stem cells by the method of the present invention, showing the process of cell aggregate formation (FIG. 1A) and 3D culture using a rotary stirrer (FIG. 1B).

[0033] [Figure 2] A graph showing the exosome yield under each culture condition.

[0034] [Figure 3] This is a graph showing the change in PDI value due to TGF-β treatment, and shows that a single peak is observed under 3D shaking culture conditions with TGF-β treatment.

[0035] Figure 4 shows the effect of TGF-β on T cell proliferation. After inducing PBMC proliferation using PHA, the T cell inhibitory effects of a negative control (untreated group), a positive control (MSC-treated group), exosomes cultured under 3D shaking conditions only (3D-EV), and exosomes obtained by adding TGF-β3 to the culture medium under 3D shaking conditions (T-3D-EV) were examined (Figure 4A). The results confirmed that exosomes obtained under 3D shaking conditions with TGF-β treatment (T-3D-EV) had the most pronounced T cell inhibitory effect (Figures 4B and 4C). This suggests that the exosomes obtained by the method of the present invention have enhanced functionality in addition to their high yield.

[0036] [Figure 5] Figure 5A shows the results of dynamic light scattering (DLS) analysis to examine exosome size. Figure 5B shows the results of transmission electron microscopy (TEM) to examine the morphology and structure of exosomes. Figure 5C shows the results of Western blotting analysis to confirm the expression of CD9, CD63, Flotillin-1, and Alix. Figure 5D shows the results of immunophenotyping of the exosome surface by flow cytometry. Figure 5E shows the results of enzyme-linked immunosorbent assay (ELISA) to confirm the TGF-β1 content of the produced exosomes.

[0037] [Figure 6] This is a photograph (left) showing the results of transwell migration analysis confirming the increase in cell migration ability of human fibroblasts (NHDF) following administration of exosomes. The right side is a graph using Image J to quantify the relative degree of staining.

[0038] Figure 7 shows the results of administering exosomes to an animal model with a wound created with a biopsy punch and examining the changes in wound healing over time. Figure 7A shows photographs of the wound site taken at regular intervals after administering exosomes to an animal model with a wound created with a biopsy punch. Figure 7B is a graph showing the size of the wound site shown in Figure 7A.

[0039] [Figure 8] Exosomes were administered to an animal model in which a wound was created with a biopsy punch, and the wound healing ability was examined over time. Histological analysis of the wound site was performed 9 days after the wound was created.

[0040] [Figure 9] Results confirming that LPS-induced inflammatory responses in Raw264.7 cells were significantly reduced by exosome administration.

[0041] [Figure 10] The results confirm that the concentrations of inflammatory cytokines TNF-α and IL-6 were significantly reduced in the culture supernatant of Raw264.7 cells cultured after administration of both LPS and exosomes.

[0042] [Fig. 11] This figure shows the results of confirming the effect of exosome administration on reducing TNF-α and IL-6 in a mouse model of endotoxemia caused by LPS toxin.

[0043] [Figure 12] This figure shows the results of examining cell proliferation after treating SV-HUC-1 (human urothelial cells) with exosomes at various concentrations. Figure 12A is a graph showing the cell proliferation rate after treating SV-HUC-1 with exosomes at various concentrations, and Figure 12B shows the results of examining the expression levels of P-AKT and P-ERK, which are associated with cell proliferation.

[0044] Figure 13 shows the results of transwell migration assays confirming the increased cell migration ability of human urothelial cells (SV-HUC-1) following exosome administration. Figure 13A shows a photograph visually confirming cells that have migrated to the opposite side of the transwell using crystal violet staining. Figure 13B shows a graph of the relative cell confluency shown in Figure 13A using Image J.

[0045] [Figure 14] An outline of the study design for producing an interstitial cystitis / bladder pain syndrome (IC / BPS) induced mouse model and evaluating the therapeutic effect of exosome administration.

[0046] Figure 15 shows the morphology of bladder tissue and the degree of inflammation after exosome administration in an interstitial cystitis / bladder pain syndrome (IC / BPS)-induced mouse model. Figure 15A shows the results of H&E staining of bladder tissue from an IC / BPS mouse model, Figure 15B shows the results of Masson's trichrome staining, Figure 15C shows the results of toluidine blue staining, and Figure 15D is a graph showing the results of confirming the degree of fibrosis and mast cell infiltration using these staining methods.

[0047] [Figure 16] mRNA was extracted from bladder tissue extracted after exosome administration in a mouse model of interstitial cystitis / bladder pain syndrome (IC / BPS). The expression levels of inflammation-related cytokines (TNFα, IL6) (Figure 16A), urothelial markers (UPK1A, UPK1B, UPK2) (Figure 16B), and genes expressed in IC / BPS (KLRB1, PSMB9, ITGAL) (Figure 16C) were confirmed.

[0048] [Figures 17 and 18] Figures showing the results of confirming the effect of exosome administration on restoring intravesical pressure and micturition cycle in an interstitial cystitis / bladder pain syndrome (IC / BPS)-induced mouse model.

[0049] Figure 19 shows the results of protein analysis of each exosome. Figure 19A shows the results of quantitative analysis of proteins contained in each exosome. Figure 19B shows a graph showing the abundance distribution of total proteins contained in each exosome. Figure 19C shows the results of gene ontology (GO) analysis of each exosome. Figure 19D shows the results of comparing the EV protein bodies discovered in this study with the dataset reported in Vesiclepedia.

[0050] Figure 20A shows proteins that are differentially expressed in the T-a3D-EV sample, which is an exosome of the present invention, among the four clusters derived by clustering analysis. Figure 20B shows the results of principal component analysis, showing the degree of separation between each exosome group as a discrimination index. Figure 20C shows the results of a two-way comparison method to determine the number of differentially expressed proteins (DEPs) depending on the culture conditions (2D and 3D) and the presence or absence of TGF-β3 treatment.

[0051] Figure 21A shows the gene sets, normalized enrichment scores (NES), and p-values ​​enriched in the PI3K-AKT signaling pathway and integrin 1 pathway of the exosome T-a3D-EV of the present invention by gene set enrichment analysis (GSEA). Figure 21B shows the enriched gene groups divided into the PI3K-AKT signaling pathway and integrin 1 pathway by gene set enrichment analysis (GSEA). Figure 21C shows the Venn diagrams generated by two-way comparison analysis (T-a3D-EV / 2D-EV, a3D-EV / 2D-EV, and T-a3D-EV / a3D-EV) showing the number of differentiated or shared proteins between each exosome. Among these, the results of Gene Ontology (GO) analysis of DEP28, a region shared between T-a3D-EV / 2D-EV and T-a3D-EV / a3D-EV. Figure 21D shows that five of the 28 DEPs (Dysregulated Epitopes) with specific characteristics in the exosome T-a3D-EV of the present invention (S100A10, SDCP, ACTG1, GIPC1, and EIF4E) were mapped with high confidence scores to 113 interactors in the Huri database. Figure 21E shows the biological functions of the exosome T-a3D-EV of the present invention and its characteristics related to the regulation of cyclin-dependent protein kinase activity.

[0052] [Mode for Carrying Out the Invention] The present invention will be described in detail below.

[0053] One aspect of the present invention relates to stem cell-derived extracellular reticulum that highly expresses one or more proteins selected from COL6A1 (Collagen alpha-1(VI) chain), COL6A3 (Collagen alpha-3(VI) chain), TNC (Tenascin), EIF4E (Eukaryotic translation initiation factor 4E), HSP90AB1 (Heat shock protein HSP90-beta), HSP90B1 (Endoplasmin), RAC1 (Ras-related C3 botulinum toxin substrate 1), TGF-β1 (Transforming growth factor-beta-induced protein ig-h3), and TGM2 (Protein-glutamine gamma-glutamyltransferase 2).

[0054] In the present invention, the term "high expression" means that the content, secretion amount, or expression amount of a specific protein, etc. in the extracellular vesicle is significantly increased to a measurable level compared to conventional extracellular vesicles derived from mesenchymal stem cells, specifically extracellular vesicles derived from stem cells cultured in two dimensions, or extracellular vesicles derived from stem cells cultured in three dimensions without adding TGF-β to the culture medium. Specifically, it means that the content, secretion amount, or expression amount is increased by 40% or more, more specifically, it means that it is increased by 50% or more, even more specifically, it means that it is increased by 60% or more, particularly specifically, it means that it is increased by 80% or more, and most specifically, it means that it is increased by 100% or more.

[0055] In the present invention, the term "extracellular vesicle" refers to small lipid bilayer membrane vesicles with diameters ranging from 30 to 1,000 nm that are secreted into the extracellular environment by fusion of the multivesicular bodies with the plasma membrane in various cells.

[0056] The stem cell-derived extracellular vesicles of the present invention particularly highly express TGF-β1. The stem cell-derived extracellular vesicles of the present invention express TGF-β1 at a concentration of 50 to 1,000 pg / 1×10 9particles, preferably 100 to 800 pg / 1 × 10 9 particles, preferably 250-400pg / 1×10 9 The amount of TGF-β1 expressed can be determined by the amount of particles. In other words, the TGF-β1 expression level in the stem cell-derived extracellular vesicles of the present invention may be at least 5-fold, preferably 5-15-fold, more preferably 6-12-fold, and even more preferably 7-10-fold, compared to the TGF-β1 expression level in the stem cell-derived extracellular vesicles obtained by two-dimensional culture. Furthermore, the TGF-β1 expression level in the stem cell-derived extracellular vesicles of the present invention may be at least 2-fold, specifically 3-fold, more specifically 3-10-fold, even more specifically 3-6-fold, and particularly specifically 3-5-fold, compared to the TGF-β1 expression level in the stem cell-derived extracellular vesicles obtained by three-dimensional culture without the addition of TGF-β.

[0057] TGF-β1 plays a crucial role in anti-inflammation and tissue regeneration to repair tissue damage. In particular, in interstitial cystitis, it plays an important role in bladder tissue anti-inflammation, urothelial cell regeneration, angiogenesis, and matrix production to prevent electrolyte permeation in urine (Ju, Cynthia, and Pranoti Mandrekar. "Macrophages and alcohol-related liver inflammation." Alcohol research: current reviews 37.2 (2015): 251.).

[0058] Furthermore, the COL6A1 expression level in the stem cell-derived extracellular vesicles of the present invention may be at least 10-fold, preferably 10- to 30-fold, more preferably 15- to 25-fold, and particularly preferably 18- to 22-fold, as compared to the COL6A1 expression level in extracellular vesicles derived from stem cells cultured in two dimensions. Furthermore, the COL6A1 expression level in the stem cell-derived extracellular vesicles of the present invention may be at least 5-fold, specifically 5- to 10-fold, and more specifically 6- to 8-fold, as compared to the COL6A1 expression level in extracellular vesicles derived from stem cells cultured in three dimensions without the addition of TGF-β.

[0059] Furthermore, the COL6A3 expression level in the stem cell-derived extracellular vesicles of the present invention may be at least 1.4-fold, preferably 1.5-fold, more preferably 1.5- to 2.5-fold, and even more preferably 1.5- to 2-fold, compared to the COL6A3 expression level in extracellular vesicles derived from stem cells cultured in two dimensions. Furthermore, the COL6A1 expression level in the stem cell-derived extracellular vesicles of the present invention may be at least 1.5-fold, specifically 1.5- to 3-fold, and more specifically 1.5- to 2.5-fold, compared to the COL6A3 expression level in extracellular vesicles derived from stem cells cultured in three dimensions without the addition of TGF-β.

[0060] Furthermore, the TNC expression level in the stem cell-derived extracellular vesicles of the present invention may be at least 1.5-fold, preferably at least 2-fold, more preferably 2- to 4-fold, and even more preferably 2.5- to 3.5-fold higher than the TNC expression level in extracellular vesicles derived from stem cells cultured in two dimensions. Furthermore, the TNC expression level in the stem cell-derived extracellular vesicles of the present invention may be at least 5-fold, specifically 5- to 10-fold, and more specifically 7- to 9-fold higher than the TNC expression level in extracellular vesicles derived from stem cells cultured in three dimensions without the addition of TGF-β.

[0061] Furthermore, the EIF4E expression level in the stem cell-derived extracellular vesicles of the present invention may be at least two-fold, preferably three-fold, more preferably three to six-fold, and even more preferably 3.5 to five-fold higher than the EIF4E expression level in stem cell-derived extracellular vesicles cultured in two dimensions or in stem cell-derived extracellular vesicles cultured in three dimensions without the addition of TGF-β.

[0062] Furthermore, the HSP90AB1 expression level in the stem cell-derived extracellular vesicles of the present invention may be at least 2-fold, preferably 2- to 4-fold, and more preferably 3- to 4-fold higher than the HSP90AB1 expression level in the stem cell-derived extracellular vesicles after two-dimensional culture. Furthermore, the HSP90AB1 expression level in the stem cell-derived extracellular vesicles of the present invention may be at least 1.5-fold, specifically 1.5- to 3-fold, and more specifically 1.5- to 2-fold higher than the HSP90AB1 expression level in the stem cell-derived extracellular vesicles after three-dimensional culture without the addition of TGF-β.

[0063] Furthermore, the HSP90B1 expression level in the stem cell-derived extracellular vesicles of the present invention may be at least 20-fold, preferably 20- to 40-fold, and more preferably 20- to 30-fold higher than the HSP90B1 expression level in the stem cell-derived extracellular vesicles after two-dimensional culture. Furthermore, the HSP90B1 expression level in the stem cell-derived extracellular vesicles of the present invention may be at least 5-fold, specifically 5- to 10-fold, and more specifically 6- to 8-fold higher than the HSP90B1 expression level in the stem cell-derived extracellular vesicles after three-dimensional culture without the addition of TGF-β.

[0064] Furthermore, the RAC1 expression level in the stem cell-derived extracellular reticulum of the present invention may be at least 2-fold, preferably 2- to 5-fold, and more preferably 3- to 4.5-fold, as compared to the RAC1 expression level in the stem cell-derived extracellular reticulum after two-dimensional culture. Furthermore, the RAC1 expression level in the stem cell-derived extracellular reticulum of the present invention may be at least 1.5-fold, specifically 1.5- to 3-fold, and more specifically 1.5- to 2-fold, as compared to the RAC1 expression level in the stem cell-derived extracellular reticulum after three-dimensional culture without the addition of TGF-β.

[0065] Furthermore, the TGM2 expression level in the stem cell-derived extracellular reticulum of the present invention may be at least 5-fold, preferably 5- to 15-fold, and more preferably 10- to 15-fold, as compared to the TGM2 expression level in the stem cell-derived extracellular reticulum after two-dimensional culture. Furthermore, the TGM2 expression level in the stem cell-derived extracellular reticulum of the present invention may be at least 2-fold, specifically 2- to 5-fold, and more specifically 3- to 5-fold, as compared to the TGM2 expression level in the stem cell-derived extracellular reticulum after three-dimensional culture without the addition of TGF-β.

[0066] The stem cell-derived extracellular vesicles of the present invention significantly reduce the levels of inflammatory cytokines such as TNF-α and IL-6 by overexpressing one or more proteins selected from COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF-β1, and TGM2, thereby preventing, alleviating, improving, or treating various inflammatory diseases, preferably cystitis, particularly interstitial cystitis. Furthermore, the stem cell-derived extracellular vesicles of the present invention significantly increase cell migration and have excellent wound healing effects.

[0067] In one embodiment, the stem cell-derived extracellular vesicles of the present invention may highly express COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF-β1, and TGM2 proteins.

[0068] In one embodiment, the extracellular vesicles of the present invention may be prepared by a method comprising: (a) culturing stem cells isolated from a subject to form cell aggregates; and (b) three-dimensionally culturing the cell aggregates in a culture medium containing TGF-β (Transforming growth factor beta).

[0069] The term "stem cell" as used herein refers to undifferentiated cells that are at a stage prior to differentiation into the cells that make up tissues and have the potential to differentiate into specific cells under specific differentiation stimuli (environments). Unlike differentiated cells in which cell division has ceased, stem cells are characterized by their ability to self-renew through cell division and their differentiation plasticity, which allows them to differentiate into various cells depending on the nature of the stimuli.

[0070] The stem cells used in the present invention can be any cells that have the properties of stem cells, i.e., undifferentiated state, unlimited proliferation, and the ability to differentiate into specific cells, and can be induced to differentiate into the tissue to be regenerated.

[0071] In one embodiment, the stem cells used in the present invention may be mesenchymal stem cells.

[0072] The term "mesenchymal stem cells" as used herein refers to stem cells with multipotency that can differentiate into adipocytes, osteocytes, chondrocytes, muscle cells, neurons, and cardiomyocytes. Mesenchymal stem cells can be identified by their spiral morphology and the expression of basic cell surface markers CD73(+), CD105(+), CD34(-), and CD45(-). In addition to their multipotency, they also have the function of regulating immune responses.

[0073] In one embodiment, step (a) may be performed by suspension culture of stem cells in a multi-well culture vessel.

[0074] The term "suspension culture" as used herein refers to culturing target cells in a floating state within a culture medium without immobilizing them on a substrate or other material. Therefore, the term "suspension culture" is used interchangeably with "3-dimensional culture." Stem cells, which are adhesion-dependent, undergo cell aggregation during suspension culture. Cells that float independently without being part of such aggregates undergo apoptosis and die. Therefore, an environment suited to their adhesion characteristics must be created. According to the present invention, stem cells are cultured in suspension in a multiwell culture system with multiple wells, forming cell aggregates of a size corresponding to the size of the wells. Therefore, the present invention allows for the production of large quantities of standardized stem cell aggregates of the same size.

[0075] The term "cell aggregate" as used herein refers to a three-dimensional structure formed by the self-aggregation of cells cultured in an environment such as suspension culture that allows three-dimensional growth rather than a monolayer. Cell aggregates formed as a result of three-dimensional culture provide an environment similar to the in vivo tissue from which stem cells are derived, and may be spherical or have a non-spherical shape depending on the size and number of self-aggregated cells. Spherical cell aggregates are called spheroids, but spheroids do not necessarily have to be perfectly spherical geometrically.

[0076] The term "cell culture medium" as used herein means a mixture for in vitro cell growth and proliferation, containing elements essential for cell growth and proliferation, such as sugars, amino acids, various nutrients, minerals, etc.

[0077] Examples of ingredients that may be further included in the cell culture medium include glycerin, L-alanine, L-arginine hydrochloride, L-cysteine ​​hydrochloride monohydrate, L-glutamine, L-histidine hydrochloride monohydrate, L-lysine hydrochloride, L-methionine, L-proline, L-serine, L-threonine, L-valine, L-asparagine monohydrate, L-aspartic acid, L-cystine 2HCl, L-glutamic acid, L-isoleucine, L-leucine, L-phenylalanine, L-tryptophan, L-tyrosine disodium salt dihydrate, i-inositol, thiamine hydrochloride, niacinamide, pyridoxine hydrochloride, biotin, and calcium D-pantothenate. Vitamin B12, folic acid, riboflavin, vitamin B12, sodium chloride (NaCl), sodium bicarbonate (NaHCO3), potassium chloride (KCl), calcium chloride (CaCl2), sodium hydrogen phosphate monohydrate (NaH2PO4-H2O), copper sulfate pentahydrate (CuSO4-5H2O), ferric sulfate heptahydrate (FeSO4-7H2O), magnesium chloride (anhydrous), magnesium sulfate (MgSO4), disodium hydrogen phosphate (Na2HPO4), zinc sulfate heptahydrate (ZnSO4-7H2O), D-glucose (dextrose), sodium pyruvate, hypoxanthine sodium, linolenic acid, lipoic acid, putrescine 2HCl, and thymidine.

[0078] The cell culture medium of the present invention can be artificially produced or commercially available. Examples of commercially available culture media include, but are not limited to, IMDM (Iscove's Modified Dulbecco's Medium), α-MEM (Alpha Modification of Eagle's Medium), F12 (Nutrient Mixture F-12), and DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12).

[0079] In one embodiment, the multi-well culture vessel may have a size of 300 to 500 μm per well, more preferably 350 to 450 μm, and most preferably about 380 to 420 μm.

[0080] In one embodiment, the suspension culture may be performed by dispensing 300 to 500 cells, preferably 350 to 450 cells, and more preferably about 380 to 420 cells per well in the multi-well culture vessel.

[0081] In one embodiment, the TGF-β may be one or more selected from TGF-β1, TGF-β2, and TGF-β3, and preferably TGF-β3.

[0082] In one embodiment, step (b) may be performed by orbital shaking culture of the cell aggregates in a suspended state.

[0083] Shaking culture, which involves rotation during cell culture, has the advantage of more smoothly supplying nutrients and oxygen to 3D cell aggregates. The rotation speed during shaking culture of 3D cell aggregates is crucial. Rotating the cell aggregates at speeds below 40 rpm can result in heterogeneous cell aggregates and induce cell apoptosis, significantly reducing the quality and yield of extracellular vesicles. Rotating the cell aggregates at speeds above 40 rpm increases the stress on the cells. However, applying TGF-β to the cell aggregates can reduce the stress on the cells by increasing the rotation speed.

[0084] The rotary shaking culture of the present invention may be carried out at a rotation speed of 50 to 70 rpm, preferably 53 to 67 rpm, more preferably 55 to 65 rpm, and even more preferably 57 to 63 rpm.

[0085] In one embodiment, the extracellular vesicles of the present invention may be obtained by a method further comprising the step of separating the extracellular vesicles from the culture medium obtained in step (b) by multiple centrifugation.

[0086] When extracellular vesicles are obtained by centrifugation from stem cells cultured by conventional methods, it is difficult to secure a sufficient amount of extracellular vesicles due to the capacity limitations of the centrifuge. However, stem cells cultured by the method of the present invention have a significantly increased amount of extracellular vesicles secreted per cell, making it easy to obtain a therapeutically effective amount of extracellular vesicles by centrifugation alone.

[0087] In one embodiment, the extracellular vesicles of the present invention have an average diameter of 100 to 250 nm, specifically 150 to 220 nm, more specifically 180 to 200 nm, and even more specifically 185 to 195 nm. Extracellular vesicles with diameters in this range are called exosomes (FIG. 5A).

[0088] According to a specific embodiment of the present invention, the stem cell-derived extracellular vesicles (e.g., exosomes) of the present invention exhibit distinct differences in protein expression profiles compared to exosomes obtained by conventional methods. As shown in the Examples below, the proteins highly expressed by the stem cell-derived extracellular vesicles of the present invention (COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF-β1, and TGM2) are significantly more highly expressed than exosomes derived from stem cells obtained by 2D culture or 3D culture alone without TGF-β3 treatment (Figure 21B). This indicates that the composition of the exosomes of the present invention is novel and not previously observed.

[0089] Furthermore, according to a specific embodiment of the present invention, the stem cell-derived extracellular vesicles of the present invention are positive for one or more proteins selected from adipocyte plasma membrane-associated protein, prolyl 3-hydroxylase 1, and prostaglandin G / H synthase 2. As shown in the Examples below, these three proteins are not detected in stem cell-derived exosomes obtained by applying only 2D culture. Furthermore, of these three proteins, prostaglandin G / H synthase 2 is not detected in stem cell-derived exosomes obtained by applying only 3D culture without 2D culture or TGF-β3 treatment, demonstrating that the exosomes of the present invention have an entirely new protein expression profile (Figure 19D).

[0090] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases or autoimmune diseases, which comprises the stem cell-derived extracellular vesicles as an active ingredient.

[0091] The stem cell-derived extracellular vesicles of the present invention have already been described in detail, so their description will be omitted to avoid excessive duplication.

[0092] The term "prevention" as used herein means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to such disease or condition.

[0093] The term "treatment" as used herein means (a) inhibiting the development of a disease, disorder, or symptom; (b) alleviating a disease, disorder, or symptom; or (c) eliminating a disease, disorder, or symptom. The composition of the present invention effectively suppresses T cell-mediated immune activity, thereby inhibiting the development, eliminating, or alleviating the symptoms of various inflammatory or autoimmune diseases caused by excessive or unwanted immune responses. Therefore, the composition of the present invention may be used as a composition for treating the disease itself, or may be administered in combination with other pharmacological ingredients that have a therapeutic effect against inflammation or autoimmune diseases, and used as an adjunct to the treatment of the disease. Thus, the terms "treatment" or "therapeutic agent" as used herein also include the meaning of "adjunct treatment" or "adjunct treatment agent."

[0094] The term "administration" as used herein refers to the direct administration of a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the subject's body, and has the same meaning as "implantation" or "injection."

[0095] The term "therapeutically effective amount" as used herein means the content of the composition of the present invention contained in an amount sufficient to provide a therapeutic or prophylactic effect to an individual to whom the composition of the present invention is to be administered, and thus includes a "prophylactically effective amount."

[0096] The term "subject" as used herein includes, but is not limited to, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus monkey. Specifically, the subject of the present invention is a human.

[0097] In one embodiment, the composition of the present invention has a preventive or therapeutic effect against various inflammatory diseases. The inflammatory diseases to which the pharmaceutical composition of the present invention can be applied include those known in the art as inflammatory diseases, and are not particularly limited. Examples of autoimmune or inflammatory diseases that may be prevented or treated with the compositions of the present invention include, but are not limited to, rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic fibrosing alveolitis, polymyositis, dermatomyositis, localized scleroderma, systemic cutaneous sclerosis, colitis, inflammatory bowel disease, Sjorgen's syndrome, Raynaud's phenomenon, Bechet's disease, Kawasaki's disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, graft-versus-host disease (GVHD), and Crohn's disease. Preferably, it may be cystitis.

[0098] In one embodiment, the cystitis prevented or treated with the composition of the present invention may be one or more selected from interstitial cystitis, chronic cystitis, and ketamine-induced cystitis, preferably interstitial cystitis.

[0099] In one example of the present invention, when the stem cell-derived extracellular vesicles of the present invention were administered to an in vivo animal model of endotoxemia, the concentrations of TNF-α and IL-6 were significantly reduced, demonstrating that the present invention is highly effective in treating endotoxemia caused by LPS toxin.

[0100] Furthermore, in one example of the present invention, it was specifically confirmed that when the stem cell-derived extracellular vesicles of the present invention were administered to an in vivo animal model of interstitial cystitis / bladder pain syndrome (IC / BPS), the bladder lining damaged during the IC / BPS induction process recovered, the degree of inflammation was alleviated, intravesical pressure was restored, and a micturition cycle similar to that of the control group was observed, thereby demonstrating excellent therapeutic efficacy against interstitial cystitis / bladder pain syndrome.

[0101] In one embodiment, the pharmaceutical composition of the present invention may contain the stem cell-derived extracellular vesicles alone, or may further contain one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0102] The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Carriers for parenteral administration may include water, a suitable oil, saline, aqueous glucose, glycol, etc., and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. Other pharmaceutically acceptable carriers may be found in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0103] The composition of the present invention may be administered to mammals, including humans, by any method. For example, it may be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous administration, intramuscular administration, intraarterial administration, intramedullary administration, intradural administration, suprachoroidal injection, transdermal administration, subcutaneous administration, intraperitoneal administration, intranasal administration, intestinal administration, topical administration, sublingual administration, or rectal administration, and preferably intravenous administration.

[0104] The pharmaceutical composition of the present invention may be formulated as a preparation for oral or parenteral administration by the administration route as described above.

[0105] For oral administration, the compositions of the present invention may be formulated into powders, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, and the like by methods known in the art. For example, oral formulations can be prepared by blending the active ingredient with a solid excipient, pulverizing the mixture, and adding appropriate additives to form a granular mixture to obtain tablets or sugar-coated tablets. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches including corn starch, wheat starch, rice starch, and potato starch; celluloses including methylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; gelatin; and fillers such as polyvinylpyrrolidone. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants. The pharmaceutical composition of the present invention may further contain an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

[0106] Preparations for parenteral administration may be formulated by methods known in the art in the form of injections, ointments, creams, lotions, oils, gels, aerosols, and nasal inhalants, as described in Remington's Pharmaceutical Sciences, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a recipe book generally known to pharmaceutical scientists.

[0107] Preferably, the pharmaceutical composition of the present invention may be prepared in any one form selected from the group consisting of an oral agent, an injection, and an ointment, and more preferably, an injection.

[0108] The pharmaceutical composition of the present invention can provide a desirable preventive, ameliorative or therapeutic effect against inflammatory diseases when it contains an effective amount of the stem cell-derived extracellular vesicles. As used herein, the term "effective amount" refers to an amount that shows a response greater than or equal to that of a negative control group, and preferably refers to an amount sufficient to ameliorate or treat inflammatory diseases, particularly interstitial cystitis. The stem cell-derived extracellular vesicles are added in an amount of 5x10 with respect to the total amount of the pharmaceutical composition. 8 ~5x10 10 particles / ml, preferably 5 x 10 9 ~5x10 10 particles / ml, more preferably 1x10 10 ~2x10 10The stem cell-derived extracellular vesicles of the present invention may be contained in an amount of 0.1 to 1.0 μg / ml. If the content of the stem cell-derived extracellular vesicles is less than the lower limit, cell viability may be excellent, but the amelioration or treatment effect for inflammatory diseases may not reach the desired level. Conversely, if the content exceeds the upper limit, the amelioration or treatment effect for inflammatory diseases may not increase proportionally with the increase in concentration, or toxicity may be exhibited. Meanwhile, in vitro experiments have shown that when the concentration of the stem cell-derived extracellular vesicles of the present invention is within the above range, significant effects on the amelioration or treatment of inflammatory diseases are exhibited without side effects such as cytotoxicity. The effective amount of the stem cell-derived extracellular vesicles contained in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is formulated.

[0109] The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may have different active ingredient contents depending on the severity of the disease.

[0110] The appropriate dosage of the pharmaceutical composition of the present invention may be variously determined depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity of the patient, etc. The preferred dosage of the pharmaceutical composition of the present invention may be in the range of 0.001 to 100 mg / kg for an adult.

[0111] Yet another aspect of the present invention relates to a pharmaceutical composition for wound healing, which contains the stem cell-derived extracellular vesicles as an active ingredient.

[0112] The stem cell-derived extracellular vesicles, pharmaceutical compositions and effective amounts of the present invention have already been described in detail, so the description will be omitted to avoid excessive duplication.

[0113] In one example of the present invention, it was specifically confirmed that when the stem cell-derived extracellular vesicles of the present invention were administered to an animal model with an in vivo wound, the area of ​​the wound site was significantly reduced (Figure 7).

[0114] The stem cell-derived extracellular vesicles of the present invention significantly increase cell migration and exhibit excellent wound healing effects, and therefore can be usefully used in wound healing.

[0115] Yet another aspect of the present invention relates to the use of the stem cell-derived extracellular vesicles in therapy.

[0116] The stem cell-derived extracellular vesicles of the present invention have already been described in detail, so their description will be omitted to avoid excessive duplication.

[0117] The therapeutic application may be for the treatment of an inflammatory disease or an autoimmune disease, preferably an inflammatory disease.

[0118] The inflammatory disease or autoimmune disease may be cystitis, rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic fibrosing alveolitis, polymyositis, dermatomyositis, localized scleroderma, systemic cutaneous sclerosis, colitis, inflammatory bowel disease, Sjorgen's syndrome, Raynaud's phenomenon, Bechet's disease, Kawasaki's disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, graft-versus-host disease (GVHD), or Crohn's disease.

[0119] The cystitis may be one or more selected from interstitial cystitis, chronic cystitis, and ketamine-induced cystitis.

[0120] Yet another aspect of the present invention relates to a method for preventing, ameliorating, or treating an inflammatory disease or an autoimmune disease, comprising administering the stem cell-derived extracellular vesicles to a subject in need thereof.

[0121] The stem cell-derived extracellular vesicles of the present invention have already been described in detail, so their description will be omitted to avoid excessive duplication.

[0122] The "subject" refers to a mammal that is the object of prevention, amelioration, treatment, observation, or experiment, and may preferably be a human or mammal in need of prevention, amelioration, and / or treatment of an inflammatory disease or an autoimmune disease.

[0123] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0124] <Example> Example 1: Three-dimensional cell culture of mesenchymal stem cells AggreWell contains approximately 5,900 400 μm microwells per well for 3D cell culture of stem cells. TM Approximately 400 umbilical cord-derived mesenchymal stem cells (KongKuk University Bioethics Committee approval number: 7001355-202010-BR-407) were seeded per well in a 400 well plate (STEMCELL Technologies; #34425) treated with F127 solution to produce uniform spheroids ranging in diameter from 120 to 200 μm. Spheroids were seeded in a non-adhesive culture dish containing TGF-β3-containing medium and then cultured for three days at 60 rpm and 37°C in an orbital shaker (INFORS HT Celtron; #69455). After three days, exosomes were isolated from the resulting culture medium.

[0125] Example 2: Exosome isolation and quantification The culture medium was centrifuged at 300g for 10 minutes to remove cell debris, then centrifuged at 2000g for 10 minutes. The supernatant was transferred to a new tube and centrifuged again at 10,000g for 30 minutes. The supernatant was then centrifuged again at 187,000g for 2 hours, and the pellet was suspended in 200µL of PBS. To purify exosomes from the suspension, a density gradient (50%, 30%, 10%) was performed using OptiPrep (BioVision; M1248). The exosome sample (suspension) to be isolated was mixed with a 50% OptiPrep solution and loaded. This was then centrifuged at 120,000g for 2 hours to obtain exosomes between 10% and 30%. The resulting exosomes were centrifuged again at 187,000g and suspended in 100µL of PBS.

[0126] After isolating exosomes from the culture medium, nanoparticle tracking analysis (NTA) was performed using an NTA device (NS300, NANOSIGHT System) according to the manufacturer's instructions to confirm the number and peak of exosomes. Results showed that 3D culture alone without shaking (60 rpm) was not significantly different from the yield in a typical 2D culture environment, indicating that the effect of 3D culture itself was insignificant. Furthermore, a significant increase in yield was observed in the group that combined 3D, shaking (60 rpm), and TGF-β3 compared to 3D culture without shaking (60 rpm) and the addition of TGF-β3 alone, or 3D shaking culture without TGF-β3 (Figure 2). Furthermore, a single peak was observed in 3D shaking culture with TGF-β treatment, confirming the production of homogeneous exosomes (Figure 3).

[0127] Example 3: PBMC proliferation assay Exosomes obtained using the method described in Example 2 were used in a PBMC proliferation assay using a previously reported method (Hsu, PJ, et al. J. Vis. Exp. (106), e53265, doi:10.3791 / 53265(2015)). This assay confirmed whether exosomes obtained from TGF-β-treated culture medium had a T cell suppressive effect compared to control culture medium (general cell culture medium). PBMCs were isolated from blood (Kongkuk University Hospital, Kongkuk University Bioethics Committee Approval Number: 7001355-201705-BR-181) using Ficoll. After 5 days of culture, PBMCs were stained with CFSE (Carboxyfluorescein succinimidyl ester, Invitrogen; #C34554). PBMC proliferation was confirmed by flow cytometry. After inducing an inflammatory environment accompanied by T cell proliferation through treatment with PHA (Phytohaemaglutinin, Sigma; #L1668), the PBMC-suppressing effect of mesenchymal stem cells themselves was used as a positive control. The PBMC-suppressing effects of exosomes (EV) obtained from general cell culture medium, exosomes obtained only under 3D shaking culture conditions (3D-EV), and exosomes obtained by adding TGF-β3 to the culture medium under 3D shaking culture conditions (T-3D-EV) were confirmed (Figure 4A).

[0128] As a result, the exosomes of the present invention (T-3aD-EV), obtained under 3D shaking culture conditions with TGF-β treatment, reduced the number of proliferating T cells in the MSC-treated group, which had reached 43.1%, to 9.6%, showing a significant reduction rate of nearly 80% compared to the positive control group, and exhibiting the most excellent T cell suppression effect (Figures 4B and 4C).

[0129] Therefore, it was found that the exosomes obtained by the method of the present invention have significantly enhanced functionality in addition to improved yield.

[0130] Example 4: Characterization of individual exosomes 4-1: Size and morphology analysis of exosomes Exosome size was examined by dynamic light scattering (DLS) analysis using a Nano Zetasizer (Malvern Instruments, Malvern, UK), and EV counts were measured using a nanoparticle tracking analyzer NS300 (Nanosight, Amesbery, UK) (Figure 5A). Exosome morphology and structure were analyzed using a transmission electron microscope (TEM, JEM-1010, Nippon Denshi, Tokyo, Japan) at 80 kV. Exosomes were observed to be cup-shaped or spherical (Figure 5B).

[0131] 4-2: Confirmation of exosome-related marker expression Exosomes were attached to a grid (Formvar / Carbon 300Mesh, Copper_FCF300-CU50 / pk) and negatively stained with 1% phosphotungstic acid hydrate (Sigma, P4006). To confirm the expression of exosome-associated positive markers, immunoblotting was performed to examine the expression of CD9 (ab263023, Abcam), CD63 (ab134045, Abcam), Flotillin-1 (#18634, CST), and Alix (#2171, CST). Western blotting revealed the expression of positive exosome markers, but not the expression of GM130 (#12480, CST), a negative exosome marker (Figure 5C).

[0132] 4-3: Immunophenotypic analysis of exosome surfaces We performed immunophenotyping of the exosome surface using flow cytometry. Because exosomes are too small to be analyzed using a flow cytometer, we first enlarged the exosomes by attaching them to 2.7 μm Dynabeads (10620D, Invitrogen, Exosome-Human CD9 Flow Detection Reagent (from cell culture)) conjugated with CD9, a positive marker for exosomes, and then labeled them with CD9-BV421 (BD Bioscience, 743047), CD63-PE (BD Bioscience, 556020), and CD81-APC (macs miltenyi biotec, M130-119-787) antibodies. We then measured the fluorescence intensity generated by the labeled antibodies using a flow cytometry analyzer (Beckman Coulter, CytoFlex Flow Cytometry Analyzer). We confirmed that the fluorescence expression of CD9, CD63, and CD81 on exosomes was over 96% (Figure 5D). The isolated exosomes expressed exosome-positive markers in 96-98% of the samples, confirming that homogeneous exosomes were isolated.

[0133] 4-4: Measurement of TGF-β1 content contained in exosomes To analyze the TGF-β1 content in exosomes, Human TGF-β1 DuoSet (R&D system, DY-240-05) Capture Ab product was coated onto Ancillary Reagent Kit 1 (R&D system, DY007). Given that TGF-β1 is activated upon oxidation, Sample Activation Kit 1 (R&D system, DY010) was used for sample acidification to precisely measure the inactive and active forms of TGF-β1.

[0134] Briefly, the experimental procedure was as follows: 100 μl of Capture Ab at a concentration of 2 μg / ml was loaded and coated overnight at room temperature. 100 μl of sample (active / latent) was loaded and incubated for 2 hours. 100 μl of Detection Ab (50 ng / ml) was added and incubated for another 2 hours, after which streptavidin-HRP B was attached. Finally, 100 μl of substrate was added and incubated. The reaction was terminated with 50 μl of stop solution, and the absorbance was measured at 450 nm using a microplate reader.

[0135] The amounts of TGF-β1 contained in exosomes grown under 2D culture conditions only (2D-EV), exosomes grown under 3D shaking culture conditions only (a3D-EV), and exosomes grown under 3D shaking culture conditions with the addition of TGF-β3 to the culture medium (T-a3D-EV) were measured by ELISA. Before Optiprep gradient purification, the amounts of TGF-β1 contained in each exosome were 86.34 pg / 1×10 9 , 306.95pg / 1×10 9 , 352.91pg / 1×10 9 After the purification process, TGF-β1 was not detected in 2D-EVs, and was 86.3 pg / 1×10 in a3D-EVs. 9 , and 310.6 pg / 1 × 10 for T-a3D-EV. 9 The TGF-β1 expression level in T-a3D-EVs was measured at a concentration of 1×10, confirming that it was approximately 3.5-fold higher than that in a3D-EVs. When the amounts of active and inactive TGF-β1 were measured using acidification of each purified EV, the TGF-β1 content in T-a3D-EVs was significantly higher than that in the other groups (Figure 5E). Specifically, the active form of TGF-β1 in a3D-EVs was 3.8 pg / 1×10. 9 , 36.9 pg / 1 × 10 in T-a3D-EV 9The concentration of TGF-β1 in T-a3D-EVs was measured at 1×10, indicating that the content of active TGF-β1 in T-a3D-EVs was approximately 10 times higher than that in a3D-EVs. In addition, the content of inactive TGF-β1 in a3D-EVs was 82.4 pg / 1×10 9 , 273.6 pg / 1 × 10 in T-a3D-EV 9 The concentration of TGF-β1 in T-a3D-EV was measured at 100 μg / mL, and the content of active TGF-β1 in T-a3D-EV was shown to be approximately 3.3 times higher than that in a3D-EV.

[0136] In summary, after the purification process, the T-a3D-EVs contained at least 2-fold, specifically 3-fold, more specifically 3-10-fold, even more specifically 3-6-fold, and particularly specifically 3-5-fold more of TGF-β1, whether active, inactive, or the sum of active and inactive forms, than a3D-EVs obtained by 3D shaking culture alone without the addition of TGF-β3. On the other hand, no TGF-β1 was detected in 2D-EVs obtained by 2D culture alone.

[0137] Therefore, it can be seen that the protein TGF-β1 is highly expressed only in T-a3D-EVs obtained by the method of the present invention.

[0138] Example 5: Wound healing assay of each exosome 5-1: Transwell migration assay We attempted to evaluate the effect of each exosome on the migration of normal human dermal fibroblasts (NHDF) (Promocell, Cat No. C-12302).

[0139] First, 5 × 10 NHDFs were placed in the upper wells of a 24-well 8.0 μm polycarbonate membrane transwell (3422, Costar). 4Cells were seeded and cultured in DMEM high glucose (D6429, Sigma) medium containing 10% FBS and 1% penicillin-streptomycin (Cat. no. 1514-163, Gibco). After 24 hours, the upper wells were filled with DMEM high glucose serum-free medium (SFM) containing 1% penicillin-streptomycin, and the lower wells were filled with serum-free medium (SFM) containing 1x10 extracellular vesicles. 9 The cells were diluted to a concentration of 100 μl per well and treated. After 24 hours, the cells were washed once with DPBS (Gibco, 10010-031) and then incubated with 4% paraformaldehyde (biosesang, P2031) at room temperature for 20 minutes. After washing once with Gibco™ DPBS, the cells were stained with 1% crystal violet (Sigma, V5265) at room temperature for 15-20 minutes. After washing two to three times with DPBS, the cells were photographed. Throughout the process, 100 μl of the solution was added to the upper well and 500 μl to the lower well. Relative cell confluency was plotted graphically using Image J.

[0140] The relative staining level was confirmed using Image J, and it was confirmed that cell migration increased approximately 4.35-fold, 7.85-fold, and 10.4-fold in the 2D-EV, a3D-EV, and T-a3D-EV groups, respectively, compared to the control group (Figure 6).

[0141] 5-2: Confirmation of in vivo wound healing ability Six-week-old female BALB / c nude mice were used for the experiment after one week of adaptation. After anesthesia, a full-thickness wound was created on the back using an 8 mm biopsy punch (Kai, BP-80F), and exosomes (1 × 10 9The group received three separate injections of 0.5mmT silicone tape with an 8mm biopsy punch around the wound, while the control group received PBS. To protect the wound, the group used silicone tape (0.5mmT) with an 8mm biopsy punch and Tegaderm tape (1622W). Photographs of the wound were taken at regular intervals, with the silicone tape with an 8mm hole used for comparison.

[0142] We compared the size of wounds created with a biopsy punch in six mice each, and confirmed that the wound area was significantly reduced in the T-a3D-EV-treated group (Figure 7). The relative wound area compared to day 0 is shown in Table 1.

[0143] [Table 1]

[0144] From Table 1 and FIG. 7 above, it can be seen that the area of ​​the wound site was significantly reduced in the T-a3D-EV treatment group.

[0145] 5-3:Histological analysis In experiment 5-2, 9 days after wound induction, tissues containing the entire circular wound were harvested and fixed in 4% paraformaldehyde solution for 24 hours. Sections were then taken through the center of the wound, dehydrated, and embedded in paraffin blocks. The tissues were cut using a tissue sectioner and mounted on polylysine-coated slides for deparaffinization and hydration, followed by H&E (Hematoxylin-Eosin) staining. For Masson's trichrome staining, slides were placed in Weigert's iron hematoxylin solution for 10 minutes, Biebrich scarlet-acid fuchsin, and aniline blue for 5 minutes each.

[0146] Histological analysis of the wound-induced site using H&E staining and Masson's trichrome staining confirmed that the wound area was significantly reduced in the T-a3D-EV-treated tissues. Furthermore, Masson's trichrome staining confirmed that ECM remodeling, the final stage of wound healing, had progressed significantly in the T-a3D-EV-treated tissues (Figure 8).

[0147] Example 6: Analysis of the anti-inflammatory efficacy of each exosome 6-1: Analysis of LPS-induced inflammatory cytokine concentrations 1.5 × 10 Raw264.7 cells in a 48-well plate 5 Cells were seeded and 12 hours later, LPS 10 ng / ml (L4391-1MG, Sigma), dexamethasone 10 μM (50002220, Biogems), and exosomes (1 × 10 9 The cells (particles) were treated with 500 μl of 10% exosome-depleted DMEM-high glucose. After 24 hours of treatment, nitric oxide was measured by reacting the culture medium with Griess reagent (0.1% N-(1-naphthyl)ethylenediamide dihydrochloride and 1% sulfanilamide in 5% phosphoric acid) and measuring absorbance at 540 nm. Raw264.7 cells were dissolved in Labozol reagent (LaboPass, CMRZ001), and total RNA was isolated according to the manufacturer's instructions. The purified RNA was quantified using a NanoDrop spectrophotometer (ND-ONE). cDNA synthesis was performed using the M-MuLV reverse transcription kit (Labopass, CMRT010) and oligo-dT primers. Real-time PCR (Amersham Pharmacia Biotech 7500) was performed using HiPi Real-Time PCR 2x Master Mix (SYBR Green, ROX, 500rxn) (ELPISBIOTECH, EBT-1802). The primer sequences used are listed in Table 2 below.

[0148] [Table 2]

[0149] To confirm the anti-inflammatory effect of each exosome, Raw264.7 cells were stimulated with LPS and treated with dexamethasone, 2D-EV, a3D-EV, and T-a3D-EV, respectively. The nitric oxide concentration was then measured in the culture medium, and the mRNA expression levels of each inflammatory response factor were measured in the cells. The results confirmed that LPS-induced inflammatory responses in Raw264.7 cells could be significantly reduced by T-a3D-EV (Figure 9).

[0150] Furthermore, the concentrations of each cytokine were measured from the culture supernatant using mTNF-alpha (BGK06804, Peprotech), mIL-6 (BGK08505, Peprotech), and mIL-10 (BGK18893, Peprotech) ELISA kits, and a significant decrease was confirmed in the T-a3D-EV group (Figure 10).

[0151] These results demonstrate that the T-a3D-EV of the present invention suppresses the expression and production of inflammatory cytokines, exhibits therapeutic effects in a mouse model of endotoxemia caused by LPS toxin, and can be usefully used for the prevention or treatment of inflammatory diseases.

[0152] 6-2: Endotoxemia mouse model Eight-week-old female C57BL / 6 mice were used in the experiment after one week of adaptation. LPS 2.5 mg / kg alone or each exosome (5 × 10 9A total of 200 μl of the solution containing 100 μg of IgG (particles) was injected into the tail vein using a 28G needle. Two hours later, the mice were sacrificed, and the spleens were removed, washed with PBS, and stored at -80°C until use. Proteins were extracted from the spleens using RIPA buffer (CBR002, LPS solution) containing a protease inhibitor cocktail (87786, Invitrogen). The concentrations of mTNF-alpha (BGK06804, Peprotech), mIL-6 (BGK08505, Peprotech), and mIL-10 (BGK18893, Peprotech) were measured using ELISA kits.

[0153] As a result, it was confirmed that the concentrations of TNF-α and IL-6 were significantly reduced in the group treated with T-a3D-EV (Figure 11).

[0154] Example 7: Analysis of the therapeutic efficacy of each exosome for interstitial cystitis 7-1: Confirmation of cell proliferation and signaling Human urothelium cells, SV-HUC-1 (ATCC, CRL-9520), were treated with exosomes at various concentrations, and cell proliferation was confirmed.

[0155] To confirm the effect of each exosome concentration, 1.5 × 10 cells were placed in a 96-well plate (30096, SPL). 4 Cells were seeded, and 12 hours later, exosomes (2D-EV, a3D-EV, T-a3D EV) were added at various concentrations (1 × 10 7 , 1×10 8 After 24 hours, the cells were treated with ez-cytoxin (EZ-3000, DOGEN), and absorbance (450 nm) was measured using a Bio-Rad x-Mark™ spectrophotometer (Bio-Rad Laboratories, USA) after 1 hour.

[0156] In addition, SV-HUC-1 cells treated with each of the exosomes were lysed using RIPA buffer (CBR002, LPS solution) containing the protease inhibitor cocktail (87786, Invitrogen), and whole cell lysates (WCLs) were isolated. Proteins were quantified using a BCA analyzer (23227, Thermo Scientific), then electrophoresed on 4-12% Bis-Tris Flus Gels (NW04125BOX, Invitrogen / NW04122BOX, Invitrogen), and transferred to NC membranes (IB23001, Invitrogen). Primary antibodies (1:1000) were incubated overnight at 4°C, followed by three washes with 1x TBST (TLP-118.1, TrnasLab). Secondary antibodies were incubated for 2 hours at room temperature, followed by washing with 1x TBST. All antibodies were diluted in 1x blocking buffer (TLP-115.1G, Translab) and used. TM iBright TM The primary and secondary antibodies used were P-AKT (sc-293125, Santa Cruz), T-AKT (CSB-PA000855, Cusabio), P-ERK (CSB-PA000749, Cusabio), T-ERK (B7074, Tebu-bio), β-actin (sc-47778, Santa Cruz), HRP-linked anti-rabbit IgG (7074, CST), and HRP-linked anti-mouse IgG (7076, CST).

[0157] We examined proliferation by treating SV-HUC-1 with various concentrations of exosomes, and found that cell proliferation increased with each concentration, with T-a3D-EV demonstrating a higher level of cell proliferation than 2D-EV or a3D-EV (Figure 12A). Furthermore, when examining AKT and ERK signaling, which are associated with cell proliferation, we confirmed that the expression levels of P-AKT and P-ERK were increased in the T-a3D-EV treatment group (Figure 12B).

[0158] 7-2: Transwell Migration Assay SV-HUC-1 cells were cultured in F-12K Nutrient Mixture (21127-022, Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin (15140-163, Gibco). To confirm the degree of cell migration according to the concentration of each exosome, 1.5×10 5 cells were seeded in the upper well of a 24-well 8.0 μm polycarbonate membrane Transwell (3422, costar), and exosomes were added to the lower well 12 hours later. After 24 hours, the cells were washed with DPBS (10010-031, Gibco), then treated with 4% paraformaldehyde (P2031, Biosesang) and fixed at room temperature for 20 minutes. After washing the fixed cells with DPBS, they were incubated with 100% methanol at room temperature for 20 minutes and then stained with 1% crystal violet (V5265, sigma) at room temperature for 15-20 minutes. After washing the staining solution with DPBS 2-3 times, the cells remaining on the upper side of the membrane were wiped off with a cotton swab. After removing cell debris wiped off by DPBS washing, the cells were observed. To confirm the degree of cell migration, the cell confluency was compared using the mean value of Image J and shown in a graph.

[0159] As a result of treating SV-HUC-1 with each exosome according to concentration, it was confirmed that the degree of migration in Transwell increased (Figure 13A). Also, among each exosome, it was confirmed that the degree of cell migration increased in the order of 2D-EV < a3D-EV < T-a3D-EV (Figure 13B).

[0160] 7-3: Interstitial Cystitis / Bladder Pain Syndrome (IC / BPS)-induced Mouse Model Eight-week-old BALB / cAnNCrlOri female mice were received and allowed to adapt for two weeks. Alfaxan and Rumpun were mixed at a 4:1 ratio and 90 μl of each mixture was injected intraperitoneally for anesthesia. A catheter (382412, BD) was inserted into the urethra to remove urine from the bladder, which was then washed with 50 μl of PBS. 5 mg / ml protamine sulfate (P3369, Sigma) was injected, followed by rinsing with PBS 30 minutes later. LPS (L4391, Sigma) was injected, followed by rinsing with PBS 30 μg / ml. Recovery of the mice was confirmed on a hot plate and the mice were housed for one week. This process was repeated four times to induce the mouse IC / BPS model. At the fifth week of the experiment, the mice were anesthetized using the same method, and the lower abdomen of the mice was incised, followed by rinsing the bladder with 5 × 10 exosomes of each type. 8 Particles were injected at a volume of 30 μl each. The lower abdomen was sutured, and the mice were allowed to recover from anesthesia before being housed. One week later, the mice were anesthetized and their bladders were removed for use in the experiment (Figure 14). Some bladders were homogenized for RNA extraction, and others were subjected to tissue selection and staining to confirm the bladder morphology and degree of inflammation.

[0161] Furthermore, H&E staining of mouse bladder tissue sections confirmed that the bladder lining was damaged in IC / BPS-induced mice, while the lining recovered in the exosome-treated groups (Figure 15A). Furthermore, Masson's trichrome staining and toluidine blue staining confirmed the degree of fibrosis and mast cell infiltration, and confirmed that the degree of inflammation was alleviated in the T-a3D-EV-treated group (Figures 15B, 15C, and 15D).

[0162] 7-4:qPCR The extracted bladders were homogenized and resuspended in Labozol reagent (CMRZ001, Cosmogenetek). Chloroform (C2432, Sigma) was mixed with Labozol at a 5:1 ratio and vortexed. After centrifugation at 13,000 rpm for 15 minutes, the supernatant containing dissolved RNA was mixed with 2-propanol (64605-0380, Junsei) at a 1:1 ratio and centrifuged at 13,000 rpm for 15 minutes. The RNA pellet was washed with 75% ethanol, centrifuged at 13,000 rpm for 10 minutes, and the RNA was resuspended in DEPC. RNA was converted to cDNA using rTaq Plus 5x PCR master mix (EBT-1319, ELPISBIO), and gene expression was confirmed using HiPi Real-Time PCR 2x Master Mix (SYBR green, ROX) (EBT-1802, ELPISBIO) (7500, Amersham Pharmacia Biotech).

[0163] We extracted mRNA from bladder tissue and examined the expression of inflammation-related cytokines (TNF-alpha, IL6). We confirmed that expression levels were significantly lower in the T-a3D-EV treatment group (Figure 16A). We also examined the expression of urothelial markers (UPK1A, UPK1B, UPK2). We confirmed that expression levels were significantly higher in the T-a3D-EV treatment group (Figure 16B). Furthermore, we confirmed that the expression of genes classified as expressed in IC / BPS by bioinformatics (KLRB1, PSMB9, ITGAL) was significantly lower (Figure 16C) (see Korean Patent Publication No. 10-2331138).

[0164] 7-5: Awake cystometry To catheterize the bladder of IC / BPS-induced mice, anesthesia was induced 3 days before bladder measurement. A cuffed polyethylene catheter (PE-50; Becton-Dickinson, Parsippany, NJ, USA) was inserted into the bladder after an abdominal incision. The catheter was then passed through the subcutaneous space and secured externally to the animal's back. For awake cystometry, the indwelling catheter was connected to a bidirectional valve and a microinfusion pump (PHD ULTRA™ Syringe; Harvard Apparatus) via a T-tube connected to a pressure transducer (Research Grade Blood Pressure Transducer; Harvard Apparatus, Holliston, MA, USA). Voiding volume was continuously recorded for 8 min using a fluid collector connected to a transducer (Research Grade Isometric Transducer; Harvard Apparatus) while sterile saline was infused into the bladder at a rate of 0.4 mL / min. IVP (intravesical pressure) and voided volume were continuously recorded using an iWork IX-RA-834 data acquisition system with LabScribe 3.637 software (iworks, 62 Littleworth Road, Dover, NH 03820, USA).

[0165] Mice with IC / BPS induced showed frequent urination due to bladder pain and decreased function, but the T-a3D-EV-treated group showed recovery of intravesical pressure and a urination cycle similar to that of the control group (Figures 17 and 18).

[0166] From the above experimental results, it can be concluded that the extracellular vesicles (T-a3D-EV) of the present invention can be usefully used in the treatment of bladder pain syndrome (BPS) such as interstitial cystitis (IC).

[0167] Example 8: Analysis of exosome-expressed proteins Experimental Method Sample preparation for proteome identification / quantitation Protein (20 μg) isolated from WJ-MSC-derived exosomes was lyophilized using a centrifugal vacuum concentrator (LABCONCO, CentriVap, Missouri, USA). The pellet was lysed in 100 mL of lysis buffer consisting of 5% sodium dodecyl sulfate and 50 mM triethylammonium bicarbonate (pH 7.55, ThermoFisher Scientific). The sample was lysed using S-Trap according to the manufacturer's protocol, except for the trypsin / LysC mixture (Promega, Madison, WI, USA). TM The solution was treated with the Micro Spin Column Digestion Protocol. The lyophilized peptides were dissolved in 0.1% formic acid. To confirm the amount of protein and peptide in the solution, the protein amount was measured after lysis using a BCA protein assay kit (Pierce). The peptide concentration was measured by absorbance at 205 nm using a NanoDrop One spectrophotometer (ThermoFisher Scientific).

[0168] Human WJ-MSC cell pellets were lysed in 5% sodium dodecyl sulfate, 50 mM triethylammonium bicarbonate (pH 7.55, ThermoFisher Scientific) with a protease and phosphatase inhibitor cocktail (Thermo Scientific Halt). The samples were transferred to Covaris microTUBE-130 AFA (Adaptive Focused Acoustics) Fiber Screw-Caps (Product No. 520216) and sonicated using a Covaris S220 AFA in a Screw-Cap microTUBE-130 holder (Product No. 500339). The AFA instrument parameters were as follows: chiller set point -5°C, maximum incident power 175 W, duty factor 10%, cycles per burst 200, duration 360 s. The digestion step was performed using S-Trap according to the manufacturer's protocol, except for the trypsin / LysC mixture (Promega, Madison, WI, USA). TM The Mini Spin Column Digestion Protocol was used. Peptides contained in elution buffer (containing 0.2% formic acid and 50% acetonitrile) were lyophilized using a centrifugal vacuum concentrator. The lyophilized peptides were resuspended in 50 mM triethylammonium bicarbonate, and peptide concentrations were determined using a NanoDrop One spectrophotometer (Thermo Fisher Scientific) at 280 nm wavelength with the sample type option set to "1 Abs = 1 mg / mL." Each sample was then labeled with 100 μg of 10-plex TMT reagent (Lot number: UH284251), excluding TMT10-130N, TMT10-130C, and TMT10-131, according to the manufacturer's instructions. After labeling and processing, the TMT-labeled samples were pooled in a 1:1:1:1:1:1 ratio before desalting by solid-phase extraction using C18 cartridges (Sep-Pak, Waters, Milford, MA, USA).

[0169] To reduce the sample complexity of the labeled peptide sample isolated from human WJ-MSCs, the bound sample was fractionated at a flow rate of 0.5 mL / min using a Shimadzu Prominence HPLC instrument (Shimadzu, Japan) equipped with an XBridge C18 column (4.6 mm id x 250 mm length, 130 Å pore size, and 5 μm particle size, Waters Corporation, USA). Mobile phase A was 10 mM trimethylamine bicarbonate (TEAB, pH 8.5), and mobile phase B was 10 mM TEAB in 90% acetonitrile (pH 10). The sample was dissolved in 200 μL of mobile phase A and injected into a 2,100 μL sample loop. Solution B was run at 5-5% for 15 min, followed by 5-45% for 62.5 min, 45-60% for 5 min, 60-60% for 12.5 min, 60-5% for 7.5 min, and 5-5% for 20 min. The column was then washed with 100% and 50% mobile phase B for 30 min each. The eluate was collected every 30 s using a Shimadzu Prominence FRC-10A fraction collector (Shimadzu, Tokyo, Japan). The 168 fractions were combined in a linked fashion to obtain 24 fractions. Each fraction was lyophilized and stored at -20°C until use.

[0170] Mass spectrometry analysis Samples were analyzed on an UltiMate 3000 RSLC nanoLC system (Thermo Scientific) coupled to a Q Exactive HF-X mass spectrometer (Thermo Scientific). For analysis, the peptides were loaded onto an Acclaim PepMap 100 trap column (100 mm × 2 cm, nanoViper, C18, 5 mm, 100 Å, Thermo Scientific) and analyzed by EASY-Spray. TMSeparation was performed on a C18 LC analytical column (PepMap RSLC, 75 mm x 50 cm, C18, 2 mm, 100 Å, Thermo Scientific). For liquid chromatography-tandem mass spectrometry analysis, each peptide was eluted with 5% dimethyl sulfoxide containing 0.1% formic acid (eluent A) and 80% acetonitrile and 5% dimethyl sulfoxide containing 0.1% formic acid (eluent B). The gradient was eluted at 50°C (5–40% over 150 min; 40–95% over 2 min; 95% held for 23 min; 95–5% over 10 min; 5% held for 15 min). The gradient was 250 nL / min.

[0171] Mass spectra were acquired in data-dependent mode with automatic switching between a full scan (MS1, m / z 350-1800) and 20 data-dependent MS / MS (MS2) scans.

[0172] The target for the whole scan mass spectrum was 3,000,000 with a maximum injection time of 100 ms and a resolution of 60,000 (m / z 400). The ion target for MS / MS was set to 100,000 with a maximum injection time of 50 ms and a resolution of 15,000 (m / z 400) using a normalized collision energy (27%) and an isolation window (1.7 m / z). Dynamic exclusion of repeating peptides was applied for 20 seconds. Three replicates were performed for each biological sample. The TMT-labeled samples were run with the same LC parameters as the exosome samples, and the MS parameters were as follows: MS1 scan range (m / z) 350–1500; MS1 resolution 120,000, MS2 resolution 45,000; MS1 AGC target 3e6, MS2 AGC target 1e5; MS1 maximum IT (ms) 50, MS2 maximum IT (ms) 96. The data-dependent mode cycles were set to trigger MS / MS for up to 20 of the most abundant precursors per cycle with an isolation window of 0.7 m / z, a fixed first mass of 110 m / z, and 32% HCD collision energy.

[0173] Database searching and label-free quantification Peptide and protein identification and quantification were performed using MaxQuant 1.6.17.0. Mass spectrometry raw files were searched against the SwissProt human database (released May 2020, http: / / www.uniprot.org) using the Andromeda search engine included in MaxQuant. The MaxQuant contaminant database was used. The following MaxQuant search parameters were used: trypsin was selected as the specific enzyme, cysteine ​​carbamidomethylation was set as the fixed variant, and N-terminal protein acetylation and oxidation (M) were set as variable variants. Protein quantification values ​​(LFQ intensities) were generated by the precursor intensity-based label-free quantification algorithm used in MaxQuant, enabling the "inter-run agreement" function. LFQ intensities were the output of the Max-LFQ algorithm for exosome samples. Reporter ions were set to 7-flex TMT corrected with a 10-flex parameter for WJ-MSC protein quantification. The FDR was set to 0.01 for all protein and peptide spectral match levels. Proteins identified by at least one unique peptide were used, while other settings were kept at baseline.

[0174] Statistical analysis of protein and experimental data The software Perseus (version 1.6.14.0) was used to perform differential analysis of protein abundance data between samples. First, various contaminants and data identified from the inverse database were removed. To assess the quality of the dataset, Perseus software was used for PCA. Normalized protein abundance values ​​for exosome EV samples and TMT-labeled samples (normalized by median rejection) were transformed to the log2 scale. Triplicate replicates of each sample were grouped, and at least three valid values ​​were required for one or more groupings. Missing values ​​were imputed with random numbers drawn from a normal distribution using base parameters (width: 0.3, downward shift: 1.8). To detect statistically significant differences between samples, a t-test was performed using the Benjamini-Hochberg FDR (0.05 cutoff). Hierarchical clustering of enriched Z-score values ​​was performed using InstantClue software. Euclidean distance was used as the metric, and hierarchical clustering was performed using the mean linkage parameter. Protein abundance values ​​(Log2 values) belonging to each cluster are displayed as box plots with the upper and lower quartiles, median, minimum and maximum values, and all individual data points along the y-axis determined by InstantClue. Data were processed using FunRich v3.1.3 and ShinyGO v0.61 for Gene Ontology (GO) analysis. Gene abundance signatures were assessed using the GSEA algorithm using 1,000 permutations and basic parameters.

[0175] For data obtained for exosomal EV yield, NTA results (mean ± standard deviation, n = 6) were analyzed using GraphPad Prism 5.0 (GraphPad Software, CA, USA) with an unpaired two-tailed Student's t-test. Experiments were repeated at least three times (n = 3 or 6) for scratch analysis. PBMCs were isolated from healthy donors, and experiments were repeated four times. Error bars for graphical data indicate the mean ± standard error of the mean (SEM). Statistical significance was set at p < 0.05.

[0176] Test results 8-1: Protein analysis of differentially expressed proteins (DEPs) from each exosome Quantitative analysis of proteins contained in each exosome To understand the potent efficacy of T-a3D-EVs on recipient cells, the protein content of 2D-EVs, a3D-EVs, and T-a3D-EVs was analyzed using LC-MS-based label-free quantification (LFQ). Proteins were extracted from the same amount of EVs (20 mg) obtained from each exosomal EV sample, and a total of 394 protein groups were identified in triplicates. Of these, 138 ± 1 protein groups were quantified in 2D-EVs, 304 ± 5 ​​protein groups in a3D-EVs, and 320 ± 14 protein groups in T-a3D-EVs. Interestingly, the amounts of EV proteins analyzed by BCA analysis after lysis were 33 μg, 26 μg, and 29 μg in 2D-EVs, a3D-EVs, and T-a3D-EVs, respectively (Figure 19A). The abundance distribution of the proteome followed the same pattern as the protein abundance. In contrast, 44 of the top 100 previously known EV marker proteins (http: / / exocarta.org / exosome_markers_new) were identified in all EV samples in this study, and the quantitative distribution of these EV marker proteins was found to be very similar between samples (Figure 19B). These results suggest that although 2D-EVs, a3D-EVs, and T-a3D-EVs each contain similar amounts of proteins, the protein complexes contained in a3D-EVs and T-a3D-EVs are significantly greater than those in 2D-EVs.

[0177] Gene ontology analysis Gene ontology (GO) analysis was performed on each sample set of 2D-EV, a3D-EV, and T-a3D-EV.

[0178] The results showed that proteins were most abundant in exosomes compared to the cytoplasm, nucleoli, ribosomes, extracellular matrix, and exosomes. In particular, 2D-EVs contained a greater proportion of proteins in exosomes than the other EV sample sets. Meanwhile, a3D-EVs and T-a3D-EVs, unlike 2D-EVs, contained more proteins in the cytoplasm, nucleoli, ribosomes, and extracellular matrix (Figure 19C).

[0179] Furthermore, when we compared the EV protein bodies discovered in this study with the dataset reported in Vesiclepedia, we found that most proteins were common to existing EV protein bodies. However, three proteins (adipocyte plasma membrane-associated protein, prolyl 3-hydroxylase 1, and prostaglandin G / H synthase 2) from the T-a3D-EVs obtained using the method of the present invention were not present in previously known EV protein bodies (Figure 19D). These three proteins were not detected in 2D-EVs, but were detected only in a3d-EVs and T-a3d-EVs. Among these three proteins, prostaglandin G / H synthase 2 was not detected in 2D-EVs or a3d-EVs, but was present only in T-a3d-EVs obtained using the method of the present invention, indicating that the T-a3d-EVs have a novel protein expression profile.

[0180] Clustering analysis Hierarchical clustering analysis was performed using a Z-score, which normalizes abundances with a Benjamini-Hochberg false discovery rate (FDR) of 0.05 or less, and the clusters were classified into four clusters (Figure 20A). As shown in Figure 20A, clusters that increased only in each sample set were found (Cluster 2: 47 protein groups in T-a3D-EVs, Cluster 3: 11 protein groups in a3D-EVs, Cluster 4: 63 protein groups in 2D-EVs). Cluster 1, which contains 82 protein groups, shows proteins that increased in both a3D-EVs and T-a3D-EVs, indicating that such proteins are characteristic of 3D culture.

[0181] Comparative analysis of biological characteristics of proteins Using gene ontology tools, we investigated the functional characteristics of the biological process categories for each cluster and specifically confirmed that proteins belonging to cluster 2 (proteins that increased only in the T-a3D-EV group) were associated with immune responses.

[0182] Principal component analysis Furthermore, principal component analysis (PCA) to identify unique trends among the three EV sample sets showed that these protein groups differed significantly depending on the culture conditions (2D vs. 3D culture) (component 1: 61.6%) (Figure 20B). The difference between TGF-β3 treatment and non-treatment was also clearly reflected in the PCA results (component 2: 27.1%), but unlike the culture method, there was no significant difference. These PCA results indicate that the protein body profiles under 3D culture conditions (a3D and T-a3D) differed significantly from those under 2D culture conditions, indicating that TGF-β3 did not significantly alter protein body profiles in 3D culture.

[0183] Two-way comparison In addition, a two-way comparison method was used to compare DEP (Differentially expressed protein) (FDR 5%, log2(ratio) 3 We sought to identify proteins with significantly higher or lower expression levels (Figure 20C). DEPs were analyzed for proteins with significantly higher or lower expression levels. The volcano plots for a3D-EV / 2D-EV and T-a3D-EV / 2D-EV showed that the number of DEPs induced by the culture conditions (2D and 3D) was greater than that induced by TGF-β3 treatment in 3D. The volcano plot for T-a3D-EV / a3D-EV also showed that 53 proteins were upregulated, while 14 proteins were downregulated, in the T-a3D-EV group after TGF-β3 treatment.

[0184] 8-2: Functional characterization of differentially expressed proteins (DEPs) from each exosome Gene set enrichment analysis To investigate the functional characteristics of EVs using EV protein body data, we first performed gene set enrichment analysis (GSEA). This can reveal whether a group of proteins that share biological properties, such as pathways or gene ontologies, can be statistically expressed. Two biological states were identified and the similarity of their expression profiles was assessed. Proteins with statistically significant expression patterns can be clustered, while other proteins can be separated by GSEA. In this study, GSEA presented a state-of-the-art subset of proteins that could explain representative pathways or gene ontologies.

[0185] The GSEA results between T-a3D-EV and other samples showed differences in the PI3K-AKT signaling pathway (24 proteins) and the integrin 1 pathway (17 proteins) (Figure 21A). Figure 21A shows the gene set enrichment analysis results for this analysis, presenting the enriched gene sets, normalized enrichment scores (NES), and p-values. The higher the NES and the lower the p-value, the greater the likelihood of a significant finding. The gene groups enriched in this analysis are shown in Figure 21B.

[0186] Of the 30 proteins identified from these pathways, 11 proteins were identified as common participants. In particular, COL6A1, COL6A3, and TNC proteins were upregulated only in the T-a3D-EV set. Furthermore, among proteins found only in the PI3AKT signaling pathway or intergrin1 pathway, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF-β1, and TGM2 were upregulated only in the T-a3D-EV set (Figure 21B, Figure 5E).

[0187] The expression levels of proteins expressed in each exosome were quantified as relative concentration ratios and are shown in Table 3 below.

[0188] [Table 3]

[0189] As shown in Table 3 above, it can be confirmed that all of the above proteins are expressed at significantly higher levels in the T-a3D-EV of the present invention than in 2D-EV and a3D-EV.

[0190] From these results, it can be inferred that the differential efficacy of the exosomes obtained by the method of the present invention compared to the exosomes of the comparison group is due to the difference in protein expression described above.

[0191] Identification of T-a3D-EV-specific proteins We also attempted to analyze the predicted role of DEPs in EVs under each culture condition when EV proteins were delivered to recipient cells. To identify protein groups that significantly changed under each culture condition, we performed two-way comparison analysis between samples (T-a3D-EV / 2D-EV, a3D-EV / 2D-EV, and T-a3D-EV / a3D-EV) and generated Venn diagrams (Figure 21C). From this analysis, 68 DEPs (40 DEPs in T-a3D-EV / 2D-EV and 28 DEPs in the region common to T-a3D-EV / 2D-EV and T-a3D-EV / a3D-EVs) could be selected as DEPs associated with TGF-β3 treatment in the T-a3D-EVs / 2D-EVs comparison, and 53 DEPs specific to TGF-β3 treatment could be identified in T-a3D-EVs / a3D-EVs (25 DEPs in T-a3D-EVs / a3D-EVs and 28 DEPs in the region common to T-a3D-EVs / 2D-EVs and T-a3D-EVs / a3D-EVs) (Figure 21B). Therefore, we speculated that the 28 proteins were unaffected by cell culture methods and strongly related to TGF-β3. Gene Ontology (GO) analysis specifically confirmed that these proteins are associated with vesicle-mediated transport, extracellular efflux, and immune responses. Interestingly, the expression of all 28 proteins, except for CAPZA1, was increased in the T-a3D-EV set. EV proteins are known to activate various pathways in vitro and in vivo after internalization into recipient cells.

[0192] HuRi (Human Reference Protein Interaction Mapping Project) database analysis To identify proteins that interact with our key proteins and predict their roles and pathways in recipient cells, we performed a network model analysis to explain the interactions between DEPs using the HuRi (Human Reference Protein Interactome Mapping Project) database for DEP28, a protein with specific characteristics in T-a3D-EVs identified above.

[0193] As a result, five proteins (S100A10, SDCP, ACTG1, GIPC1, and EIF4E) out of the 28 DEPs had high confidence scores. 3 0.9, and mapped to 113 interactors in the Huri database (Figure 21D). Search results for the five DEPs with interactors in the annotation database (ShinyGO v0.61) specifically identified various signaling pathways that may be activated in recipient cells (Figure 21D). One of these signaling pathways was the regulation of cyclin-dependent protein kinase activity, which supports the potent function of T-a3D-EVs (Figure 21E). Furthermore, among the DEPs observed in the T-a3D-EVs of the present invention, SDCBP is a protein that primarily interacts with various proteins, and this protein is known to be involved in immune regulation, exosome biogenesis, and tumorigenesis. In conclusion, the specific proteins discovered in the T-a3D-EVs support the results confirmed by animal and cell experiments.

[0194] Although the present invention has been described with reference to the preferred embodiments, various modifications and variations can be made without departing from the spirit and scope of the invention, and the appended claims include such modifications and variations that fall within the spirit and scope of the invention. [Brief explanation of the drawings]

[0195] [Figure 1] 1A and 1B are diagrams showing the process of 3D culture of mesenchymal stem cells according to the method of the present invention, showing the process of cell aggregate formation (FIG. 1A) and 3D culture using a rotary stirrer (FIG. 1B). [Figure 2] FIG. 1 shows the exosome yield under each culture condition. [Figure 3] FIG. 1 shows the change in PDI value due to TGF-β treatment, showing that a single peak was observed under 3D shaking culture conditions with TGF-β treatment. [Figure 4]Figure 4A shows the effect of TGF-β on T cell proliferation. After inducing PBMC proliferation using PHA, the T cell inhibitory effects of a negative control (untreated group), a positive control (MSC-treated group), exosomes cultured under 3D shaking conditions only (3D-EV), and exosomes obtained by adding TGF-β3 to the culture medium under 3D shaking conditions (T-3D-EV) were examined (Figure 4A). The results confirmed that exosomes obtained under 3D shaking conditions with TGF-β treatment (T-3D-EV) had the most pronounced T cell inhibitory effect (Figures 4B and 4C). The exosomes obtained by the method of the present invention exhibited enhanced functionality in addition to improved yield. [Figure 5] Figure 5A shows the results of dynamic light scattering (DLS) analysis to examine exosome size. Figure 5B shows the results of transmission electron microscopy (TEM) to examine the morphology and structure of exosomes. Figure 5C shows the results of Western blotting analysis to confirm the expression of CD9, CD63, Flotillin-1, and Alix. Figure 5D shows the results of immunophenotyping of the exosome surface by flow cytometry. Figure 5E shows the results of enzyme-linked immunosorbent assay (ELISA) to confirm the TGF-β1 content of the produced exosomes. [Figure 6] The photograph (left) shows the results of transwell migration analysis confirming the increase in cell migration ability of human fibroblasts (NHDF) following administration of exosomes, and the graph (right) shows the relative degree of staining quantified using Image J. [Figure 7] Figure 7 shows the results of administering exosomes to an animal model with wounds created with a biopsy punch and examining the changes in wound healing over time. Figure 7A shows photographs of the wound site taken at regular intervals after administering exosomes to an animal model with wounds created with a biopsy punch. Figure 7B is a graph showing the size of the wound site shown in Figure 7A. [Figure 8] Exosomes were administered to an animal model in which a wound was created using a biopsy punch, and the wound healing ability was examined over time. Histological analysis of the wound site was performed 9 days after the wound was induced. [Figure 9] The results confirmed that the LPS-induced inflammatory response in Raw264.7 cells was significantly reduced by administration of exosomes. [Figure 10] The results confirmed that the concentrations of inflammatory cytokines TNF-α and IL-6 were significantly reduced in the supernatant of the culture medium of Raw264.7 cells cultured with both LPS and exosomes. [Figure 11] FIG. 1 shows the results of confirming the TNF-α and IL-6 reducing effect of exosome administration in a mouse model of endotoxemia caused by LPS toxin. [Figure 12] Figure 12 shows the results of examining cell proliferation after treating SV-HUC-1 (human urothelial cells) with exosomes at various concentrations. Figure 12A is a graph showing the cell proliferation rate after treating SV-HUC-1 with exosomes at various concentrations, and Figure 12B shows the results of examining the expression levels of P-AKT and P-ERK, which are associated with cell proliferation. [Figure 13] Figure 13 shows the results of transwell migration assays confirming the increased cell migration ability of human urothelial cells (SV-HUC-1) following exosome administration. Figure 13A shows a photograph visually confirming cells that had migrated to the opposite side of the transwell using crystal violet staining. Figure 13B shows a graph of the relative cell confluency shown in Figure 13A using Image J. [Figure 14] Fig. 1 shows an outline of the study design for producing an interstitial cystitis / bladder pain syndrome (IC / BPS) induced mouse model and evaluating the therapeutic effect of exosome administration. [Figure 15]These figures show the morphology and degree of inflammation of bladder tissue after exosome administration in an interstitial cystitis / bladder pain syndrome (IC / BPS)-induced mouse model. Figure 15A shows the results of H&E staining of bladder tissue from an IC / BPS mouse model, Figure 15B shows the results of Masson's trichrome staining, Figure 15C shows the results of toluidine blue staining, and Figure 15D is a graph showing the results of confirming the degree of fibrosis and mast cell infiltration using these staining methods. [Figure 16] In a mouse model of interstitial cystitis / bladder pain syndrome (IC / BPS), mRNA was extracted from bladder tissue extracted after exosome administration, and the expression levels of inflammation-related cytokines (TNFα, IL6) (Figure 16A), urothelial markers (UPK1A, UPK1B, UPK2) (Figure 16B), and genes expressed in IC / BPS (KLRB1, PSMB9, ITGAL) (Figure 16C) were confirmed. [Figure 17] FIG. 1 shows the results of confirming the effect of exosome administration on the recovery of intravesical pressure and micturition cycle in an interstitial cystitis / bladder pain syndrome (IC / BPS)-induced mouse model. [Figure 18] FIG. 1 shows the results of confirming the effect of exosome administration on the recovery of intravesical pressure and micturition cycle in an interstitial cystitis / bladder pain syndrome (IC / BPS)-induced mouse model. [Figure 19] Figure 19 shows the results of protein analysis of each exosome. Figure 19A shows the results of quantitative analysis of proteins contained in each exosome. Figure 19B shows a graph showing the abundance distribution of total proteins contained in each exosome. Figure 19C shows the results of gene ontology (GO) analysis of each exosome. Figure 19D shows the results of comparing the EV protein bodies discovered in this study with the dataset reported in Vesiclepedia. [Figure 20]Figure 20A shows proteins that are differentially expressed in the T-a3D-EV sample, which is the exosome of the present invention, among the four clusters derived by clustering analysis. Figure 20B shows the results of principal component analysis, showing the degree of separation between each exosome group as a discrimination index. Figure 20C shows the results of a two-way comparison method to determine the number of differentially expressed proteins (DEPs) depending on the culture conditions (2D and 3D) and the presence or absence of TGF-β3 treatment. [Figure 21] Figure 21A shows the gene sets, normalized enrichment scores (NES), and p-values ​​enriched in the PI3K-AKT signaling pathway and integrin 1 pathway of the exosome T-a3D-EV of the present invention by gene set enrichment analysis (GSEA). Figure 21B shows the enriched gene groups divided into the PI3K-AKT signaling pathway and integrin 1 pathway by gene set enrichment analysis (GSEA). Figure 21C shows the Venn diagrams generated by two-way comparison analysis (T-a3D-EV / 2D-EV, a3D-EV / 2D-EV, and T-a3D-EV / a3D-EV) showing the number of differentiated or shared proteins between each exosome. Among these, the Gene Ontology (GO) analysis results for DEP28, a region shared between T-a3D-EV / 2D-EV and T-a3D-EV / a3D-EV. Figure 21D shows that five of the 28 DEPs (Dysregulated Epitopes) with specific characteristics in the exosome T-a3D-EV of the present invention (S100A10, SDCP, ACTG1, GIPC1, and EIF4E) were mapped with high confidence scores to 113 interactors in the Huri database. Figure 21E shows the biological functions of the exosome T-a3D-EV of the present invention and its characteristics related to the regulation of cyclin-dependent protein kinase activity.

Claims

1. A stem cell-derived extracellular endoplasmic reticulum that highly expresses COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF-β1, and TGM2 proteins, the stem cell-derived extracellular vesicles express the protein at a level 40% or more higher than that of stem cell-derived extracellular vesicles cultured in two dimensions or stem cell-derived extracellular vesicles cultured in three dimensions without adding TGF-β to the culture medium; The stem cell-derived extracellular vesicle is characterized in that the stem cell is a mesenchymal stem cell.

2. The stem cell-derived extracellular vesicles are The stem cell-derived extracellular vesicle according to claim 1, characterized by highly expressing TGF-β1.

3. The stem cell-derived extracellular vesicles are TGF-β1 50-1,000 pg / 1×10 ９ The stem cell-derived extracellular vesicle according to claim 2, characterized in that it is expressed in particulate amounts.

4. A method for producing the stem cell-derived extracellular vesicles according to claim 1, comprising: (a) culturing stem cells isolated from a subject to form cell aggregates; and (b) three-dimensionally culturing the cell aggregates in a culture medium containing TGF-β3.

5. 5. The method for producing stem cell-derived extracellular vesicles according to claim 4, wherein step (a) is performed by suspension culture of stem cells in a multi-well culture vessel.

6. 5. The method for producing stem cell-derived extracellular vesicles according to claim 4, wherein step (b) is performed by orbital shaking culture of the cell aggregates in a suspended state.

7. The method for producing stem cell-derived extracellular vesicles according to claim 6, wherein the rotary shaking culture is carried out at a rotation speed of 50 to 70 rpm.

8. The method for producing stem cell-derived extracellular vesicles according to claim 4, wherein the stem cell-derived extracellular vesicles have an average diameter of 30 to 150 nm.

9. A pharmaceutical composition for preventing or treating inflammatory diseases or autoimmune diseases, comprising the stem cell-derived extracellular vesicles of claim 1 as an active ingredient.

10. The inflammatory disease or autoimmune disease includes cystitis, rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic fibrosing alveolitis, polymyositis, dermatomyositis, localized scleroderma, systemic cutaneous sclerosis, colitis, inflammatory bowel disease, Sjögren's syndrome, Raynaud's phenomenon, Behcet's disease, Kawasaki's disease, primary biliary sclerosis, and primary sclerosing cholangitis.

10. The pharmaceutical composition according to claim 9, wherein the disease is selected from the group consisting of ulcerative colitis, graft-versus-host disease (GVHD), and Crohn's disease.

11. The pharmaceutical composition according to claim 10, wherein the cystitis is at least one selected from the group consisting of interstitial cystitis, chronic cystitis, and ketamine-induced cystitis.

12. A pharmaceutical composition for wound healing comprising the stem cell-derived extracellular vesicles of claim 1 as an active ingredient.

13. 10. Use of the stem cell-derived extracellular vesicles of claim 1 for the manufacture of a pharmaceutical composition for the prevention or treatment of inflammatory diseases or autoimmune diseases.

14. A method for preventing or treating an inflammatory disease or an autoimmune disease, comprising administering the stem cell-derived extracellular vesicles of claim 1 to a non-human subject in need thereof.

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