Mesenchymal stem cells, extracellular vesicles isolated therefrom, and their uses

Abstract

The present invention relates to mesenchymal stem cells, extracellular vesicles isolated therefrom, and uses thereof. The mesenchymal stem cells of the present invention exhibit the characteristics of increasing the expression level of one or more markers selected from SPP1, PLAU, ITGA6, CENPI, and BUB1 and / or decreasing the expression level of the PTGS2 marker, thereby significantly reducing the amount of inflammatory cytokines such as TNF-α and IL-6, and are therefore effective in preventing, alleviating, improving, or treating various inflammatory diseases. In particular, when the mesenchymal stem cells of the present invention or the extracellular vesicles derived from the stem cells isolated therefrom are administered to an animal model of interstitial cystitis / bladder pain syndrome (IC / BPS), they restore the bladder inner wall that has collapsed during the IC / BPS induction process and alleviate the degree of inflammation, thereby having excellent therapeutic efficacy against interstitial cystitis / bladder pain syndrome. In addition, the mesenchymal stem cells of the present invention or the extracellular vesicles derived from the stem cells isolated therefrom have excellent wound healing effects and can be usefully used for wound healing.

Description

Detailed description of the invention

[0001] [Technical Field] This invention relates to mesenchymal stem cells, extracellular vesicles isolated therefrom, and their uses.

[0002] [Background technology] Generally, the inflammatory response is a biological defense mechanism that repairs and regenerates damage caused by invasive factors that result in structural changes in the cells and tissues of the body. This response involves the action of local blood vessels, various tissue cells in body fluids, and immune cells. While a normal inflammatory response induced by invading bacteria is a defense system that protects the body, abnormally excessive inflammatory responses can lead to a variety of diseases, collectively known as inflammatory diseases. These inflammatory diseases are those in which various inflammatory mediators secreted from target cells activated by external stimuli amplify and prolong inflammation, threatening human life. They include acute inflammation, diseases of the bladder such as cystitis, diseases of the joints such as rheumatoid arthritis, skin diseases that manifest as psoriasis, and allergic inflammatory diseases such as bronchial asthma.

[0003] In particular, interstitial cystitis (IC) is a chronic bladder disease of unknown cause characterized by pain, such as pelvic pain, and lower urinary tract symptoms (LUTS), such as increased frequent urination / urinary urgency. More recent terminology, to collectively disclose this complex symptom, includes painful bladder syndrome (PBS) ([MacDiarmid SA et al. Rev Urol 2007;9(1):9-16]) or bladder pain syndrome (BPS) ([(van der Merve et al. European Urology 53(2008)60-67]) along with IC, i.e., it has evolved into IC / BPS or IC / PBS / BPS.

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

[0005] The aforementioned IC / PBS / BPS has no single identified etiology and primarily causes hyperalgesia, chronic bladder pain, and urinary dysfunction (Forrest JB et al. Clinical Courier 2006;24(3):1-8).

[0006] On the other hand, mesenchymal stem cells (MSCs) are pluripotent progenitor cells that have the ability to differentiate into mesenchymal tissue lineages. These cells are emerging as a new alternative for treating autoimmune diseases because they can regulate adaptive and innate immune responses in a variety of cells and mediate potential immunomodulatory effects. In addition, these cells are known to have immunosuppressive and anti-inflammatory effects (European Patent Registration No. 02298861, US Published Patent No. 2012-0269774), as well as to suppress T cell activation and proliferation (Li ZJ et al., PloS ONE 8(10):77159, 2013).

[0007] However, until now, only the general anti-inflammatory effects of MSCs have been publicly known, and MSC stem cell therapies optimized to have more potent effects against inflammation-related diseases are still underdeveloped. Therefore, there is an urgent need for the development of MSC therapies suitable for the prevention or treatment of inflammatory diseases.

[0008] Therefore, the inventors diligently conducted research on MSC stem cells optimized to have an even stronger effect against inflammation-related diseases. Through this research, they confirmed that increasing the expression level of one or more markers selected from SPP1, PLAU, ITGA6, CENPI, and BUB1, and decreasing the expression level of the PTGS2 marker in mesenchymal stem cells (MSCs) is effective in preventing, alleviating, improving, or treating a variety of inflammatory diseases, including interstitial cystitis, and thus completed the present invention.

[0009] [Summary of the Invention] [Problems the invention aims to solve] The object of the present invention is to provide mesenchymal stem cells that are effective in preventing, alleviating, improving, or treating a variety of inflammatory diseases, including interstitial cystitis.

[0010] Another object of the present invention is to provide a composition for the prevention or treatment of inflammatory diseases or autoimmune diseases, comprising the mesenchymal stem cells or extracellular vesicles isolated therefrom as an active ingredient.

[0011] Another object of the present invention is to provide a wound healing composition containing the mesenchymal stem cells or extracellular vesicles isolated therefrom as an active ingredient.

[0012] [Means for solving the problem] To achieve the above objectives, the present invention provides mesenchymal stem cells exhibiting one or more characteristics selected from (A) increased expression levels of one or more markers selected from SPP1, PLAU, ITGA6, CENPI, and BUB1; and (B) decreased expression levels of the PTGS2 marker.

[0013] According to one preferred embodiment of the present invention, the mesenchymal stem cells may exhibit increased expression levels of the SPP1, PLAU, ITGA6, CENPI, and BUB1 markers.

[0014] According to a preferred embodiment of the present invention, the mesenchymal stem cells are produced by a method including: (1) a step of forming an embryoid body (EB) by culturing totipotent stem cells isolated from a subject; (2) a step of forming a spheroid by three-dimensional culturing of the embryoid body in a bioreactor under microgravity; and (3) a step of differentiating the spheroid into mesenchymal stem cells by adhering and culturing the spheroid in a culture vessel coated with an adhesive polymer on the culture surface.

[0015] According to a preferred embodiment of the present invention, the totipotent stem cells may be embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC).

[0016] According to a preferred embodiment of the present invention, the totipotent stem cells may be induced pluripotent stem cells.

[0017] According to a preferred embodiment of the present invention, the step (1) may be performed by three-dimensional culturing of the totipotent stem cells in a multi-well culture vessel.

[0018] According to a preferred embodiment of the present invention, the step (1) may further include a step of inducing cell aggregation through centrifugation during the three-dimensional culturing.

[0019] According to a preferred embodiment of the present invention, the microgravity in the step (2) may be induced by a microgravity simulator that cancels the gravity applied to the bioreactor by rotating the bioreactor.

[0020] According to a preferred embodiment of the present invention, the step (2) may be performed by culturing for 3 to 8 days while rotating the microgravity simulator at 15 rpm to 80 rpm.

[0021] According to a preferred embodiment of the present invention, the step (2) can be performed by rotating the microgravity simulator starting from 40 rpm to 60 rpm and increasing it by 5 rpm every day.

[0022] According to a preferred embodiment of the present invention, the adhesive polymer can be any one selected from hyaluronic acid, alginate, heparin, fucoidan, cellulose, dextran, chitosan, albumin, fibrin, collagen, and gelatin.

[0023] According to a preferred embodiment of the present invention, the adhesive polymer can be gelatin.

[0024] In addition, in order to achieve other objects of the present invention, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or autoimmune diseases containing the mesenchymal stem cells or extracellular vesicles isolated therefrom as an active ingredient.

[0025] 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 fibrous alveolitis, polymyositis, dermatomyositis, focal scleroderma, systemic scleroderma, colitis, inflammatory bowel disease, Sjögren's syndrome, Raynaud's phenomenon, Behçet's disease, Kawasaki's disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, graft-versus-host disease (GVHD), or Crohn's disease.

[0026] According to one preferred embodiment of the present invention, the cystitis may be one or more selected from interstitial cystitis, chronic cystitis, and ketamine-induced cystitis.

[0027] Furthermore, in order to achieve yet another objective of the present invention, the present invention provides a wound healing pharmaceutical composition comprising the mesenchymal stem cells or extracellular vesicles isolated therefrom as an active ingredient.

[0028] [Effects of the invention] The mesenchymal stem cells of the present invention exhibit increased expression levels of one or more markers selected from SPP1, PLAU, ITGA6, CENPI, and BUB1, and / or decreased expression levels of the PTGS2 marker, thereby significantly reducing the levels of inflammatory cytokines such as TNF-α and IL-6, and are therefore effective in preventing, alleviating, improving, or treating a variety of inflammatory diseases.

[0029] In particular, when administered to an animal model of interstitial cystitis / bladder pain syndrome (IC / BPS), the mesenchymal stem cells of the present invention, or stem cell-derived extracellular vesicles isolated therefrom, restore the bladder wall damaged during the IC / BPS induction process and alleviate the degree of inflammation, thus exhibiting excellent therapeutic efficacy against interstitial cystitis / bladder pain syndrome.

[0030] Furthermore, the mesenchymal stem cells of the present invention, or the stem cell-derived extracellular vesicles isolated therefrom, exhibit excellent wound healing effects and can therefore be usefully utilized for wound healing.

[0031] [Brief explanation of the drawing] Figure 1 is a schematic diagram summarizing the protocol of the present invention for differentiating iPSCs from MSCs.

[0032] Figure 2 shows the morphology of embryoid bodies (EBs) formed on Aggrewell.

[0033] Figure 3 shows the morphology of spheroids formed through a BAM (Bio Array Matrix) microgravity biological culture device (top) and the staining results using OCT4 and DAPI antibodies (bottom).

[0034] Figure 4 shows the external shape of mesenchymal stem cells derived from spheroids, confirming that they take on a spindle shape after passage (right side).

[0035] Figure 5 shows the external shape of iMSCs separated by the method of the present invention at each passage.

[0036] Figure 6 shows the cumulative cell proliferation curve of mesenchymal stem cells differentiated by the method of the present invention, and indicates the CPD (Cummulative Population Doubling) (Figure 6a), doubling time (Figure 6b), and log cell number (Figure 6c) for each passage, respectively.

[0037] Figure 7 shows the results of confirming the expression of cell surface markers in mesenchymal stem cells differentiated by the method of the present invention using FACS analysis.

[0038] Figure 8 shows the results of confirming, using immunocytochemical staining, that induced pluripotent stem cells lost their totipotency and differentiated into cells expressing mesenchymal stem cell markers through the method of the present invention.

[0039] Figure 9 is a schematic diagram showing the experimental procedure for confirming the inflammatory control effect of stem cells differentiated by the present invention method in inflammation-induced cells using LPS.

[0040] Figure 10 shows the results of confirming the expression of inflammatory markers via RT-PCR.

[0041] Figure 11 shows the results of staining cells in which inflammation was induced by LPS treatment using immunocytochemistry (left side) and the results of counting the TRAP-stained cells (right side).

[0042] Figure 12 shows the results of measuring the size (left) and concentration (right) of exosomes isolated from mesenchymal stem cells according to the present invention.

[0043] Figure 13a is a photograph showing the degree of cell migration over time after treating NHDF cells that have been scratched with exosomes isolated from mesenchymal stem cells according to the present invention, and Figure 13b is a graph showing the degree of cell migration quantified.

[0044] Figure 14a shows the results of a network analysis using the database-based String-db tool to confirm the protein-protein interactions of each upward regulatory gene contained in the mesenchymal stem cells of the present invention, and Figure 14b shows the results of a network analysis of each downward regulatory gene.

[0045] Figures 15a to 15d are Venn diagrams generated using the predicted functions of each upward regulatory gene, which was analyzed using DAVID analysis for each of the mesenchymal stem cells contained in the present invention.

[0046] Figures 16a to 16d are Venn diagrams generated using the predicted functions of each downward regulatory gene, which was subjected to DAVID analysis for each of the mesenchymal stem cells contained in the present invention.

[0047] Figure 17a shows the morphology and degree of inflammation of bladder tissue after administration of mesenchymal stem cells (iMSCs) of the present invention in a mouse model of interstitial cystitis / bladder pain syndrome (IC / BPS). Figure 17a shows the results of staining the bladder tissue of the IC / BPS mouse model with H&E, Masson's trichrome, and toluidine blue, respectively. Figure 17b is a graph showing the degree of fibrosis (%) and mast cell infiltration confirmed through the aforementioned staining.

[0048] Figure 18 shows the results of extracting mRNA from bladder tissue after administration of mesenchymal stem cells (iMSCs) of the present invention in a mouse model of interstitial cystitis / bladder pain syndrome (IC / BPS), and confirming the expression levels of inflammation-related cytokines (TNFα, IL6) (Figure 18a), urothelial markers (UPK1A, UPK1B, UPK2) (Figure 18b), and genes expressed in IC / BPS (KLRB1, PSMB9, ITGAL) (Figure 18c).

[0049] [Modes for carrying out the invention] The present invention will be described in detail below.

[0050] One aspect of the present invention relates to mesenchymal stem cells having one or more characteristics selected from (A) increased expression levels of one or more markers selected from SPP1 (secreted phosphoprotein 1; osteopontin), PLAU (Plasminogen Activator, Urokinase), ITGA6 (integrin subunit alpha 6), CENPI (Centromere Protein I), and BUB1 (Budding Uninhibited By Benzimidazoles 1 Homolog; Mitotic checkpoint serine / threonine-protein kinase); and (B) decreased expression levels of the PTGS2 (Prostaglandin-Endoperoxide Synthase 2, or COX2;) marker.

[0051] In the present invention, the terms "increased expression level" or "decreased expression level" mean that the expression level of a specific marker expressed by mesenchymal stem cells has increased or decreased to a measurable degree compared to conventional mesenchymal stem cells, preferably general Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs). Specifically, this means an increase or decrease of 40% or more in expression, more specifically an increase or decrease of 50% or more, more specifically an increase or decrease of 60% or more, even more specifically an increase or decrease of 80% or more, and most specifically an increase or decrease of 100% or more.

[0052] In this invention, the term "stem cell" refers collectively to undifferentiated cells that are in the stage before differentiation into the various cells that make up a tissue, and which have the ability to differentiate into specific cells under specific differentiation stimuli (environments). Unlike differentiated cells whose cell division has stopped, stem cells are characterized by their ability to self-renew by producing cells identical to themselves through cell division, and by their ability to differentiate into various cells depending on the nature of the differentiation stimuli applied, thus possessing differentiation flexibility (plasticity).

[0053] In this invention, the term "mesenchymal stem cells" refers to stem cells that have multipotency and are capable of differentiating into adipocytes, osteocytes, chondrocytes, muscle cells, nerve cells, and cardiomyocytes. Mesenchymal stem cells can be identified through their spiral morphology and the degree of expression of basic cell surface markers CD73(+), CD105(+), CD34(-), and CD45(-), and they also have the function of regulating immune responses along with their multipotency.

[0054] The mesenchymal stem cells of the present invention can regulate or increase the expression level of SPP1 to at least three times, more specifically 3.0 to 4.0 times, more specifically 3.50 to 4.00 times, and more specifically 3.80 to 3.90 times, compared to conventional mesenchymal stem cells, preferably general hWJ-MSCs (control group).

[0055] Furthermore, the mesenchymal stem cells of the present invention can regulate or increase the expression level of PLAU by at least 1.5 times, more specifically 1.5 to 2.2 times, more specifically 1.80 to 2.10 times, and even more specifically 1.90 to 2.00 times, compared to conventional mesenchymal stem cells, preferably general hWJ-MSCs (control group).

[0056] Furthermore, the mesenchymal stem cells of the present invention can adjust or increase the expression level of ITGA6 to at least 2.1 times, more specifically 2.1 to 3.0 times, more specifically 2.30 to 2.60 times, and even more specifically 2.40 to 2.50 times, compared to conventional mesenchymal stem cells, preferably general hWJ-MSCs (control group).

[0057] Furthermore, the mesenchymal stem cells of the present invention can regulate or increase the expression level of CENPI to at least 1.5 times, more specifically 1.5 to 2.0 times, more specifically 1.60 to 1.90 times, and even more specifically 1.70 to 1.80 times, compared to conventional mesenchymal stem cells, preferably general hWJ-MSCs (control group).

[0058] Furthermore, the mesenchymal stem cells of the present invention can regulate or increase the expression level of BUB1 to at least 1.4 times, more specifically 1.4 to 2.1 times, more specifically 1.50 to 1.90 times, and even more specifically 1.65 to 1.75 times, compared to conventional mesenchymal stem cells, preferably general hWJ-MSCs (control group).

[0059] Furthermore, the mesenchymal stem cells of the present invention can be used to downwardly regulate or reduce the expression level of PTGS2 by at least 2.0 times, more specifically 2.0 to 3.0 times, more specifically 2.30 to 2.90 times, and even more specifically 2.50 to 2.70 times, compared to conventional mesenchymal stem cells, preferably general hWJ-MSCs (control group).

[0060] The mesenchymal stem cells of the present invention have the characteristic of increasing the expression level of one or more markers selected from SPP1, PLAU, ITGA6, CENPI, and BUB1; and / or decreasing the expression level of the PTGS2 marker, thereby significantly reducing the amount of inflammatory cytokines such as TNF-α and IL-6, and can prevent, alleviate, improve, or treat various inflammatory diseases, preferably cystitis, especially interstitial cystitis. Furthermore, the mesenchymal stem cells of the present invention significantly increase cell migration and have excellent wound healing effects.

[0061] In one embodiment, the mesenchymal stem cells of the present invention may exhibit increased expression levels of the SPP1, PLAU, ITGA6, CENPI, and BUB1 markers.

[0062] In one embodiment, the mesenchymal stem cells of the present invention may be produced by a method comprising: (1) forming an embryoid body (EB) by culturing fully differentiated stem cells isolated from a target organism; (2) forming a spheroid by three-dimensionally culturing the embryoid body in a bioreactor under microgravity; and (3) differentiating the spheroid into mesenchymal stem cells by attaching and culturing it in a culture vessel coated with an adhesive polymer on its culture surface.

[0063] In the present invention, the term "pluripotent stem cell" refers to a cell that has developed from a fertilized egg and is capable of differentiating into all cells that constitute the endoderm, mesenchyme, and ectoderm. In specific examples of the present invention, the pluripotent stem cells used in the present invention are embryonic stem cells (ESCs), embryonic germ cells, embryonic carcinoma cells, or induced pluripotent stem cells (iPSCs), more specifically embryonic stem cells or induced pluripotent stem cells, and most specifically induced pluripotent stem cells.

[0064] In this invention, the term "induced pluripotent stem cell" refers to a type of pluripotent stem cell artificially derived by inserting specific genes associated with an undifferentiated or pluripotent phenotype into non-pluripotent cells (e.g., somatic cells). Induced pluripotent stem cells are considered in the industry to have the same phenotype, physiological characteristics, and developmental characteristics as naturally occurring pluripotent stem cells such as embryonic stem cells, in terms of stem cell gene and protein expression, chromosome methylation, doubling time, embryoid body formation, teratoma formation, viable chimera formation, hybridization, and differentiation.

[0065] In the present invention, the term "stem cell differentiation" includes not only the complete differentiation of undifferentiated stem cells into specific cells, but also the formation of precursor cells, which are formed in an intermediate stage before complete differentiation of stem cells into specific cells.

[0066] In one embodiment, the cell culture medium used in each of the above steps is a mixture for in vitro cell growth and proliferation that contains elements essential for cell growth and proliferation, such as sugars, amino acids, various nutrients, and minerals. Additional components that may be included in the cell culture medium include, for example, 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, calcium D-pantothenate, folic acid, and riboflavin. This product contains, but is not limited to, 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, sodium hypoxanthine, linolenic acid, lipoic acid, putrescine 2HCl, and thymidine.

[0067] In one embodiment, the cell culture medium may be artificially manufactured and used, or it may be purchased and used from a commercially available source. 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).

[0068] In one embodiment, step (1) can be carried out by three-dimensionally culturing the fully differentiated stem cells in a multi-well culture vessel.

[0069] In this invention, the term "three-dimensional culture" is a concept opposite to two-dimensional culture, and refers to culturing target cells in a state of floating in a culture medium without fixing them to a substrate or the like. Therefore, the term "three-dimensional culture" is used with the same meaning as "suspension culture." Adhesion-dependent stem cells undergo cell aggregation during suspension culture, and cells that are not included in such aggregation and float alone undergo apoptosis and die. Therefore, an environment that matches the adhesion characteristics of the cells must be created. According to this invention, by suspension culture of pluripotent stem cells in a multi-well system having multiple wells, pluripotent stem cell aggregates, i.e., embryoid bodies (EBs), with a diameter corresponding to the size of the wells, are formed in proportion to the number of wells. Therefore, in step (1) of this invention, a large number of standardized embryoid bodies having the same size and shape can be obtained.

[0070] In one embodiment, the multiwell culture vessel may be a microwell plate having a size of 350 μm × 350 μm to 450 μm × 450 μm per well.

[0071] In one example, the suspension culture was performed at a rate of 0.5 × 10 per well in the multi-well culture vessel. 5 Individual to 1.5×10 5 pieces, preferably 0.7 × 10 5 Individual to 1.3×10 5 pieces, more preferably about 0.9 × 10 5 Individual to 1.1×10 5 This can be done by dispensing individual cells.

[0072] In one embodiment, step (1) may further include the step of inducing cell aggregation by centrifugation during the suspension culture.

[0073] In this invention, the term "cell aggregation" refers to the formation of three-dimensional cell aggregates through self-aggregation of cells cultured in an environment such as suspension culture that allows for three-dimensional growth rather than monolayer growth. The cell aggregates produced as a result of three-dimensional culture provide an environment similar to in vivo tissues derived from stem cells, and may be spherical or non-spherical in shape depending on their size and the number of self-aggregated cells.

[0074] In one embodiment, the microgravity in step (2) can be induced by a microgravity simulator that counteracts the gravity applied to the biological reactor by rotating the biological reactor.

[0075] In this invention, the term "microgravity" means that gravity is absent, present only at a level below measurable, or present only to the extent that no biological or physiological effects of gravity are observed, specifically 1 × 10⁻⁶ 6 It refers to an environment with a minimum of g. Therefore, the term "microgravity" can also be expressed as "weightlessness."

[0076] In the present invention, the term "microgravity simulator" refers to a device that induces a microgravity environment by artificially counteracting gravity within a normal gravity environment or an environment where significant gravity exists. Examples of such microgravity simulators include clinostats, RPMs (Random Positioning Machines), and RWVs (Rotating Wall Vessels), but the invention is not limited to these. Any device that can counteract gravity for a specified time within the culture environment of the biological reactor of the present invention by applying an appropriate external force can be used without limitation.

[0077] In one embodiment, the microgravity simulator is a clinostat. A clinostat is a device that is coupled to a culture vessel such as a biological reactor and rotates while continuously changing direction randomly or according to an instructed (input) pattern, constantly changing its three-dimensional orientation, thereby causing continuous fluctuations in the direction of gravity and counteracting gravity.

[0078] In one embodiment, step (2) can be carried out by culturing the microgravity simulator for 3 to 8 days while rotating it at 15 rpm to 80 rpm, preferably 40 rpm to 80 rpm, more preferably 4 to 7 days, and even more preferably 5 days.

[0079] Preferably, step (2) can be carried out by rotating the microgravity simulator starting at 40 rpm to 60 rpm and increasing by 5 rpm each day. More preferably, it can be cultured for 5 days starting at 50 rpm and increasing by 5 rpm each day.

[0080] In the present invention, the term "bioreactor" means a biological sample culture apparatus or system that includes a culture space for creating a biologically active culture environment and a series of mechanical devices that operate in conjunction with it.

[0081] In step (2) above, spheroids are formed by three-dimensional culture of the embryoid bodies generated in step (1) in a bioreactor under microgravity. A spheroid is a spherical cell aggregate, but it does not need to be geometrically perfect sphere.

[0082] After carrying out three-dimensional suspension culture twice in steps (1) and (2) above, the spheroids formed through this process can be differentiated into mesenchymal stem cells by attaching and culturing them in a culture vessel coated with an adhesive polymer.

[0083] In the present invention, the term "polymer" refers to a synthetic or natural polymer compound in which the same or different types of monomers are continuously bonded together. Thus, polymers include homopolymers (polymers polymerized from one type of monomer) and hybrid polymers produced by polymerization of at least two different monomers, and hybrid polymers include both copolymers (polymers produced from two different monomers) and polymers produced from more than two different monomers.

[0084] In the present invention, the term "adhesive polymer" means a natural or artificial polymer that forms crosslinks through covalent or non-covalent bonds between the culture surface and cells or their aggregates (e.g., spheroids), thereby enabling the cells or their aggregates to remain attached to the bottom or sides of the culture vessel during cultivation.

[0085] In one embodiment, the adhesive polymer may be any one selected from hyaluronic acid, alginic acid, heparin, fucoidan, cellulose, dextran, chitosan, albumin, fibrin, collagen, and gelatin, and is preferably gelatin.

[0086] According to a specific embodiment of the present invention, the mesenchymal stem cells of the present invention show clear differences in gene marker expression characteristics compared to conventional mesenchymal stem cells, particularly general hWJ-MSCs (control group). As can be seen in the following example, the mesenchymal stem cells of the present invention show a significantly increased expression level of one or more markers selected from SPP1, PLAU, ITGA6, CENPI, and BUB1, and a significantly decreased expression level of the PTGS2 marker, compared to general hWJ-MSCs (control group).

[0087] Another aspect of the present invention relates to a composition for the prevention or treatment of inflammatory diseases or autoimmune diseases, comprising the mesenchymal stem cells or extracellular vesicles isolated therefrom as an active ingredient.

[0088] Since the mesenchymal stem cells of the present invention have already been described, a further description will be omitted to avoid excessive repetition.

[0089] In this invention, the term "extracellular vesicle" refers to a lipid bilayer vesicle having a diameter in the range of 30 nm to 1,000 nm that is secreted into the extracellular environment through the fusion of the polyspleen and the plasma membrane in various cells.

[0090] In one embodiment, the extracellular vesicles of the present invention may be separated from the culture medium of the mesenchymal stem cells through multiple centrifugations.

[0091] In one embodiment, the extracellular vesicles of the present invention have an average diameter of 100 nm to 250 nm, more specifically 150 nm to 220 nm, even more specifically 180 nm to 200 nm, and even more specifically 185 nm to 195 nm. Extracellular vesicles having such a fine diameter range are called exosomes.

[0092] In this invention, the term "prevention" means suppressing the occurrence of a disease or illness in an object that has not been diagnosed with having such a disease or illness, but is at risk of developing such a disease or illness.

[0093] In the present invention, the term “treatment” means (a) suppression of the development of a disease, illness or symptom; (b) alleviation of a disease, illness or symptom; or (c) elimination of a disease, illness or symptom. The compositions of the present invention play a role in suppressing, eliminating, or alleviating the development of symptoms of various inflammatory or autoimmune diseases caused by excessive or unwanted immune responses by efficiently suppressing T cell-mediated immune activity. Therefore, the compositions of the present invention may constitute a treatment composition for these diseases on their own, or they may be administered together with other pharmacological components having therapeutic effects against inflammatory or autoimmune diseases and applied as adjuncts to the treatment of said diseases. Accordingly, the terms “treatment” or “therapeutic agent” as used herein include the meaning of “adjunct” or “adjunct to treatment.”

[0094] In the present invention, the term "administration" means directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed within the subject's body, and has the same meaning as "transplantation" or "injection."

[0095] In the present invention, the term "therapeutic effective amount" means the amount of the composition contained in an amount sufficient to provide a therapeutic or preventive effect to an individual to whom the composition of the present invention is to be administered, and therefore includes the meaning of "preventive effective amount".

[0096] In the present invention, the term "subject" includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the subject of the present invention is humans.

[0097] In one embodiment, the composition of the present invention has preventive or therapeutic effects against a variety of inflammatory diseases. The inflammatory diseases to which the pharmaceutical composition of the present invention can be applied are not particularly limited, as long as they are known in the art as inflammatory diseases. Autoimmune or inflammatory diseases that can be prevented or treated with the pharmaceutical composition 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 fibrous alveolitis, polymyositis, dermatomyositis, focal scleroderma, systemic scleroderma, colitis, inflammatory bowel disease, Sjögren's syndrome, Raynaud's phenomenon, Behçet's disease, Kawachi disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, graft-versus-host disease (GVHD), and Crohn's disease, and are preferably cystitis.

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

[0099] In one embodiment of the present invention, when mesenchymal stem cells or their culture medium, or extracellular vesicles isolated therefrom, are administered to an anti-inflammatory cell model, the concentrations of TNF-α and IL-6 are significantly reduced, thus specifically confirming that the anti-inflammatory therapeutic effect is excellent.

[0100] Furthermore, in one embodiment of the present invention, it was specifically confirmed that administering the mesenchymal stem cells of the present invention to an animal model of interstitial cystitis / bladder pain syndrome (IC / BPS) in vivo resulted in the restoration of the bladder wall damaged during the IC / BPS induction process and the reduction of inflammation, demonstrating excellent therapeutic efficacy against interstitial cystitis / bladder pain syndrome.

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

[0102] Pharmaceutically acceptable carriers may further include, for example, oral or parenteral carriers. Oral carriers may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Parenteral carriers may include water, suitable oils, saline solutions, aqueous glucose and 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 propylparaben, and chlorobutanol. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. For other pharmaceutically acceptable carriers, please refer to the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0103] The composition of the present invention can be administered to mammals, including humans, by any method, for example, orally or parenterally. Parenteral administration methods are not limited to intravenous, intramuscular, intra-arterial, intramuscular, intradural, intradural, suprachoroidal injection, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal administration, and are preferably intravenous.

[0104] The pharmaceutical composition of the present invention can be formulated into oral or parenteral formulations via the above-described route of administration.

[0105] In the case of orally administered formulations, the compositions of the present invention can be formulated into dosage forms such as powders, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions using methods known to the art. For example, oral formulations can be obtained by compounding the active ingredient with a solid excipient, grinding the mixture, adding appropriate adjuvants, and then processing it into a granular mixture to obtain tablets or sugar-coated tablets. Examples of appropriate excipients may include sugars such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches such as corn starch, wheat starch, rice starch, and potato starch; celluloses such as cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose; and fillers such as gelatin and polyvinylpyrrolidone. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants. Furthermore, the pharmaceutical composition of the present invention may further contain an anti-coagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, and a preservative.

[0106] For parenteral formulations, dosage forms can be developed using methods known to the industry, including injections, ointments, creams, lotions, oils, gels, aerosols, and nasal inhalants. These dosage forms are described in the generally known prescription literature for all pharmaceutical chemistry (Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).

[0107] Preferably, the pharmaceutical composition of the present invention may be manufactured in any one form selected from the group consisting of oral preparations, injectable preparations, and ointments, and more preferably injectable preparations.

[0108] When the pharmaceutical composition of the present invention contains an effective amount of the mesenchymal stem cells or extracellular vesicles isolated therefrom, it can provide a preferable preventive, ameliorative or therapeutic effect on inflammatory diseases. As used herein, "effective amount" refers to an amount showing a greater response as compared with a negative control group, preferably an amount sufficient to ameliorate or treat an inflammatory disease, particularly interstitial cystitis.

[0109] The mesenchymal stem cells may be contained at 5×10 4 cell / ml to 2×10 5 cell / ml, preferably 7.5×10 4 cell / ml to 1.5×10 5 cell / ml, more preferably 8×10 4 cell / ml to 1.2×10 5 cell / ml. At this time, when the content of the mesenchymal stem cells is less than the lower limit value, although the cell viability is excellent, there is a possibility that the ameliorative or therapeutic effect on inflammatory diseases may not appear to the desired degree. On the other hand, when it exceeds the upper limit value, the ameliorative or therapeutic effect on inflammatory diseases may not increase or may be toxic as the concentration increases. On the other hand, as a result of in vitro experiments, when the concentration of the mesenchymal stem cells of the present invention is within the above range, a significant effect on the amelioration or treatment of inflammatory diseases appears and side effects such as cytotoxicity do not appear.

[0110] In addition, the extracellular vesicles isolated from the mesenchymal stem cells of the present invention are 5×10 8 particles / ml to 5×10 10 particles / ml, preferably 5×10 9 particles / ml to 5×10 10 particles / ml, more preferably 1×10 10 particles / ml to 2×10 10It may be contained in particles / ml. In this case, if the content of the stem cell-derived extracellular vesicles is below the lower limit, cell viability will be excellent, but the improvement or therapeutic effect of inflammatory diseases may not be as desired. On the other hand, if it exceeds the upper limit, the improvement or therapeutic effect of inflammatory diseases may not increase by the amount of the concentration increase, or it may be toxic. On the other hand, 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, a significant effect on the improvement or treatment of inflammatory diseases is observed, and no side effects such as cytotoxicity occur.

[0111] The effective amount of mesenchymal stem cells contained in the pharmaceutical composition of the present invention, or extracellular vesicles isolated therefrom, may vary depending on the form in which the composition is formulated.

[0112] 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 involving multiple doses over a long period. The pharmaceutical composition of the present invention can have different active ingredient contents depending on the severity of the disease.

[0113] The appropriate dosage of the pharmaceutical composition can be formulated in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, administration route, excretion rate, and response sensitivity. The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg / kg to 100 mg / kg based on an adult.

[0114] Another aspect of the present invention relates to a wound healing composition comprising the mesenchymal stem cells or extracellular vesicles isolated therefrom as an active ingredient.

[0115] Since the mesenchymal stem cells, extracellular vesicles isolated therefrom, pharmaceutical compositions, and effective amounts of the present invention have already been described, a further description will be omitted to avoid excessive repetition.

[0116] In one embodiment of the present invention, it was specifically confirmed that when mesenchymal stem cells of the present invention, or extracellular vesicles isolated therefrom, are administered to a cell model that has induced a wound, cell migration increases significantly (Figures 13a and 13b).

[0117] The mesenchymal stem cells of the present invention, or extracellular vesicles isolated therefrom, significantly increase cell migration and exhibit excellent wound healing effects, and therefore can be usefully utilized for wound healing.

[0118] Another aspect of the present invention relates to the therapeutic use of the mesenchymal stem cells or extracellular vesicles isolated therefrom.

[0119] Since the mesenchymal stem cells of the present invention, or the extracellular vesicles isolated therefrom, have already been described, a further description will be omitted to avoid excessive redundancy.

[0120] The aforementioned therapeutic use may be for the treatment of inflammatory diseases or autoimmune diseases, preferably inflammatory diseases.

[0121] The aforementioned inflammatory or autoimmune diseases may include cystitis, rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic fibrous alveolitis, polymyositis, dermatomyositis, focal scleroderma, systemic scleroderma, colitis, inflammatory bowel disease, Sjögren's syndrome, Raynaud's phenomenon, Behçet's disease, Kawachi disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, graft-versus-host disease (GVHD), or Crohn's disease.

[0122] The aforementioned cystitis may be one or more selected from interstitial cystitis, chronic cystitis, and ketamine-induced cystitis.

[0123] Another aspect of the present invention relates to a method for preventing, improving or treating an inflammatory or autoimmune disease, comprising the step of administering the mesenchymal stem cells or extracellular vesicles isolated therefrom to a subject in need;

[0124] Since the mesenchymal stem cells of the present invention, or the extracellular vesicles isolated therefrom, have already been described, a further description will be omitted to avoid excessive redundancy.

[0125] The aforementioned "subject" refers to a mammal that is the subject of prevention, improvement, treatment, observation, or experimentation, and is preferably a human or mammal that requires prevention, improvement, and / or treatment of an inflammatory disease or autoimmune disease.

[0126] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0127] <Examples> Experimental method Culture of a single colony Induced pluripotent stem cells (iPSCs) were cultured for one week in iPSC medium attached as single cells to 96-well plates coated with Matrigel (354234, Corning, USA) to generate single colonies. Each generated colony was then sequentially passaged into 24-well and 6-well dishes coated with Matrigel, followed by 1 × 10⁶ cells. 5 Once the cell count increased to a certain extent, it was used for spheroid experiments (Figure 1).

[0128] Spheroid generation using a BAM system iPSCs were placed in Aggrewell plates (34460, stemcell, Canada) in 1x10⁶ plates. 5After seeding to a certain extent and centrifugation by 300g for 5 minutes, the cells were cultured for 24 hours in a 5% CO2 incubator to generate embryoid bodies (EBs). After 24 hours, the generated EBs were carefully transferred to a bioreactor (CelVivo, Denmark), which was then mounted on a microgravity chamber (BAM system, CelVivo, Denmark) and rotated for 5 days. Rotation was started at 50 rpm and increased by 5 rpm daily (Figure 1).

[0129] Generation of induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) Spheroids grown in the BAM system were transferred to 6-well culture dishes coated with 0.1% gelatin and cultured in a medium of DMEM / F12 supplemented with 10% FBS and 1% P / S, with the medium changed every 2-3 days. After the cells emerged from the spheroids attached to the coated bottom, they were subcultured using Tryple when they reached 70-80% confluence. The first subgeneration was named P0 and subcultured until the cell shape became homogenous.

[0130] Confirmation of pluripotency and mesenchymal stem cells using immunocytochemistry (ICC) staining. Spheroids generated during the iPSC stage were partially removed from the bioreactor and fixed with 4% paraformaldehyde (PFA). iPSC MSCs made from iPSC spheroids were placed in a confocal dish (101350, SPL, Korea) at a density of 1 × 10⁶ 5After seeding with the specified concentration, the cells were fixed with 4% PFA when they reached 60%-70% confluence. The fixed spheroids and iPSC-MSCs were washed three times with DPBS for 5 minutes each, and then the surface was permeated with 0.3% Triton X-100 to allow good antibody permeability. Subsequently, they were washed three times with DPBS for 5 minutes each. The washed spheroids were cultured at room temperature for 1 hour in 3% BSA / PBS for blocking. After 1 hour, the 3% BSA / PBS was removed, and the primary antibodies (1:200) Anti-OCT4, Anti-SSEA4, and Anti-PDGFRβ were added, followed by incubation in the refrigerator for 12 hours. After 12 hours, they were removed at room temperature and washed three times with DPBS for 5 minutes each.

[0131] After washing with water, the samples were cultured at room temperature for 1 hour with the secondary antibody (1:200) goat anti-mouse 488. After 1 hour, the secondary antibody was removed, and the nuclei were stained with DAPI or Topro3 for 20 minutes, followed by three 5-minute washes with DPBS. Antifade Mounting Medium (H-1000, VECTOR LABORATORY, UK) was used to prevent the loss of fluorescence in the washed samples.

[0132] iPSC-MSC passage and cell proliferation curves Cell morphology was recorded from the first passage when the shape of the iPSC-MSC became homogenized, and the proliferation curve was plotted while calculating the number of cells. From P5 onwards, iPSC-MSCs were placed in 2 × 10⁶ 60 mm culture dishes. 5After seeding with the specified cell count, the cells were cultured for 5 days, with the culture medium changed once every 5 days. Cell proliferation was shown using three growth curves. First, the cell count for the cumulative population doubling level (CPD) was calculated as "CPD = log(later cell count / initial cell count) / log(2)". Next, the doubling time, which is the time it takes for cells to double in number, was calculated as "doubling time = culture days × log(2) / (log(later cell count) - log(initial cell count))". Subsequently, the cell count at the time of passage was calculated, and the logarithm of this value was calculated as "cell count = log(cell count)". The calculated values ​​are shown in line graphs, and AD-MSC (mesenchymal stem cells) and hWJ-MSC (human Wharton jelly-derived mesenchymal stem cells) were used as control groups.

[0133] Immunophenotyping using flow cytometry (FACS) analysis Cells in culture are LE TM Cells were separated using Express (10624013, Gibco, USA), centrifuged at 1,500 rpm for 5 minutes, the supernatant was removed, and the cells were suspended in FACS buffer (D-PBS containing 2% FBS). Primary reactions were then performed with mouse anti-CD34, mouse anti-CD45, mouse anti-CD73, and sheep anti-CD90 antibodies. These primary antibodies were diluted 1:500, and 200 μl was added to each cell, followed by incubation at 4°C for 30 minutes. Next, the cells were washed with D-PBS, centrifuged at 1,500 rpm for 5 minutes, the supernatant was removed, and 200 μl of either rabbit anti-mouse 488 or donkey anti-sheep PE, diluted 1:500, was added to each cell, followed by incubation at 4°C for 20 minutes. Subsequently, the stained cells were suspended in 500 μL of FACS buffer and flow cytometry analysis was performed using a FACS Calibur (Becton Dickinson, Heidelberg, Germany), followed by analysis using Cell Quest pro software.

[0134] Anti-inflammatory cell model To evaluate the anti-inflammatory capacity of the iPSC-MSCs of the present invention, Raw 264.7 cells, a macrophagee used in inflammatory cell models, were employed. Raw 264.7 cells were grown in α-MEM (Minimum Essential Medium) medium containing 10% FBS and 1% P / S. First, hWJ-MSCs (control group) or iPSC-MSCs (the present invention) were cultured in culture vessels. When the vessels reached 50%, fresh medium was supplied, and the cultures were incubated for 48 hours. After 48 hours, the condition medium was collected, filtered using a 0.20 μm syringe filter, and stored in a 4°C refrigerator.

[0135] Raw 264.7 cells, 3.75 × 10⁶ cells per well. 5 The cells were divided into 6-well culture dishes and inoculated according to the cell count. After 12 hours, when the cells had attached, the medium was replaced with 100% of the prepared condition medium. After another 12 hours, as shown in Figure 9, all groups, including the condition medium group, except for the control group, were treated with 200 ng / ml of LPS (lipopolysaccharide, Sigma, USA). The positive control group was treated with 1 μM of DEX (Dexamathasone, Peprotech, USA). After 7 hours, images were recorded, and after fixing the cells with 4% PFA, immunocytochemical staining was performed.

[0136] Total RNA isolation, RT-PCR Total RNA was extracted from Raw264.7 cells using the Labo Pass Kit and TRIzol (Cosmogenetech, Seoul, Korea) according to the manufacturer's instructions. Total RNA concentration was measured using a Nanodrop (ND1000) spectrophotometer (Nanodrop Technologies Inc., Wilmington DE, USA). cDNA was synthesized using 2 μg of total RNA and M-MLV reverse transcriptase (Promega) according to the manufacturer's instructions. RT-PCR reactions were analyzed on a 2% agarose gel after completion. The sequences of the primers used are listed in Table 1:

[0137] [Table 1]

[0138] Immunocytochemical staining The fixed cells were washed twice with PBS. Infiltration into the cell nuclei was performed using 0.3% Triton X-100 for 10 minutes, followed by washing with PBS. The cells were then incubated at room temperature for 1 hour with 10% normal goat serum to block nonspecific antibody binding. The cells were reacted with the primary antibody, TRAP mouse monoclonal antibody (Santacruz, USA), at 4°C for 12 hours. Afterward, the cells were washed three times with DPBS and reacted with the secondary antibody, Alexa-488 conjugated goat anti-mouse antibody, at room temperature for 1 hour. After 1 hour, the cells were incubated in DPBS, and the cell nuclei were reacted with ToPro3 (Thermo Fisher Scientific, USA) for 15 minutes at room temperature for staining. After 15 minutes, the cells were washed three times with water in DPBS and then mounted using Antifade mounting solution (H-1000, VECTOR LABORATORY, UK).

[0139] Exosome isolation and NTA analysis iMSC according to the present invention has a density of 5,000 cells / cm². 2After seeding and culturing, when the medium reached approximately 70%-80% capacity, it was replaced with fresh DMEM-F12 medium (D8437, Sigma) (containing 10% exosome depletion FBS and 1% P / S). After 48 hours, the condition medium was collected in a 50 ml tube and centrifuged at 300 g for 3 minutes. After centrifugation, it was transferred to a new 50 ml tube (50050, SPL) and centrifuged at 2,000 g for 10 minutes at 4°C. After centrifugation, it was transferred to a new 50 ml tube and centrifuged at 10,000 g for 30 minutes at 4°C. After centrifugation, it was transferred to a new 50 ml tube. 32 ml-36 ml of condition medium was transferred to an ultracentrifuge tube (344058, Beckman) and centrifuged at 32,000 rpm (187,000 g, Beckman, SW 32Ti rotor) for 2 hours at 4°C. After centrifugation, 200 μl of exosomes were collected in a single ultracentrifuge tube using 0.2 μm filtered (S6534-FMOSK, Sartorious) 1X PBS (10010-031, Gibco).

[0140] The exosomes obtained as described above were diluted 500- to 2000-fold with 1x PBS filtered through a 0.2 μm filter, and then the size and concentration (particles / ml) of the exosomes were measured using an NTA-equipped (Zetaview) analyzer.

[0141] Wound healing ability analysis To confirm the migration effect of control extracellular vesicles (Cont EVs) and mesenchymal stem cell-derived extracellular vesicles (iMSCEVs) of the present invention on NHDF cells, NHDF cells were seeded at 2.5E+05 cells / well in 6-well plates (30006, SPL) and incubated until approximately 100% confluence was reached. Once the cells reached confluence, 1 ml / well of Mitomycin C (M4287, Sigma-Aldrich), diluted to 10 μg / ml, was added to Serum-free medium (SFM) containing 1% penicillin / streptomycin (1514-163, Gibco) in DMEM high glucose (D6429, Sigma). After incubation for 2 hours and washing with DPBS, cells were scraped longitudinally in the wells using a 1000 μl pipette tip. After washing with DPBS and removing cell debris, each extracellular vesicle was treated with DMEM high SFM at a rate of 1E+09 particles / well. Following the treatment, the degree of cell migration was confirmed by taking photographs at a fixed position at 0, 12, 24, and 48 hours. The degree of migration was analyzed and compared using TScratch software.

[0142] RNA sequencing analysis Human Wharton jelly-derived mesenchymal stem cells (UC-MSCs) (ATCC:PCS-500-010) and iMSC cells of the present invention were provided to LAS (Gimpo, South Korea) in pellet form to isolate RNA, perform QC testing, and then obtain volcano plot data through microarray analysis. Through this volcano plot, it was confirmed that there were 183 upward-regulating genes and 322 downward-regulating genes. Furthermore, data predicting the major function (role) of each gene was obtained through DA VID analysis. The analyzed data was first subjected to network analysis using the database-based String-db tool (https: / / string-db.org / ) to confirm the protein-protein interactions of each gene. For the major functions of the genes predicted by the aforementioned DAVID analysis, we used a Venn diagram (https: / / bioinfogp.cnb.csic.es / tools / venny / ) to search for overlapping gene functions.

[0143] Interstitial cystitis / bladder pain syndrome (IC / BPS) induced mouse model Eight-week-old BALB / cAnNCrlOri female mice were received and allowed to undergo a two-week adaptation period. Mice were anesthetized by intraperitoneal injection of 90 μl / mouse mixed with alpha-xane and rompun in a 4:1 ratio. Urine was removed from the bladder by inserting a catheter (382412, BD) into the urethra, and the bladder was washed with 50 μl of PBS. 5 mg / ml protamine sulfate (P3369, Sigma) was injected, and the bladder was washed with PBS after 30 minutes. 30 μg / ml LPS (L4391, Sigma) was injected, and the bladder was washed with PBS. Recovery of the mice was confirmed using a hot plate, and they were housed for one week. This process was repeated four times to induce a mouse IC / BPS model. In the fifth week of the experiment, mice were anesthetized using the same method, the lower abdomen was incised, and 1 × 10⁶ iMSCs of the present invention were placed on the surface of the bladder. 5 The cells were suspended in PBS (10 μl) and injected. The lower abdomen was sutured, and the mice were reared after confirming that they had recovered from anesthesia. One week later, the mice were anesthetized, and their bladders were removed and used in the experiment. RNA was extracted from some of the bladders after homogenization, and the morphology and degree of inflammation of the bladder were examined through tissue dissection and staining of some of the bladders.

[0144] qPCR After homogenizing the extracted bladder, it was resuspended in Labozol reagent (CMRZ001, Cosmogenetech). Chloroform (C2432, Sigma) was mixed with Labozol in a 5:1 ratio and vortexed. The mixture was centrifuged at 13,000 rpm for 15 minutes, and the supernatant containing the dissolved RNA was slowly transferred to a new tube, into which an equal volume of 2-propanol (64605-0380, Junsei, 1:1) was added. The tube containing the supernatant and 2-propanol was inverted once or twice, and then centrifuged at 13,000 rpm for 15 minutes. The RNA pellet was washed with 75% EtoH, centrifuged at 13,000 rpm for 10 minutes, and the RNA was resuspended in DEPC. RNA was used to synthesize cDNA using rTaq Plus 5x PCR master mix (EBT-1319, ELPISBIO), and gene expression levels were confirmed using HiPi Real-Time PCR 2x Master Mix (SYBR green, ROX) (EBT-1802, ELPISBIO) (7500, Amersham Phamacia Biotech).

[0145] Experimental results Spheroid generation using a BAM system iPSCs placed on Aggrewell plates were confirmed to form spherical EBs with uniform shape and size after 24 hours (Figure 2).

[0146] Confirmation of pluripotent cells in spheroids by immunocytochemical staining. When spheroids were stained with OCT4, a pluripotency marker, they were stained green. When stained with DAPI, a nucleus-staining marker, the nuclear regions were specifically stained green and blue. This revealed that OCT4 is expressed in the nucleus, and that iPSC-derived spheroids maintain their pluripotency (Figure 3).

[0147] iPSC-MSC generation In culture dishes coated with 0.1% gelatin, spheroids (black arrows) adhered to the bottom, and cells protruded (white arrows). Over time, the number of protruding cells increased, and the cultures were passed through at 70%–80% confluence. As the passage progressed, the cell shape became homogenized into a spindle shape (white dotted arrow) (Figure 4).

[0148] iPSC-MSC passage and cell proliferation curves iPSC-MSC cells developed a spindle shape from P5 and could be passaged up to P13 (Figure 5). Subsequently, the cells were stored in LN2 in a storage solution. The cell proliferation curves were compared with those of the control groups hWJ-MSC and AD-MSC. The results showed that AD-MSC cells increased in number up to P9 and then decreased, while hWJ-MSC cells steadily proliferated even at P13. iPSC-MSC cells had significantly higher CPD and cumulative cell counts than the control groups, and their doubling time was also faster than that of the control groups (Figure 6).

[0149] Immunophenotyping using flow cytometry (FACS) FACS analysis revealed that iPSC-MSCs were positive for anti-CD73 and anti-CD90, and negative for anti-CD34 and anti-CD45, compared to the control group of AD-MSCs (Figure 7). This indicates that iPSC-MSCs produced through the BAM system possess distinct characteristics of mesenchymal stem cells.

[0150] Confirmation of iPSC-MSC mesenchymal stem cells by immunocytochemical staining. ICC was promoted in iPSC-MSC cells using the pluripotency markers OCT4 and SSEA4, and the mesenchymal stem cell marker PDGFRβ. In iPSC-MSC cells, the green-stained OCT4 and SSEA4 markers resulted in fewer green-stained cells, while the green PDGFRβ marker resulted in a larger distribution of green-stained cells (Figure 8). This indicated that the iPSC-MSC cells were converted into mesenchymal stem cells while losing their overall differentiation potential.

[0151] Anti-inflammatory cell model The experiment was conducted according to a schematic diagram of the experimental procedure to confirm the inflammation-controlling effect of stem cells (Figure 9). As a result of the inflammation experiment, large multinucleated cells were observed in the LPS group, in which Raw 264.7 cells were treated with LPS to induce inflammation. Multinucleated cells were not observed in the positive control group treated with DEX along with LPS (LPS+DEX), the test groups treated with hWJ-MSC conditioning medium along with LPS (LPS+hWJ-MSCCM), and the test group treated with iMSC conditioning medium (LPS+iMSCCM) (Figure 10). Furthermore, RT-PCR was performed on each experimental group and it was confirmed that IL-6 expression was highest in the LPS group, and that IL-6 expression was significantly reduced in the test groups treated with hWJ-MSC conditioning medium along with LPS (LPS+hWJ-MSCCM) and iMSC conditioning medium (LPS+iMSCCM) compared to the LPS group (Figure 10). On the other hand, in the positive control group treated with both LPS and DEX (LPS+DEX), IL-6 expression was significantly lower.

[0152] Confirmation of the presence or absence of multinucleated cells by immunocytochemical staining After testing the anti-inflammatory cell model described above, the presence or absence of multinucleated cells using TRAP antibodies was confirmed in each test group. Multinucleated cells are generated as cells aggregate during the progression of an inflammatory response, and the presence or absence of multinucleated cells can be used to confirm the presence or absence of inflammation. The experimental results showed that the proportion of TRAP-stained cells (TRAP Positive) was very high in the LPS group, while the test groups treated with hWJ-MSC conditioning medium along with LPS (LPS+hWJ-MSC CM) and iMSC conditioning medium (LPS+iMSC CM) had significantly fewer TRAP-positive cells compared to the LPS group (Figure 11).

[0153] Exosome isolation and NTA analysis Using NTA equipment, we measured the size and concentration (particles / ml) of exosomes. The results showed that the particle size of exosomes isolated from iPSC-MSCs of the present invention was approximately 100 nm, and the total number of particles was about four times greater than that of exosomes isolated from WJ-MSCs (Figure 12).

[0154] Wound healing ability analysis When exosomes isolated from iPSC-MSCs of the present invention were placed in scratched normal human dermal fibroblasts (NHDF) and observed for 48 hours, approximately 50% of wounds in the control group (PBS group) healed after 48 hours. However, in the test group treated with exosomes isolated from iPSC-MSCs of the present invention (Exosome), more than 90% of wounds healed (Figures 13a and 13b). These results indicate that the wound-healing ability of exosomes isolated from iPSC-MSCs of the present invention is significantly higher than that of the control group (PBS).

[0155] RNA sequencing analysis By plotting volcano plots using RNA sequencing data, we were able to identify 183 superior regulatory genes and 322 inferior regulatory genes. These were then presented as string data and Venn diagrams, respectively.

[0156] First, in string data using 183 top regulatory genes, it was found that many genes, including SPP1 (secreted phosphoprotein 1; osteopontin), PLAU (Plasminogen Activator, Urokinase), ITGA6 (integrin subunit alpha 6), CENPI (Centromere Protein I), and BUB1 (Budding Uninhibited By Benzimidazoles 1 Homolog; Mitotic checkpoint serine / threonine-protein kinase), which are up-regulated genes, were interacting with each other (Figure 14a). The differences in expression of these genes in the volcano plot are as follows. SPP1 expression increased by approximately 3.0 to 4.0 times, specifically 3.50 to 4.00 times, and more specifically 3.80 to 3.90 times in weight, compared to the control group (hWJ-MSC). PLAU expression increased by approximately 1.5 to 2.2 times, specifically 1.80 to 2.10 times, and more specifically 1.90 to 2.00 times in weight. ITGA6 expression increased by approximately 2.1 to 3.0 times, specifically The weight of each marker (Up-regulated Genes) increased by 2.30 to 2.60 times, and more specifically by 2.40 to 2.50 times. CENPI expression increased by approximately 1.5 to 2.0 times, specifically by 1.60 to 1.90 times, and more specifically by 1.70 to 1.80 times. BUB1 expression increased by approximately 1.4 to 2.1 times, specifically by 1.50 to 1.90 times, and more specifically by 1.65 to 1.75 times. Using GOTERM, the volcano plot data of each marker whose expression increased (Up-regulated Genes) was shown in a Venn diagram. The results confirmed that the ITGA6 gene overlaps with the genes related to transcriptional regulation by positive RNA polymerization promoters and the genes related to cell adhesion and proliferation (Figure 15a).SSP1 is used as a marker for osteogenic differentiation, and it can be observed that osteogenic differentiation is promoted. PLAU is an enzyme isolated from urine and can be used as a therapeutic agent to restore blood flow to venous pathways blocked by coagulated blood or fibrin. ITGA6 plays an important structural role in hemidesmosomes as a receptor for laminin in epithelial cells. CENPI is involved in the response of gonadal tissue to follicle-stimulating hormone, and BUB1 is known to play an important role in the mitotic spindle checkpoint and chromosome condensation.

[0157] Next, in string data using 322 subgenes, we confirmed that PTGS2 (Prostaglandin-Endoperoxide Synthase 2, or COX2), a down-regulated gene, interacted frequently (Figure 14b). The expression differences in the volcano plot of the PTGS2 gene were as follows: PTGS2 expression decreased by approximately 2.0 to 3.0 times compared to the control group (hWJ-MSC), specifically by 2.30 to 2.90 times, and even more specifically by 2.50 to 2.70 times in weight. Using GOTERM, we plotted the volcano data of the down-regulated marker in a Benn diagram, confirming that PTGS2 overlapped with genes related to negative cell proliferation regulation, LPS response, and inflammation (Figure 16a). PTGS2 is an enzyme responsible for prostaglandin production during inflammatory responses.

[0158] The difference in the relative expression levels of the five upward regulatory genes and the one downward regulatory gene was quantified and is shown in Table 2 below.

[0159] [Table 2]

[0160] Examining Table 2, it can be confirmed that the expression of the five upward regulatory genes was significantly increased in the iMSCs of the present invention compared to hWJ-MSCs, and the expression of the one downward regulatory gene was significantly decreased in the iMSCs of the present invention compared to hWJ-MSCs.

[0161] From these results, it can be inferred that the differentiated efficacy of the iMSCs of the present invention compared to conventional hWJ-MSCs is due to the difference in gene expression.

[0162] Interstitial cystitis / bladder pain syndrome (IC / BPS) induced mouse model Incisions were made in mouse bladder tissue and H&E staining was performed. The results showed that the bladder wall was disintegrated in IC / BPS-induced mice, while the bladder wall was restored in the groups treated with iMSCs and hWJ-MSCs. Furthermore, the degree of fibrosis (%) and mast cell infiltration were examined through Masson's trichrome staining and toluidine blue staining, and it was confirmed that the degree of inflammation was alleviated in the groups treated with iMSCs and hWJ-MSCs (Figures 17a and 17b). In particular, the group treated with the iMSCs of the present invention showed a significant reduction in the degree of fibrosis (%) and mast cell infiltration to approximately 50% compared to the group treated with hWJ-MSCs, indicating that the iMSCs of the present invention are more effective than hWJ-MSCs in preventing, improving, or treating inflammation.

[0163] qPCR mRNA was extracted from bladder tissue of IC / BPS-induced mice, and it was confirmed that the expression levels of inflammation-related cytokines (IL6, TNF-alpha) were lower in the group treated with the iMSC of the present invention (Figure 18a). Furthermore, the expression of urothelial markers (UPK1A, UPK1B, UPK2) was confirmed to be higher in the group treated with the iMSC of the present invention (Figure 18b). Finally, it was confirmed that the IC / BPS markers (KLRB1, PSMB9, ITGAL) discovered in the inventors' prior research were expressed at a lower level in the group treated with the iMSC of the present invention (Figure 18c) (see Korean Patent Publication No. 10-2331138).

[0164] From the experimental results described above, it can be inferred that the iMSC of the present invention can be usefully used in the treatment of bladder pain syndromes (BPS), such as interstitial cystitis (IC).

[0165] Although the present invention has been described above as a preferred embodiment, various modifications and variations are possible without departing from the spirit and scope of the invention. Furthermore, the appended claims include such modifications and variations that fall within the spirit of the invention. [Brief explanation of the drawing]

[0166] [Figure 1] Figure 1 is a schematic diagram summarizing the protocol of the present invention for differentiating iPSCs from MSCs. [Figure 2] Figure 2 shows the morphology of embryoid bodies (EBs) formed on Aggrewell. [Figure 3] Figure 3 shows the morphology of spheroids formed through a BAM (Bio Array Matrix) microgravity biological culture device (top) and the staining results using OCT4 and DAPI antibodies (bottom). [Figure 4] Figure 4 shows the external shape of mesenchymal stem cells derived from spheroids, confirming that they take on a spindle shape after passage (right side). [Figure 5] Figure 5 shows the external shape of iMSCs separated by the method of the present invention at each passage. [Figure 6] Figure 6 shows the cumulative cell proliferation curve of mesenchymal stem cells differentiated by the method of the present invention, and indicates the CPD (Cummulative Population Doubling) (Figure 6a), doubling time (Figure 6b), and log cell number (Figure 6c) for each passage, respectively. [Figure 7] Figure 7 shows the results of confirming the expression of cell surface markers in mesenchymal stem cells differentiated by the method of the present invention using FACS analysis. [Figure 8]Figure 8 shows the results of confirming, using immunocytochemical staining, that induced pluripotent stem cells lost their totipotency and differentiated into cells expressing mesenchymal stem cell markers through the method of the present invention. [Figure 9] Figure 9 is a schematic diagram showing the experimental procedure for confirming the inflammatory control effect of stem cells differentiated by the present invention method in inflammation-induced cells using LPS. [Figure 10] Figure 10 shows the results of confirming the expression of inflammatory markers via RT-PCR. [Figure 11] Figure 11 shows the results of staining cells in which inflammation was induced by LPS treatment using immunocytochemistry (left side) and the results of counting the TRAP-stained cells (right side). [Figure 12] Figure 12 shows the results of measuring the size (left) and concentration (right) of exosomes isolated from mesenchymal stem cells according to the present invention. [Figure 13] Figure 13a is a photograph showing the degree of cell migration over time after treating NHDF cells that have been scratched with exosomes isolated from mesenchymal stem cells according to the present invention, and Figure 13b is a graph showing the degree of cell migration quantified. [Figure 14] Figure 14a shows the results of a network analysis using the database-based String-db tool to confirm the protein-protein interactions of each upward regulatory gene contained in the mesenchymal stem cells of the present invention, and Figure 14b shows the results of a network analysis of each downward regulatory gene. [Figure 15] Figures 15a to 15d are Venn diagrams generated using the predicted functions of each upward regulatory gene, which was analyzed using DAVID analysis for each of the mesenchymal stem cells contained in the present invention. [Figure 16] Figures 16a to 16d are Venn diagrams generated using the predicted functions of each downward regulatory gene, which was subjected to DAVID analysis for each of the mesenchymal stem cells contained in the present invention. [Figure 17] Figure 17a shows the morphology and degree of inflammation of bladder tissue after administration of mesenchymal stem cells (iMSCs) of the present invention in a mouse model of interstitial cystitis / bladder pain syndrome (IC / BPS). Figure 17a shows the results of staining the bladder tissue of the IC / BPS mouse model with H&E, Masson's trichrome, and toluidine blue, respectively. Figure 17b is a graph showing the degree of fibrosis (%) and mast cell infiltration confirmed through the aforementioned staining. [Figure 18] Figure 18 shows the results of extracting mRNA from bladder tissue after administration of mesenchymal stem cells (iMSCs) of the present invention in a mouse model of interstitial cystitis / bladder pain syndrome (IC / BPS), and confirming the expression levels of inflammation-related cytokines (TNFα, IL6) (Figure 18a), urothelial markers (UPK1A, UPK1B, UPK2) (Figure 18b), and genes expressed in IC / BPS (KLRB1, PSMB9, ITGAL) (Figure 18c).

Claims

1. Compared to general Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs), these mesenchymal stem cells exhibit the following characteristics: (A) increased expression levels of SPP1, PLAU, ITGA6, CENPI, and BUB1 markers, and (B) decreased expression levels of the PTGS2 marker. The aforementioned mesenchymal stem cells are (1) The step of forming an embryoid body (EB) by culturing fully differentiated stem cells isolated from the target organism; (2) The step of forming a spheroid by three-dimensional culture of the embryoid body in a bioreactor under microgravity; and (3) Differentiating the spheroid into mesenchymal stem cells by a method comprising the step of (3) attaching and culturing the spheroid in a culture vessel coated with an adhesive polymer on its culture surface.

2. The mesenchymal stem cell according to claim 1, characterized in that the fully differentiated stem cell is an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC).

3. The mesenchymal stem cell according to claim 1, characterized in that the fully differentiated stem cell is an induced pluripotent stem cell.

4. The mesenchymal stem cell according to claim 1, characterized in that step (1) is performed by three-dimensionally culturing the fully differentiated stem cells in a multi-well culture vessel.

5. The mesenchymal stem cell according to claim 1, characterized in that step (1) further comprises a step of inducing cell aggregation by centrifugation during the three-dimensional culture.

6. The mesenchymal stem cell according to claim 1, characterized in that the microgravity in step (2) is induced by a microgravity simulator that cancels out the gravity applied to the bioreactor by rotating the bioreactor.

7. The mesenchymal stem cells according to claim 6, characterized in that step (2) is performed by culturing the microgravity simulator for 3 to 8 days while rotating it at 15 rpm to 80 rpm.

8. The mesenchymal stem cell according to claim 7, characterized in that step (2) is performed by rotating the microgravity simulator starting at 40 rpm to 60 rpm and increasing it by 3 rpm to 7 rpm each day.

9. The mesenchymal stem cell according to claim 1, characterized in that the adhesive polymer is one selected from hyaluronic acid, alginic acid, heparin, fucoidan, cellulose, dextran, chitosan, albumin, fibrin, collagen, and gelatin.

10. The mesenchymal stem cell according to claim 9, characterized in that the adhesive polymer is gelatin.

11. A pharmaceutical composition for the prevention or treatment of inflammatory diseases or autoimmune diseases, comprising mesenchymal stem cells as an active ingredient, as described in claim 1.

12. The pharmaceutical composition according to claim 11, characterized in that the inflammatory disease or autoimmune disease is cystitis, rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic fibrous alveolitis, polymyositis, dermatomyositis, focal scleroderma, systemic scleroderma, colitis, inflammatory bowel disease, Sjögren's syndrome, Raynaud's phenomenon, Behçet's disease, Kawaki disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, graft-versus-host disease (GVHD), or Crohn's disease.

13. The pharmaceutical composition according to claim 12, characterized in that the cystitis is one or more selected from interstitial cystitis, chronic cystitis, and ketamine-induced cystitis.

14. A wound healing pharmaceutical composition comprising mesenchymal stem cells as an active ingredient, as described in claim 1.

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