WO2023282424A1 - Vésicules extracellulaires dérivées de cellules souches et leur utilisation - Google Patents

Vésicules extracellulaires dérivées de cellules souches et leur utilisation Download PDF

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WO2023282424A1
WO2023282424A1 PCT/KR2022/001339 KR2022001339W WO2023282424A1 WO 2023282424 A1 WO2023282424 A1 WO 2023282424A1 KR 2022001339 W KR2022001339 W KR 2022001339W WO 2023282424 A1 WO2023282424 A1 WO 2023282424A1
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stem cell
extracellular vesicles
derived extracellular
tgf
cell
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PCT/KR2022/001339
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Korean (ko)
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조쌍구
임경민
데이엠 아메드 아브달
이수빈
최유진
강근호
김아람
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건국대학교 산학협력단
스템엑소원 주식회사
건국대학교 글로컬산학협력단
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Priority claimed from KR1020220011023A external-priority patent/KR20230007922A/ko
Publication of WO2023282424A1 publication Critical patent/WO2023282424A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to stem cell-derived extracellular vesicles and uses thereof.
  • the inflammatory response is a biological defense reaction process for repairing and regenerating damage caused by invasion that causes organic changes in cells or tissues of a living body, and this reaction process includes local blood vessels, various tissue cells of body fluids, and immune cells, etc.
  • the inflammatory response normally induced by external invading bacteria is a defense system for protecting the living body, whereas when an abnormally excessive inflammatory response is induced, various diseases appear. These diseases are collectively referred to as inflammatory diseases.
  • the inflammatory diseases are diseases in which various inflammatory mediators secreted from target cells activated by external stimuli amplify and sustain inflammation and threaten the life of the human body, such as acute inflammation, diseases in the bladder such as cystitis, and rheumatoid arthritis. It includes diseases within the joints, skin diseases in the form of psoriasis, and allergic inflammatory diseases such as bronchial asthma.
  • interstitial cystitis is a chronic bladder disease of unknown cause, characterized by symptoms of pain, eg, pelvic pain, and lower urinary tract syndrome (LUTS), eg, increased urinary frequency/urgency.
  • PBS painful bladder syndrome
  • BPS bladder pain syndrome with IC to collectively describe this complex symptom.
  • BPS [(van der Merve et al . European Urology 53(2008) 60-67]
  • IC/BPS IC/PBS/BPS.
  • IC/PBS/BPS IC/PBS/BPS
  • IC significantly affects quality of life, affects 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).
  • the IC/PBS/BPS has not been identified as a single etiology, and mainly causes hyperalgesia, chronic bladder pain and urination disorders (Forrest JB et al . Clinical Courier 2006; 24(3):1-8).
  • Extracellular vesicles are lipid bilayer structured vesicles of various sizes secreted by various eukaryotic cells such as humans and animals as well as insects, plants, and microorganisms. do. Exosomes contain specific molecules such as proteins, nucleic acids, lipids, and carbohydrates contained in cells, stably protect these molecules with a lipid bilayer, and play a role in transmitting information to other cells after secretion.
  • Exosomes are extracellular vesicles with a size of tens to hundreds of nanometers composed of a double phospholipid membrane identical to the structure of a cell membrane, and contain proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo inside. It is known that the exosome cargo contains a wide range of signaling factors, and these signaling factors are cell type-specific and differently regulated depending on the environment of the secreting cell. Exosomes are intercellular signaling mediators secreted by cells, and various cell signals transmitted through them regulate cell behavior including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. It is known.
  • Exosomes contain specific genetic materials and bioactive factors depending on the nature and state of the derived cell. In the case of proliferating stem cell-derived exosomes, cell behaviors such as cell migration, proliferation, and differentiation are regulated, and characteristics of stem cells related to tissue regeneration are reflected (Nature Review Immunology 2002 (2) 569-579).
  • the exosomes isolated from the stem cell culture medium were COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF- ⁇ 1 and High expression of one or more proteins selected from TGM2 can solve the problem of safety of the stem cell itself or the stem cell culture medium, and it is confirmed that it is effective in preventing, alleviating, improving, or treating various inflammatory diseases including interstitial cystitis, and the present invention has been completed.
  • An object of the present invention is to provide stem cell-derived extracellular vesicles effective for the prevention, alleviation, improvement or treatment of various inflammatory diseases including interstitial cystitis.
  • Another object of the present invention is to provide a composition for preventing or treating inflammatory diseases or autoimmune diseases comprising the stem cell-derived extracellular vesicles as an active ingredient.
  • Another object of the present invention is to provide a composition for wound healing comprising the stem cell-derived extracellular vesicles as an active ingredient.
  • the present invention provides stem cell-derived extracellular vesicles highly expressing at least one protein selected from COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF- ⁇ 1 and TGM2.
  • the stem cell-derived extracellular vesicles are compared to stem cell-derived extracellular vesicles cultured in two dimensions or stem cell-derived extracellular vesicles cultured in three dimensions without adding TGF- ⁇ to the culture medium.
  • the protein can be highly expressed.
  • the stem cell-derived extracellular vesicles can express TGF- ⁇ 1 at a high level.
  • the stem cell-derived extracellular vesicles can express TGF- ⁇ 1 in an amount of 50 to 1,000 pg/1x10 9 particles.
  • the stem cell-derived extracellular vesicles can highly express COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF- ⁇ 1 and TGM2 proteins.
  • the stem cell-derived extracellular vesicles include: (a) culturing the stem cells isolated from the subject to form a cell aggregate; and (b) three-dimensionally culturing the cell aggregate in a culture medium containing transforming growth factor beta (TGF- ⁇ ).
  • TGF- ⁇ transforming growth factor beta
  • the stem cells may be mesenchymal stem cells.
  • step (a) may be performed by suspension culture of stem cells in a multi-well culture vessel.
  • the TGF- ⁇ may be TGF- ⁇ 3.
  • step (b) may be performed by orbital shaking culture of the cell aggregate in a floating state.
  • the rotary shaking culture may be performed at a rotational speed of 50 - 70 rpm.
  • the stem cell-derived extracellular vesicles may have an average diameter of 30 to 150 nm.
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or autoimmune diseases comprising the stem cell-derived extracellular vesicles as an active ingredient.
  • the inflammatory disease or autoimmune disease is cystitis, rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic fibrosing alveolitis, polymyositis , dermatomyositis, localized dermatosclerosis, systemic scleroderma, colitis, inflammatory bowel disease, Sjorgen's syndrome, Raynaud's phenomenon, Bechet's disease, Kawasaki's disease, primary biliary It may be primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, graft-versus-host disease (GVHD) or Crohn's disease.
  • cystitis cystitis, rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic fibrosing alveolitis
  • the cystitis may be at least one selected from interstitial cystitis, chronic cystitis, and ketamine-induced cystitis.
  • the present invention provides a pharmaceutical composition for wound healing comprising the stem cell-derived extracellular vesicles as an active ingredient.
  • the stem cell-derived extracellular vesicles of the present invention express high levels of one or more proteins selected from COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF- ⁇ 1 and TGM2, thereby reducing inflammation such as TNF- ⁇ and IL-6. Since it significantly reduces the amount of cytokines, it is effective in preventing, alleviating, improving or treating various inflammatory diseases.
  • the stem cell-derived extracellular vesicles of the present invention are administered to an interstitial cystitis/bladder pain syndrome (IC/BPS) animal model, the bladder inner wall collapsed during the IC/BPS induction process is restored, the degree of inflammation is alleviated, Since the intravesical pressure is restored and the voiding cycle is similar to that of the control group, it has excellent therapeutic efficacy for interstitial cystitis/bladder pain syndrome.
  • IC/BPS interstitial cystitis/bladder pain syndrome
  • stem cell-derived extracellular vesicles of the present invention significantly increase cell migration and have excellent wound healing effects, they can be usefully used for wound healing.
  • FIG. 1 is a picture showing the 3D culture process of mesenchymal stem cells according to the method of the present invention, showing the cell aggregate formation (FIG. 1a) and 3D culture using a rotary stirrer (FIG. 1b), respectively.
  • 2 is a diagram showing the yield of exosomes according to each culture condition.
  • Figure 3 is a diagram showing the change in PDI value by TGF- ⁇ treatment, showing that a single peak appears in the 3D shaking culture condition treated with TGF- ⁇ .
  • FIG. 4 is a picture showing the effect of TGF- ⁇ on T cell proliferation.
  • Figure 5a is a picture showing the results of examining the size of exosomes through dynamic light scattering (DLS) analysis.
  • Figure 5b is the result of observing the morphology and structure of exosomes with a transmission electron microscope (TEM).
  • 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 analysis of the surface of exosomes through flow cytometry.
  • Figure 5e shows the result of confirming the TGF- ⁇ 1 content of the produced exosomes through enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Figure 6 is a photograph showing the result of confirming the increase in cell migration ability according to exosome administration in human fibroblasts (NHDF) through transwell migration analysis (left), and the right is expressed by quantifying the relative staining degree using Image J it's a graph
  • FIG. 7 is a picture showing the results of confirming the wound healing ability over time by administering exosomes to an animal model in which a wound was created by a biopsy punch.
  • FIG. 7A is a photograph of a wound site at regular intervals after administration of exosomes to an animal model in which a wound was created by a biopsy punch.
  • Figure 7b is a graph showing the size of the wound area of Figure 7a.
  • TNF- ⁇ and IL-6 which are inflammatory cytokines, were significantly decreased in the culture supernatant of Raw264.7 cells cultured by co-administration of LPS and exosomes.
  • 11 is a picture showing the results of confirming the effect of reducing TNF- ⁇ and IL-6 according to the administration of exosomes in a mouse model of endotoxemia caused by LPS toxin.
  • FIG. 12 is a picture showing the result of confirming cell proliferation by treating SV-HUC-1 (human urothelial cells) with exosomes at each concentration.
  • Figure 12a is a graph showing the cell proliferation rate after treatment with exosomes by concentration in SV-HUC-1
  • Figure 12b is the result of confirming the expression level of P-AKT and P-ERK associated with cell proliferation.
  • FIG. 13 is a picture showing the result of confirming the increase in cell migration ability according to exosome administration in human urinary tract epithelial cells (SV-HUC-1) through transwell migration assay.
  • 13a is a photograph visually confirming the migration of cells to the opposite side of the transwell through Crystal violet staining.
  • FIG. 13b is a graph showing the relative cell confluency shown using Image J of FIG. 13a.
  • FIG. 14 is a schematic diagram of a test design for preparing an interstitial cystitis/bladder pain syndrome (IC/BPS) induced mouse model and evaluating the therapeutic effect of exosome administration.
  • IC/BPS interstitial cystitis/bladder pain syndrome
  • FIG. 15 is a diagram confirming the morphology and inflammation level of bladder tissue after administration of exosomes in an interstitial cystitis/bladder pain syndrome (IC/BPS) induced mouse model.
  • Figure 15a is the result of H&E staining of the bladder tissue of the IC/BPS mouse model
  • Figure 15b is the result of masson's trichome staining
  • Figure 15c is the result of toluidine blue staining
  • Figure 15d is the degree of fibrosis and mast cell staining through the above staining. The result of confirming the penetration of is shown in a graph.
  • FIG. 16 is an expression level of inflammation-related cytokines (TNF ⁇ , IL6) by extracting mRNA from bladder tissue extracted after administration of exosomes in an interstitial cystitis/bladder pain syndrome (IC/BPS) induced mouse model (FIG. 16a); The expression levels of urinary epithelial markers (UPK1A, UPK1B, UPK2) (FIG. 16b) and the expression levels of genes (KLRB1, PSMB9, ITGAL) expressed in IC/BPS (FIG. 16c) were confirmed.
  • IC/BPS interstitial cystitis/bladder pain syndrome
  • 17 and 18 are pictures showing the results confirming the recovery effect of intravesical pressure and voiding cycle according to exosome administration in interstitial cystitis/bladder pain syndrome (IC/BPS) induced mouse model.
  • IC/BPS interstitial cystitis/bladder pain syndrome
  • 19 is a diagram showing the results of proteomic analysis contained in each exosome.
  • 19a is a result of quantitative analysis of proteins contained in each exosome.
  • 19B is a graph showing the abundance distribution of total proteins contained in each exosome.
  • 19c shows the results of gene ontology (GO) analysis for each exosome.
  • 19d is a comparison result of the data set reported in Vesiclepedia and the EV proteomes discovered in this study.
  • FIG. 20a is a diagram showing proteins that specifically change in the T-a3D-EV sample, which is the exosome of the present invention, among four clusters derived through clustering analysis.
  • FIG. 20B is a diagram showing a result showing the degree of separation between each exosome group using a discrimination index through principal component analysis.
  • FIG. 20C is a picture showing the result of confirming the number of differentially expressed proteins (DEPs) according to culture conditions (2D and 3D) and TGF- ⁇ 3 treatment through a two-way comparison method.
  • DEPs differentially expressed proteins
  • Figure 21a shows enriched gene sets, normalized enrichment scores (NES) and p-values in the PI3K-AKT signaling pathway and integrin1 pathway of the exosome T-a3D-EV of the present invention through gene set enrichment analysis (GSEA) show each 21B is a diagram illustrating a gene group enriched through gene set enrichment analysis (GSEA) divided into a PI3K-AKT signaling pathway and an integrin1 pathway.
  • Figure 21c shows the number of differentiated or common proteins between each exosome through binary comparison analysis (T-a3D-EV/2D-EV, a3D-EV/2D-EV, and T-a3D-EV/a3D-EV).
  • FIG. 21d shows that 5 proteins (S100A10, SDDCP, ACTG1, GIPC1 and EIF4E) among 28 DEP proteins, which are proteins having specific characteristics in the exosome T-a3D-EV of the present invention, were found in 113 interactions of the Huri database with high confidence scores. This is a picture showing the results mapped with interactors. 21E is a diagram showing the biological function of the exosome T-a3D-EV of the present invention and its characteristics related to the regulation of cyclin-dependent protein kinase activity.
  • One aspect of the present invention is COL6A1 (collagen alpha-1 (VI) chain), COL6A3 (collagen alpha-3 (VI) chain), TNC (Tenascin), EIF4E (Eukaryotic translation initiation factor 4E), HSP90AB1 (heat shock protein HSP) 90-beta), HSP90B1 (Endoplasmin), RAC1 (Ras-related C3 botulinum toxin substrate 1), TGF- ⁇ 1 (Transforming growth factor-beta-induced protein ig-h3) and TGM2 (Protein-glutamine gamma-glutamyltransferase 2) It relates to stem cell-derived extracellular vesicles that highly express one or more selected proteins.
  • high expression in the present invention refers to conventional mesenchymal stem cell-derived extracellular vesicles, specifically stem cell-derived extracellular vesicles cultured in two dimensions or stem cells cultured in three dimensions without adding TGF- ⁇ to the culture medium.
  • content, secretion or expression level of a specific protein in extracellular vesicles has increased significantly to a measurable extent, specifically, it means an increase of 40% or more in content, secretion or expression level, and more Specifically means an increase of 50% or more, more specifically means an increase of 60% or more, more specifically means an increase of 80% or more, and most specifically means an increase of 100% or more.
  • extracellular vesicle refers to a lipid bilayer structured vesicle with a diameter in the range of 30-1,000 nm that is secreted into the extracellular environment through fusion of the polycystic body and the plasma membrane in various cells.
  • the stem cell-derived extracellular vesicles of the present invention particularly highly express TGF- ⁇ 1.
  • the stem cell-derived extracellular vesicles of the present invention contain TGF- ⁇ 1 in an amount of 50 to 1,000 pg/1x10 9 particles, preferably 100 to 800 pg/1x10 9 particles, more preferably 250 to 400 pg/1x10 9 particles. can manifest.
  • the expression level of TGF- ⁇ 1 in the stem cell-derived extracellular vesicles of the present invention is at least 5 times higher, preferably 5 to 15 times higher than the expression level of TGF- ⁇ 1 in the two-dimensionally cultured stem cell-derived extracellular vesicles. , more preferably 6 to 12 times, more preferably 7 to 10 times.
  • the expression level of TGF- ⁇ 1 in the stem cell-derived extracellular vesicles of the present invention is at least twice higher than the expression level of TGF- ⁇ 1 in the 3-dimensional cultured stem cell-derived extracellular vesicles without the addition of TGF- ⁇ , specifically may be 3 times or more, more specifically 3 to 10 times, more specifically 3 to 6 times, and more specifically 3 to 5 times.
  • the TGF- ⁇ 1 plays a very important role in anti-inflammatory and tissue regeneration to restore the tissue when tissue damage occurs.
  • it can play important roles such as anti-inflammation in bladder tissue, regeneration of urinary epithelial cells, formation of blood vessels, and generation of matrix to prevent permeation of urine electrolytes (Ju, Cynthia, and Pranoti Mandrekar. "Macrophages and alcohol-related liver inflammation.” Alcohol research: current reviews 37.2 (2015): 251.).
  • the expression level of COL6A1 in the stem cell-derived extracellular vesicles of the present invention is at least 10-fold, preferably 10 to 30-fold, more preferably 15-fold higher than the COL6A1 expression level in the two-dimensionally cultured stem cell-derived extracellular vesicles. to 25 times, more preferably 18 to 22 times.
  • the expression level of COL6A1 in the stem cell-derived extracellular vesicles of the present invention is at least 5 times greater than that of the COL6A1 expression level in the 3-dimensionally cultured stem cell-derived extracellular vesicles without the addition of TGF- ⁇ , specifically 5 to 10 times, more specifically 6 to 8 times.
  • the expression level of COL6A3 in the stem cell-derived extracellular vesicles of the present invention is at least 1.4 times higher than that of the COL6A3 expression level in the two-dimensionally cultured stem cell-derived extracellular vesicles, preferably 1.5 times higher, more preferably 1.5 to 10 times higher. 2.5 times, more preferably 1.5 to 2 times.
  • the expression level of COL6A1 in the stem cell-derived extracellular vesicles of the present invention is at least 1.5 times greater than the expression level of COL6A3 in the 3-dimensionally cultured stem cell-derived extracellular vesicles without the addition of TGF- ⁇ , specifically 1.5 to 3 times. times, more specifically, it may be 1.5 to 2.5 times.
  • the TNC expression level in the stem cell-derived extracellular vesicles of the present invention is at least 1.5 times higher than the TNC expression level in the two-dimensionally cultured stem cell-derived extracellular vesicles, preferably 2 times higher, more preferably 2 to 2 times higher. It may be 4 times, more preferably 2.5 to 3.5 times.
  • the TNC expression level in the stem cell-derived extracellular vesicles of the present invention is at least 5 times greater than the TNC expression level in the 3-dimensional cultured stem cell-derived extracellular vesicles without the addition of TGF- ⁇ , specifically 5 to 10 times, more specifically 7 to 9 times.
  • the expression level of EIF4E in stem cell-derived extracellular vesicles of the present invention is higher than that in stem cell-derived extracellular vesicles cultured in two dimensions or stem cell-derived extracellular vesicles cultured in three dimensions without adding TGF- ⁇ . It may be at least 2 times or more, preferably 3 times or more, more preferably 3 to 6 times, and even more preferably 3.5 to 5 times.
  • the HSP90AB1 expression level in the stem cell-derived extracellular vesicles of the present invention is at least 2-fold, preferably 2 to 4-fold, more preferably 3-fold higher than the HSP90AB1 expression level in the two-dimensionally cultured stem cell-derived extracellular vesicles. It can be up to 4 times.
  • the expression level of HSP90AB1 in the stem cell-derived extracellular vesicles of the present invention is at least 1.5 times higher than that of the HSP90AB1 expression level in the 3-dimensionally cultured stem cell-derived extracellular vesicles without the addition of TGF- ⁇ , specifically 1.5 to 3 times. times, more specifically, it may be 1.5 to 2 times.
  • the expression level of HSP90B1 in the stem cell-derived extracellular vesicles of the present invention is at least 20 times higher, preferably 20 to 40 times, more preferably 20 times higher than the expression level of HSP90B1 in the two-dimensionally cultured stem cell-derived extracellular vesicles. to 30 times.
  • the expression level of HSP90B1 in the stem cell-derived extracellular vesicles of the present invention is at least 5 times higher than that of the HSP90B1 expression level in the 3-dimensionally cultured stem cell-derived extracellular vesicles without the addition of TGF- ⁇ , specifically 5 to 10 times, more specifically 6 to 8 times.
  • the expression level of RAC1 in the stem cell-derived extracellular vesicles of the present invention is at least 2-fold, preferably 2 to 5-fold, more preferably 3-fold higher than that of the RAC1 expression level in the two-dimensionally cultured stem cell-derived extracellular vesicles. to 4.5 times.
  • the expression level of RAC1 in the stem cell-derived extracellular vesicles of the present invention is at least 1.5 times higher than that of the RAC1 expression level in the 3-dimensionally cultured stem cell-derived extracellular vesicles without the addition of TGF- ⁇ , specifically 1.5 to 3 times higher. times, more specifically, it may be 1.5 to 2 times.
  • the expression level of TGM2 in the stem cell-derived extracellular vesicles of the present invention is at least 5-fold, preferably 5 to 15-fold, more preferably 10-fold higher than the TGM2 expression level in the two-dimensionally cultured stem cell-derived extracellular vesicles. to 15 times.
  • the expression level of TGM2 in the stem cell-derived extracellular vesicles of the present invention is at least twice as high as that of the TGM2 expression level in the 3-dimensionally cultured stem cell-derived extracellular vesicles without the addition of TGF- ⁇ , specifically 2 to 5 times, more specifically, it may be 3 to 5 times.
  • the stem cell-derived extracellular vesicles of the present invention highly express one or more proteins selected from COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF- ⁇ 1 and TGM2, such as TNF- ⁇ , IL-6, etc. significantly reduces the amount of inflammatory cytokines, and can prevent, alleviate, improve or treat various inflammatory diseases, preferably cystitis, especially interstitial cystitis.
  • the stem cell-derived extracellular vesicles of the present invention significantly increase cell migration and have excellent wound healing effects.
  • the stem cell-derived extracellular vesicles of the present invention may be those that express high levels of COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF- ⁇ 1 and TGM2 proteins.
  • the extracellular vesicles of the present invention include: (a) culturing stem cells isolated from a subject to form cell aggregates; and (b) three-dimensionally culturing the cell aggregate in a culture medium containing transforming growth factor beta (TGF- ⁇ ).
  • TGF- ⁇ transforming growth factor beta
  • stem cell is an undifferentiated cell at a stage before differentiation into each cell constituting a tissue, and has the ability to differentiate into a specific cell under a specific differentiation stimulus (environment).
  • Stem cells unlike differentiated cells in which cell division is suspended, can produce (self-renewal) cells identical to themselves by cell division, and can differentiate into various cells depending on the nature of the stimulus when a differentiation stimulus is applied. , it is characterized by the plasticity of differentiation.
  • Stem cells used in the present invention can be used without limitation as long as they have the characteristics of stem cells, that is, undifferentiated, indefinitely proliferating, and differentiated into specific cells, and can induce differentiation into tissues to be regenerated.
  • the stem cells used in the present invention may be mesenchymal stem cells.
  • meenchymal stem cells refers to stem cells having multipotency capable of differentiating into adipocytes, bone cells, chondrocytes, muscle cells, nerve cells, and cardiomyocytes.
  • Mesenchymal stem cells can be identified through their swirl-shaped morphology and the level of expression of the basic cell surface markers CD73(+), CD105(+), CD34(-), and CD45(-), and they exhibit pluripotency and immune response. It also has a control function.
  • step (a) may be performed by suspension culture of stem cells in a multi-well culture vessel.
  • the term “suspension culture” refers to culturing cells to be cultured in a floating state in a culture medium without being immobilized on a substrate or the like. Accordingly, the term “suspension culture” is used in the same sense as “3-dimensional culture”. Adhesion-dependent stem cells cause cell aggregation during suspension culture, and cells floating alone that are not included in this aggregation die by inducing apoptosis. do. According to the present invention, by suspension culture of stem cells in multiple wells having a plurality of wells, cell aggregates having a size according to the size of the wells are formed. Thus, the present invention can obtain a large amount of standardized stem cell aggregates having the same size and shape.
  • cell aggregate refers to a three-dimensional structure formed by self-aggregation of cells cultured in an environment such as a suspension culture in which three-dimensional growth is permitted rather than a monolayer. of cell aggregates.
  • Cell aggregates produced as a result of three-dimensional culture provide an environment similar to in vivo tissues from which stem cells are derived, and may be spherical or non-spherical depending on the size and number of self-assembled cells.
  • Spheroidal cell aggregates are called spheroids, but spheroids do not have to be geometrically perfect spheres.
  • cell culture medium refers to a mixture for cell growth and proliferation in vitro, including elements essential for cell growth and proliferation, such as sugars, amino acids, various nutrients, and minerals.
  • Components that may be additionally included in the cell culture medium are, 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, D-calcium pantothenate, folic acid, riboflavin,
  • the medium for cell culture according to the present invention may be artificially prepared and used, or a commercially available medium may be purchased and used.
  • Examples of commercially available culture media are Iscove's Modified Dulbecco's Medium (IMDM), Alpha Modification of Eagle's Medium ( ⁇ -MEM), Nutrient Mixture F-12 (F12) and Dulbecco's Modified Eagle Medium: Nutrient Mixture (DMEM/F12). F-12), but is not limited thereto.
  • the multi-well culture vessel may have a size of 300 - 500 ⁇ m per well. More preferably, it has a size of 350-450 ⁇ m, and most preferably, it may have a size of about 380-420 ⁇ m.
  • the suspension culture is achieved by dispensing 300-500, preferably 350-450, more preferably about 380-420 cells per well in the multi-well culture vessel.
  • the TGF- ⁇ may be at least one selected from TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3, preferably TGF- ⁇ 3.
  • the step (b) may be performed by orbital shaking culture of the cell aggregate in a floating state.
  • Shaking culture in which rotation is applied during cell culture has the advantage of being able to more smoothly supply nutrients and oxygen to the three-dimensional cell aggregate.
  • the rotational speed when culturing the three-dimensional cell aggregate with shaking is very important.
  • the quality and yield of extracellular vesicles may be very deteriorated, such as inducing apoptosis with the appearance of non-homogeneous cell aggregates, and cell aggregates at a speed of 40 rpm or more
  • the stress applied to the cells increases.
  • TGF- ⁇ is applied to cell aggregates, there is an effect of suppressing stress applied to cells due to an increase in rotation speed.
  • Rotation shaking culture of the present invention can be carried out at a rotational speed of 50 - 70 rpm, preferably 53 - 67 rpm, more preferably 55 - 65 rpm, more preferably 57 - 63 rpm.
  • the extracellular vesicles of the present invention may be obtained by a method further comprising separating the extracellular vesicles from the culture solution obtained in step (b) by centrifugation a plurality of times.
  • the extracellular vesicles of the present invention have an average diameter of 100 - 250 nm, more specifically 150 - 220 nm, more specifically 180 - 200 nm, more specifically 185 - 195 nm have an average diameter. Extracellular vesicles having a fine diameter in this range are called exosomes (FIG. 5a).
  • stem cell-derived extracellular vesicles for example, exosomes of the present invention show a clear difference in protein expression profile compared to exosomes obtained by conventional methods.
  • the proteins (COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF- ⁇ 1 and TGM2) highly expressed by the stem cell-derived extracellular vesicles of the present invention are obtained by two-dimensional culture or TGF- ⁇ 1.
  • the composition of the exosomes of the present invention itself has a novel composition that has not previously existed.
  • the stem cell-derived extracellular vesicles of the present invention are positive for at least one protein selected from adipocyte plasma membrane-related proteins, prolyl 3-hydroxylase 1 and prostaglandin G/H synthase 2. .
  • the three proteins are not detected in stem cell-derived exosomes obtained by applying only two-dimensional culture.
  • prostaglandin G/H synthase 2 is a protein that is not detected in stem cell-derived exosomes obtained by applying only two-dimensional culture or three-dimensional culture without TGF- ⁇ 3 treatment. It can be seen that has a completely new protein expression profile (FIG. 19d).
  • Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases or autoimmune diseases comprising the stem cell-derived extracellular vesicles as an active ingredient.
  • stem cell-derived extracellular vesicles of the present invention Since the stem cell-derived extracellular vesicles of the present invention have already been described in detail, the description thereof is omitted to avoid excessive redundancy.
  • prevention means suppressing the occurrence of a disease or disease in a subject who has never been diagnosed with the disease or disease, but is likely to suffer from such disease or disease.
  • the term “treatment” refers to (a) inhibition of the development of a disease, disorder or symptom; (b) alleviation of the disease, condition or symptom; or (c) eliminating the disease, disorder or condition.
  • the compositions of the present invention serve to suppress, eliminate or alleviate 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 composition of the present invention may be a composition for treating these diseases by itself, or may be administered together with other pharmacological ingredients having a therapeutic effect on inflammatory or autoimmune diseases and applied as a therapeutic adjuvant for these diseases. Accordingly, the term “treatment” or “therapeutic agent” in the present specification includes the meaning of "therapeutic aid” or "therapeutic aid”.
  • the term “administration” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the body of the subject, and has the same meaning as “implantation” or “infusion”. .
  • the term "therapeutically effective amount” refers to an amount of the composition contained in an amount sufficient to provide a therapeutic or prophylactic effect to a subject to whom the composition of the present invention is to be administered, and thus “prophylactically effective amount” meaning to include
  • the term “subject” includes, without limitation, 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.
  • the composition of the present invention has a preventive or therapeutic effect on various inflammatory diseases.
  • Inflammatory diseases to which the pharmaceutical composition of the present invention can be applied are not particularly limited as long as they are known as inflammatory diseases in the art.
  • Autoimmune diseases or inflammatory diseases prevented or treated by the composition of the present invention are, for example, rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic fibrosing alveolitis, polymyositis, Dermatomyositis, localized dermatosclerosis, systemic dermatosclerosis, colitis, inflammatory bowel disease, Sjorgen's syndrome, Raynaud's phenomenon, Bechet's disease, Kawasaki's disease, primary biliary sclerosis (primary biliary sclerosis), primary sclerosing cholangitis, ulcerative colitis, graft-versus-host disease (GVHD) and
  • the cystitis prevented or treated with the composition of the present invention may be at least one selected from interstitial cystitis, chronic cystitis, and ketamine-induced cystitis, preferably interstitial cystitis.
  • the stem cell-derived extracellular vesicles of the present invention when administered to an in vivo endotoxemia animal model, since the concentrations of TNF- ⁇ and IL-6 are significantly reduced, It was specifically confirmed that the treatment effect of toxemia was excellent.
  • the stem cell-derived extracellular vesicles of the present invention when administered to an in vivo interstitial cystitis/bladder pain syndrome (IC/BPS) animal model, the bladder inner wall collapsed during the IC/BPS induction process It was specifically confirmed that the treatment showed excellent therapeutic efficacy for interstitial cystitis/bladder pain syndrome, such as recovery, alleviation of inflammation, recovery of intravesical pressure, and similar urination cycle to that of the control group.
  • IC/BPS interstitial cystitis/bladder pain syndrome
  • the pharmaceutical composition of the present invention may include the stem cell-derived extracellular vesicles alone, or may further include one or more pharmaceutically acceptable carriers, excipients, or diluents.
  • a 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 and the like.
  • the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol, and the like, and may further include a stabilizer and a preservative.
  • Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, and the like in addition to the above components.
  • a lubricant for example, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, and the like in addition to the above components.
  • composition of the present invention can be administered to mammals including humans by any method.
  • parenteral administration methods include, but are not limited to, intravenous administration, intramuscular administration, intraarterial administration, intramedullary administration, intrathecal administration, suprachoroidal injection, transdermal administration, subcutaneous administration, intraperitoneal It may be administration, intranasal administration, enteral administration, topical administration, sublingual administration or intrarectal administration, preferably intravenous administration.
  • composition of the present invention may be formulated into a preparation for oral administration or parenteral administration according to the administration route as described above.
  • composition of the present invention may be formulated into powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, etc. using a method known in the art.
  • preparations for oral use may be obtained by combining the active ingredient with a solid excipient, which is then milled and, after adding suitable auxiliaries, processed into a mixture of granules to obtain tablets or dragees.
  • excipients examples include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, and the like, fillers such as gelatin, polyvinylpyrrolidone, and the like may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant, if desired. Furthermore, the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, and a preservative.
  • sugars including lactose, dextrose, sucrose, sorbitol, mannitol,
  • preparations for parenteral administration they may be formulated in the form of injections, ointments, creams, lotions, oils, gels, aerosols and nasal inhalations by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, which is a generally known formula for all pharmaceutical chemistry.
  • the pharmaceutical composition of the present invention may be prepared in any one form selected from the group consisting of oral preparations, injections and ointments, more preferably injections.
  • the pharmaceutical composition of the present invention contains the stem cell-derived extracellular vesicles in an effective amount, it can provide desirable effects of preventing, improving or treating inflammatory diseases.
  • the term 'effective amount' refers to an amount that exhibits a higher response than that of the negative control group, and preferably refers to an amount sufficient to improve or treat inflammatory diseases, particularly interstitial cystitis.
  • the stem cell-derived extracellular vesicles may be included at 5x10 8 to 5x10 10 particles/ml, preferably 5x10 9 to 5x10 10 particles/ml, and more preferably 1x10 10 to 2x10 10 particles/ml, based on the total content of the pharmaceutical composition. there is.
  • the content of the stem cell-derived extracellular vesicles is less than the lower limit, the cell viability is excellent, but the effect of improving or treating inflammatory diseases may not appear to a desired extent.
  • the concentration exceeds the upper limit the improvement or treatment effect of inflammatory diseases does not increase as much as the concentration increases, or toxicity may occur.
  • 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 shown, but side effects such as cytotoxicity are not shown.
  • the effective amount of the stem cell-derived extracellular vesicles included in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is formulated.
  • the total effective amount of the pharmaceutical composition of the present invention may be administered to the patient in a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time.
  • the pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease.
  • a suitable dosage of the pharmaceutical composition of the present invention will be variously prescribed depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, route of administration, excretion rate and reaction sensitivity.
  • a preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg/kg for adults.
  • Another aspect of the present invention relates to a pharmaceutical composition for wound healing comprising the stem cell-derived extracellular vesicles as an active ingredient.
  • the stem cell-derived extracellular vesicles of the present invention when administered to an animal model that produced an in vivo wound, it was specifically confirmed that the area of the wound site was significantly reduced (FIG. 7).
  • stem cell-derived extracellular vesicles of the present invention significantly increase cell migration and have excellent wound healing effects, they can be usefully used for wound healing.
  • Another aspect of the present invention relates to a therapeutic use of the stem cell-derived extracellular vesicles (for use in therapy).
  • stem cell-derived extracellular vesicles of the present invention Since the stem cell-derived extracellular vesicles of the present invention have already been described in detail, the description thereof is omitted to avoid excessive redundancy.
  • the therapeutic use may be for the treatment of an inflammatory disease or an autoimmune disease, preferably an inflammatory disease.
  • the inflammatory disease or autoimmune disease is cystitis, rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic fibrosing alveolitis, polymyositis, dermatomyositis, localized scleroderma, systemic skin Sclerosis, colitis, inflammatory bowel disease, Sjorgen's syndrome, Raynaud's phenomenon, Bechet's disease, Kawasaki's disease, primary biliary sclerosis, primary sclerosing cholangitis (primary sclerosing cholangitis), ulcerative colitis, graft-versus-host disease (GVHD) or Crohn's disease.
  • cystitis cystitis, rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic fibrosing alve
  • the cystitis may be at least one selected from interstitial cystitis, chronic cystitis, and ketamine-induced cystitis.
  • another aspect of the present invention relates to a method for preventing, improving or treating an inflammatory disease or an autoimmune disease, comprising administering the stem cell-derived extracellular vesicles to a subject in need thereof.
  • stem cell-derived extracellular vesicles of the present invention Since the stem cell-derived extracellular vesicles of the present invention have already been described in detail, the description thereof is omitted to avoid excessive redundancy.
  • the “subject” refers to a mammal that is a subject of prevention, improvement, treatment, observation, or experimentation, and is preferably a human or mammal in need of prevention, improvement, and/or treatment of an inflammatory disease or an autoimmune disease.
  • Example 1 3-dimensional cell culture of mesenchymal stem cells
  • AggreWell TM 400 (STEMCELL Technologies; #34425) containing about 5900 microwells of 400 ⁇ m per well was treated with F127 solution, and about 400 umbilical cord-derived mesenchymal stem cells per well (Konkuk University) Bioethics Committee Approval No.: 7001355-202010-BR-407) was seeded to produce uniform spheroids ranging in diameter from 120 to 200 ⁇ m. After seeding spheroids in a culture solution containing TGF- ⁇ 3 in a non-adsorption culture dish, shaking culture was performed at 60 rpm and 37 ° C. with an orbital shaker (INFORS HT Celtron; # 69455) for 3 days. . After 3 days, exosomes were isolated from the obtained culture medium.
  • the culture solution was centrifuged at 300g for 10 minutes to remove cell debris, centrifuged at 2000g for 10 minutes, the supernatant was transferred to a new tube, centrifuged again at 10,000g for 30 minutes, and the supernatant was centrifuged at 187,000g for 2 hours.
  • the pellet was suspended in 200 ⁇ l of PBS.
  • concentration gradient centrifugation 50%, 30%, 10%
  • concentration gradient centrifugation was performed using optiprep (BioVision; M1248).
  • An exosome sample (suspension) for isolation was loaded by mixing with a 50% optiprep solution, and centrifuged at 120,000g for 2 hours to obtain between 10% and 30% exosomes. The obtained exosomes were centrifuged once more at 187,000g and suspended in 100 ⁇ l of PBS.
  • NTA nanoparticle tracking analysis
  • PBMC peripheral blood mononuclear cells
  • the exosome (T-3aD-EV) of the present invention obtained through 3D shaking culture conditions treated with TGF- ⁇ reduced the number of proliferating T cells in the MSC-treated group from 43.1% to 9.6%, making it a positive control group. showed the most excellent T cell inhibitory effect while showing a remarkable reduction rate close to 80% compared to (FIGS. 4b and 4c).
  • the size of exosomes was investigated through dynamic light scattering (DLS) analysis using a Nano Zetasizer (Malvern Instruments, Malvern, UK), and the number of EVs was measured using a nanoparticle tracking analyzer NS300 (Nanosight, Amesbery, UK). and measured (FIG. 5a).
  • the shape and structure of exosomes were analyzed using a transmission electron microscope (TEM, JEM-1010, Nippon Denshi, Tokyo, Japan) at 80 kV, and as a result of observation, the shape of exosomes was cup or spherical (Fig. 5b).
  • Exosomes were attached on a grid (Formvar/Carbon 300 Mesh, Copper_FCF300-CU 50/pk), and negative staining was performed using 1% phosphotungstic acid hydrate (sigma, P4006).
  • CD9 ab263023, abcam
  • CD63 ab134045, abcam
  • Flotillin-1 Flotillin-1
  • Alix a2171, CST proteins
  • the fluorescence intensity generated from the labeled antibody was measured using a flow cytometry (Beckman Coulter, CytoFlex Flow Cytometry Analyzer). As a result, it was confirmed that more than 96% of the fluorescence expression levels of CD9, CD63, and CD81 were expressed in the exosomes (FIG. 5d). Judging from the fact that 96-98% of the exosome-positive marker was expressed in the isolated exosome, it can be confirmed that a homogeneous exosome was isolated.
  • 2D-EV Exosomes using only 2D culture conditions
  • a3D-EV exosomes using only 3D shaking culture conditions
  • T-a3D-EV exosomes obtained by adding TGF- ⁇ 3 to the culture medium under 3D shaking culture conditions
  • TGF- ⁇ 1 was not detected in 2D-EV, and it was measured at a concentration of 86.3 pg/1x10 9 in a3D-EV and 310.6 pg/1x10 9 in T-a3D-EV, resulting in T-a3D-EV It was confirmed that the TGF- ⁇ 1 expression level of the a3D-EV increased by about 3.5 times or more.
  • the contents of active and inactive forms of TGF- ⁇ 1 were measured through acidication using each EV that had undergone the purification process, the content of TGF- ⁇ 1 in T-a3D-EV was significantly higher than that of other groups (Fig. 5e). ).
  • the TGF- ⁇ 1 active form was measured at a concentration of 3.8 pg/1x10 9 in a3D-EV and 36.9 pg/1x10 9 in T-a3D-EV, indicating that the TGF- ⁇ 1 active content of T-a3D-EV was a3D It was found to be about 10 times higher than that of -EV.
  • TGF- ⁇ 1 inactive form was measured at a concentration of 82.4 pg/1x10 9 in a3D-EV and 273.6 pg/1x10 9 in T-a3D-EV, indicating that the content of TGF- ⁇ 1 active form in T-a3D-EV was a3D-EV. It was found to be about 3.3 times higher than that of EV.
  • the TGF- ⁇ 1 content was obtained by applying only 3D shaking culture conditions without the addition of TGF- ⁇ 3.
  • TGF- ⁇ 1 was not detected in 2D-EVs obtained only by 2D culture.
  • the protein TGF- ⁇ 1 is highly expressed only in the T-a3D-EVs obtained by the method of the present invention.
  • NHDF was seeded with 5x10 4 cells in the upper well of a 24 well 8.0 ⁇ m polycarbonate membrane Transwell (3422, costar), and DMEM high glucose (D6429, sigma) was mixed with 10% FBS and 1% penicillin streptomycin (Cat no. 1514- 163, gibco) was cultured in media. After 24 hours, DMEM high glucose SFM (Serum free medium) containing 1% penicillin streptomycin was added to the upper well, and each extracellular vesicle was diluted to a concentration of 1x10 9 particles/well in SFM (Serum free medium) to the lower well. processed.
  • SFM Serum free medium
  • Raw264.7 cells were seeded 1.5x10 5 cells in a 48well plate and after 12 hours, LPS 10 ng/ml (L4391-1MG, Sigma) and Dexamethasone 10 uM (50002220, biogems), each exosome (1x10 9 particles) It was treated with 500 ⁇ l of 10% exosome depleted DMEM-high glucose. Nitric oxide was measured by measuring absorbance at 540 nm after treatment with the culture medium and reaction with Griess reagent (0.1% N-(1-naphthyl) ethylenediamide dihydrochloride and 1% sulfanilamide in 5% phosphoric acid) from the culture medium after 24 hours.
  • Raw264.7 was stimulated with LPS, treated with Dexamethasone, 2D-EV, a3D-EV, and T-a3D-EV, respectively, and then the concentration of nitric oxide was checked in the culture medium. And, as a result of confirming the mRNA expression level of each inflammatory response factor from the cells, it was confirmed that the inflammatory response induced by LPS in Raw264.7 cells could be significantly reduced by T-a3D-EV (FIG. 9).
  • the T-a3D-EV of the present invention inhibits 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 preventing or treating inflammatory diseases. know that it can.
  • mice Eight-week-old female C57BL/6 mice were acclimatized for one week and then used in the experiments. A total of 200 ⁇ l of LPS 2.5 mg/kg alone or together with each of the exosomes (5x10 9 particles) was injected into the tail-vein using a 28G needle. After 2 hours, the mice were sacrificed, the spleens were removed, washed with PBS, and stored at -80 °C until use.
  • Protein was extracted from the spleen using RIPA buffer (CBR002, LPS solution) containing protease inhibitor cocktail (87786, Invitrogen), and mTNF-alpha (BGK06804, peprotech), mIL-6 (BGK08505, peprotech), mIL- 10 (BGK18893, peprotech) ELISA kit was used to measure the concentration of each cytokine.
  • RIPA buffer CBR002, LPS solution
  • protease inhibitor cocktail 87786, Invitrogen
  • mTNF-alpha BGK06804, peprotech
  • mIL-6 BGK08505, peprotech
  • mIL- 10 BGK18893, peprotech
  • Human urothelium cells SV-HUC-1 (ATCC, CRL-9520), were treated with exosomes at different concentrations, and cell proliferation was confirmed.
  • the SV-HUC-1 cells treated with each of the exosomes were lysed using RIPA buffer (CBR002, LPS solution) containing protease inhibitor cocktail (87786, Invitrogen), and whole cell lysate (WCL) was separated.
  • the obtained protein was quantified through BCA analysis (23227, Thermo), electrophoresed with 4-12% Bis-Tris Flus Gels (NW04125BOX, Invitrogen/NW04122BOX, Invitrogen), and transferred to NC membrane (IB23001, Invitrogen).
  • the primary antibody (1:1000) was incubated overnight at 4°C and washed three times with 1x TBST (TLP-118.1, TrnasLab).
  • the secondary antibody was reacted at room temperature for 2 hours and washed with 1x TBST. All antibodies were diluted in 1x blocking buffer (TLP-115.1G, Translab) and confirmed using InvitrogenTM iBrightTM Imagers (CL-1000).
  • the primary and secondary antibodies used were the following products: P-AKT (sc-293125, santa cruz), T-AKT (CSB-PA000855, cusabio), P-ERK (CSB-PA000749, cusabio), T-ERK (B7074, Tebu-bio), ⁇ -actin (sc-47778, santa cruz), HRP linked anti-rabbit IgG (7074, CST), and HRP linked anti-mouse IgG (7076, CST).
  • SV-HUC-1 cells were cultured in F-12K Nutrient Mixture (21127-022, Gibco) in media containing 10% FBS and 1% Penicillin streptomycin (15140-163, GIbco).
  • F-12K Nutrient Mixture 21127-022, Gibco
  • Penicillin streptomycin 15140-163, GIbco
  • 1.5x10 5 cells were seeded in the upper well of a 24 well 8.0 ⁇ m polycarbonate membrane Transwell (3422, costar), and the exosomes were treated in the lower well after 12 hours. After 24 hours, it was washed with DPBS (10010-031, GIbco), treated with 4% paraformaldehyde (P2031, Biosesang), and fixed at room temperature for 20 minutes.
  • the fixed cells were washed with DPBS, incubated with 100% methanol at room temperature for 20 minutes, and then stained with 1% crystal violet (V5265, sigma) for 15-20 minutes at room temperature. After washing the staining solution 2-3 times with DPBS, the cells remaining on the top of the membrane were wiped off using a cotton swab. Observations were made after removing cell debris wiped off with DPBS wash. In order to confirm the degree of cell migration, the mean value of image J was compared with the confluency of the cells and displayed as a graph.
  • V5265 crystal violet
  • mice 8-week-old BALB/cAnNCrlOri female mice were given an adaptation period of 2 weeks.
  • Alphaxane and Rumpun were mixed at a ratio of 4:1 and anesthetized by intraperitoneal injection of 90 ul per mouse.
  • a catheter 382412, BD was inserted into the urethra to remove urine from the inside of the bladder, and washed with 50 ⁇ l of PBS.
  • 5 mg/ml of protamine sulfate (P3369, Sigma) was injected and washed with PBS after 30 minutes. After injecting 30 ⁇ g/ml of LPS (L4391, Sigma), the cells were washed with PBS. After confirming the recovery of the mice on the hot plate, they were bred for one week.
  • RNA cDNA was synthesized using rTaq Plus 5x PCR master mix (EBT-1319, ELPISBIO), and gene expression was performed using HiPi Real-Time PCR 2x Master Mix (SYBR green, ROX) (EBT-1802, ELPISBIO). The amount was confirmed (7500, Amersham Phamacia Biotech).
  • a polyethylene catheter PE-50; Becton-Dickinson, Parsippany, NJ, USA
  • PE-50 Becton-Dickinson, Parsippany, NJ, USA
  • the catheter was then pierced through the animal's back through the subcutaneous space and fixed externally.
  • an indwelling catheter to the bladder was connected via a T-tube to a pressure transducer (Research Grade Blood Pressure Transducer; Harvard Apparatus, Holliston, MA, USA) with a bidirectional valve and a microinfusion pump (PHD ULTRATM Syringe; Harvard Apparatus) was used.
  • the amount of urine output was continuously recorded for 8 minutes by a fluid collector connected to a transducer (Research Grade Isometric Transducer; Harvard Apparatus) while sterile saline was injected into the bladder at a rate of 0.4 mL/min.
  • IVP intraavesical pressure
  • output volume were continuously recorded using the iWork IX-RA-834 data acquisition system with LabScribe 3.637 software (iworks, 62 Littleworth Road Dover, NH 03820, USA).
  • mice induced by IC/BPS show frequent urination due to bladder pain and decreased function.
  • intravesical pressure was recovered and urination cycles similar to those of the control group were recovered (Fig. 17 and FIG. 18).
  • T-a3D-EV extracellular vesicles
  • BPS bladder pain syndrome
  • IC interstitial cystitis
  • Protein (20 ⁇ g) isolated from WJ-MSC-derived exosomes was freeze-dried using a centrifugal vacuum concentrator (LABCONCO, CentriVap, Missouri, USA). The pellet was lysed with 100 mL lysis buffer consisting of 5% sodium dodecyl sulfate and 50 mM triethylammonium bicarbonate (pH 7.55, ThermoFisher Scientific). Samples were processed with the S-TrapTM Micro Spin Column Digestion Protocol according to the manufacturer's protocol except for the trypsin/LysC mixture (Promega, Madison, WI, USA). The lyophilized peptide was dissolved in 0.1% formic acid.
  • the protein amount was measured with a BCA protein assay kit (Pierce), and the peptide concentration was measured by absorbance at 205 nm using a NanoDrop One spectrophotometer (ThermoFisher Scientific).
  • Digestion steps were performed with the S-TrapTM Mini Spin Column Digestion Protocol according to the manufacturer's protocol, except for the trypsin/LysC mixture (Promega, Madison, WI, USA).
  • Peptides in elution buffer water with 0.2% formic acid and water with 50% acetonitrile
  • each sample was labeled with 100 ⁇ g of 10-plex TMT reagent (Lot number: UH284251), excluding TMT10-130N, TMT10-130C and TMT10-131, according to the instructions of the manufacturer (Thermo Scientific).
  • TMT-labeled samples were pooled at a 1:1:1:1:1:1 ratio before desalting by solid phase extraction (Sep-Pak, Waters, Milford, MA, USA) using a C18 cartridge. .
  • the sample was dissolved in 200 ⁇ L mobile phase A and injected into a 2,100 ⁇ L sample loop.
  • Solution B at 5-5% for 15 minutes, again at 5-45% for 62.5 minutes, at 45-60% for 5 minutes, at 60-60% for 12.5 minutes, at 60-5% for 7.5 minutes, and at 5-5%
  • the column was washed with 100% and 50% mobile phase B for 30 minutes each.
  • the eluate was collected every 30 seconds using an FRC-10A fraction collector from Shimadzu Prominence (Shimadzu, Tokyo, Japan). 24 fractions were obtained by mixing the resulting 168 fractions in a concatenated manner. Each fraction was lyophilized and stored at -20 °C until use.
  • eluent A was 5% dimethyl sulfoxide with 0.1% formic acid
  • eluent B was 80% acetonitrile with 0.1% formic acid and 5% dimethyl sulfoxide at 50 °C.
  • Eluent B 5-40% over 150 min; 40-95% over 2 min; 23 min hold at 95%; 95-5% over 10 min; 15 min hold 5%
  • Elution was performed with a 150 minute gradient at a flow rate of /min.
  • 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.
  • the target value for the full scan mass spectrum was 3,000,000 at a maximum injection time of 100 ms and a resolution of 60,000 (m/z 400).
  • the ion target value of MS/MS was set to 100,000 at a maximum injection time of 50 ms and a resolution of 15,000 (m/z 400) by applying a normalized collision energy (27%) and an isolation window (1.7 m/z).
  • Dynamic exclusion of repeated peptides was applied for 20 seconds. Three replicates were performed for each biological sample.
  • LC parameters were performed in the same way as for exosome samples and MS parameters were as follows: scan range (m/z) 350-1500 in MS1; 120,000 resolution on MS1, 45,000 resolution on MS2; AGC target 3e6 of MS1, AGC target 1e5 of MS2; The maximum IT(ms) in MS1 is 50, and the maximum IT(ms) in MS2 is 96.
  • the period of the data-dependent mode is an isolation window (0.7 m/z), a fixed first mass of 110 m/z and a period of 32%. It was set to trigger MS/MS up to the 20 most abundant precursors per cycle at HCD collision energies of .
  • LFQ intensity is the output of the Max-LFQ algorithm for exosome samples.
  • the reporter ion was set to a modified 7plex TMT at 10plex parameters for WJ-MSC proteomic quantification.
  • FDR was set to 0.01 at both protein and peptide spectral match levels. Proteins identified by at least one unique peptide were used. Other settings were kept at their default values.
  • the software Perseus (version 1.6.14.0) was used for differential analysis of proteomic data between samples. First, all data identified from various contaminants and station databases were removed. Perseus software was used for PCA to assess the quality of the data set. Normalized protein abundance (abundance) values for exosomal EV samples and TMT labeled samples (normalized via median subtraction) were converted to a log2 scale. Three replicates of each sample were grouped and a minimum of three valid values were required for at least one grouping. Missing values were replaced with random numbers drawn from a normal distribution with default parameters (width: 0.3, downward shift: 1.8). A t-test was performed using Benjamini-Hochberg FDR (0.05 cut-off) to find statistically significant differences between samples.
  • Hierarchical clustering of enriched Z-score values was performed using InstantClue software. Hierarchical clustering was performed using the Euclidean distance as the metric and the average linkage parameter. Protein abundance (Log2 value) values belonging to each cluster are presented as box plots with upper and lower quartiles, median, minimum and maximum values, all individual data points, along with the y-axis determined by InstantClue. Data were processed using FunRich v3.1.3 and ShinyGO v0.61 to perform gene ontology (GO). Evaluation of the enriched signature genes was performed with the GSEA algorithm using 1,000 permutations and default parameters.
  • T-a3D-EVs the protein contents of 2D-EVs, a3D-EVs and T-a3D-EVs were analyzed using LC-MS-based label-free quantification (LFQ). Proteins were extracted from the same amount of EV (20 mg) obtained from each exosomal EV sample, and a total of 394 protein groups were identified in triplicate. Among them, 138 ⁇ 1 protein groups were quantified for 2D-EVs, 304 ⁇ 5 protein groups for a3D-EVs, and 320 ⁇ 14 protein groups for T-a3D-EVs, respectively.
  • LFQ label-free quantification
  • FIG. 20A A hierarchical clustering analysis using Z-scores normalizing the abundance with a Benjamini-Hochberg false discovery rate (FDR) of 0.05 or less was performed and classified into four clusters (FIG. 20a).
  • FDR Benjamini-Hochberg false discovery rate
  • FIG. 20A clusters that increase only in each sample set can be found (Cluster 2: 47 protein groups of T-a3D-EVs, Cluster 3: 11 protein groups of a3D-EVs, Cluster 4: 47 protein groups of 2D-EVs). 63 protein groups each).
  • Cluster 1 with 82 protein groups showed increased proteins in both a3D-EVs and T-a3D-EVs, suggesting that these proteins are characteristic of 3D culture.
  • PCA principal component analysis
  • DEP Densired protein
  • FDR 5%, log2(ratio) 31 or £ -1 a binary comparison method
  • FIG. 20c a binary comparison method
  • DEP is an analysis of proteins with significantly high or low expression.
  • Volcano plots of a3D-EV/2D-EV and T-a3D-EV/2D-EV showed that the number of DEPs induced by culture conditions (2D and 3D) was greater than that induced by TGF- ⁇ 3 treatment in 3D. appear.
  • the volcano plot of T-a3D-EV/a3D-EV showed that 53 proteins were up-regulated while 14 proteins were down-regulated in the T-a3D-EV group after TGF- ⁇ 3 treatment.
  • GSEA Gene Set Enrichment Analysis
  • FIG. 21a shows the gene set enrichment analysis results in this analysis, and the enriched gene set, standardized enrichment score (NES) and p-value are presented. The higher the NES and the lower the p-value, the more likely the finding is to be significant. big. Gene groups enriched in this analysis are shown in FIG. 21B.
  • the exosomes obtained by the method of the present invention have differentiated efficacy from the exosomes of the comparative group due to the difference in protein expression.
  • 68 DEPs (40 DEPs in T-a3D-EV/2D-EVs, and 28 DEPs in the region common between T-a3D-EVs/2D-EVs and T-a3D-EVs/a3D-EVs) dogs) could be selected as DEPs related to TGF- ⁇ 3 treatment in T-a3D-EVs/2D-EVs comparison, and 53 DEPs specific to TGF- ⁇ 3 treatment were found in T-a3D-EVs/a3D-EVs (T-a3D-EVs).
  • HuRi Human Reference Protein Interaction Mapping Project
  • HuRi Human Reference Protein Interactome Mapping Project
  • SDCBP is a protein that mainly interacts with various proteins, and this protein is known to be involved in immune regulation, exosome biosynthesis, and tumorigenesis.
  • the specific proteins found in the T-a3D-EVs support the results confirmed in animal and cell experiments.

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Abstract

La présente invention concerne des vésicules extracellulaires dérivées de cellules souches, et leur utilisation. Les vésicules extracellulaires dérivées de cellules souches de la présente invention permettent à une ou plusieurs protéines choisies parmi COL6A1, COL6A3, TNC, EIF4E, HSP90AB1, HSP90B1, RAC1, TGF-β1 et TGM2 d'être fortement exprimées, de manière à réduire remarquablement les quantités de cytokines inflammatoires telles que TNF-α et IL-6, et sont donc efficaces dans la prévention, la réduction, le soulagement ou le traitement de diverses maladies inflammatoires. En particulier, lorsqu'elle sont administrées à un modèle animal de cystite interstitielle/syndrome de douleur de la vessie (IC/BPS), les vésicules extracellulaires dérivées de cellules souches de la présente invention permettent le revêtement de la vessie, endommagé lors de l'induction de CI/BPS, d'être restauré, le degré d'inflammation d'être réduit, et la pression interne de la vessie d'être restauré, et présentent un cycle de vide similaire à celui d'un groupe témoin, et ainsi les vésicules extracellulaires ont l'excellent effet de traitement de la cystite interstitielle/du syndrome de douleur de la vessie. De plus, les vésicules extracellulaires dérivées de cellules souches de la présente invention augmentent remarquablement la migration de cellules, et ont un excellent effet de cicatrisation des plaies, et peuvent ainsi être efficacement utilisées pour la cicatrisation de plaies.
PCT/KR2022/001339 2021-07-06 2022-01-25 Vésicules extracellulaires dérivées de cellules souches et leur utilisation WO2023282424A1 (fr)

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WO2014042292A1 (fr) * 2012-09-12 2014-03-20 연세대학교 산학협력단 Composition comprenant un activateur de la protéine kinase c destiné à favoriser l'adhérence de cellules souches et méthode destinée à favoriser l'adhérence de cellules souches
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