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WO2022108165A1 - Procédé de production d'exosomes isolés à partir de cellules souches mésenchymateuses dérivées de cellules souches pluripotentes induites, et utilisation associée - Google Patents

Procédé de production d'exosomes isolés à partir de cellules souches mésenchymateuses dérivées de cellules souches pluripotentes induites, et utilisation associée Download PDF

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WO2022108165A1
WO2022108165A1 PCT/KR2021/015286 KR2021015286W WO2022108165A1 WO 2022108165 A1 WO2022108165 A1 WO 2022108165A1 KR 2021015286 W KR2021015286 W KR 2021015286W WO 2022108165 A1 WO2022108165 A1 WO 2022108165A1
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cells
stem cells
minutes
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mesenchymal stem
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Korean (ko)
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이미연
나득채
김경운
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Oncoinsight Co Ltd
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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Definitions

  • It relates to a method for producing an exosome isolated from dedifferentiated stem cell-derived mesenchymal stem cells and a use thereof.
  • Mesenchymal stromal cells can be obtained from adult tissues such as bone marrow, adipose tissue, umbilical cord blood, and umbilical cord, and have the form of fibroblasts.
  • the stem cells can proliferate indefinitely in vitro, and unlike blood stem cells, they can be differentiated into various types of important cell lines such as fat, osteocytes, chondrocytes, cardiomyocytes, and nerve cells. Research is being done.
  • autoimmune disease is a disease in which immune cells that defend the body from external invaders such as bacteria, viruses, and foreign substances attack themselves. Since autoimmune diseases can appear in all organs and tissues of the human body, all cells in the body are attacked or only cells in specific organs are destroyed. In addition, autoimmune diseases selectively attack specific organs or the whole body, such as rheumatoid arthritis.
  • autoimmune diseases As the types and symptoms of autoimmune diseases are diverse, the treatment methods for autoimmune diseases are also diverse.
  • the goal of treatment for autoimmune diseases is to relieve symptoms, preserve function, or block the pathogenesis of the disease.
  • Drugs for this purpose include steroids, nonsteroidal anti-inflammatory drugs, immunosuppressants, and the like, but long-term administration of these drugs often causes serious side effects.
  • One aspect comprises the steps of isolating cells from a biological sample; preparing dedifferentiated stem cells from the isolated cells; Inducing mesenchymal stem cells from the prepared dedifferentiated stem cells; And to provide a method for producing an exosome comprising the step of isolating the exosome from the induced mesenchymal stem cells.
  • Another aspect is to provide a pharmaceutical composition for preventing or treating an autoimmune disease comprising the exosome prepared by the method for producing the exosome.
  • Another aspect is to provide a method for preventing or treating an autoimmune disease comprising administering the exosome prepared by the method for producing the exosome.
  • One aspect comprises the steps of isolating cells from a biological sample; preparing dedifferentiated stem cells from the isolated cells; Inducing mesenchymal stem cells from the prepared dedifferentiated stem cells; And it provides a method for producing an exo-some comprising the step of isolating the exosome from the induced mesenchymal stem cells.
  • the "biological sample” refers to a sample derived from an organism.
  • the organism may be a mammal including a human.
  • the biological sample may be derived from a tissue or body fluid of the body.
  • the body fluids include blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, intestinal fluid, genitourinary fluid, breast milk, lymphatic fluid, semen, cerebrospinal fluid, intratracheal fluid , ascites, cystic tumor body fluid, amniotic fluid, or a combination thereof.
  • the blood may be derived from peripheral blood.
  • the blood may include blood cells.
  • the blood cells may include one or more of red blood cells, white blood cells, and platelets.
  • the white blood cells may include eosinophils, basophils, neutrophils, lymphocytes, monocytes, macrophages, and the like.
  • the cells isolated from the biological cells may be mononuclear cells.
  • the “induced pluripotent stem cell (iPSC)” is also referred to as an induced pluripotent stem cell, and refers to a cell having pluripotency obtained by dedifferentiation from a differentiated cell (eg, a somatic cell). do.
  • the dedifferentiated stem cells can differentiate into various organ cells.
  • the dedifferentiated stem cells can be obtained by reprogramming the cells differentiated by the dedifferentiation inducing factors.
  • the "mesenchymal stem cell (MSC)” refers to a stem cell present in cartilage, bone tissue, adipose tissue, bone marrow stroma, etc. differentiated from mesoderm generated by division of a fertilized egg.
  • the mesenchymal stem cells may be differentiated into osteoblasts, adipocytes, chondrocytes, etc. in vitro, and may express CD73, CD90 and CD105.
  • the mesenchymal stem cells may not express c-kit, CD11b, CD19, CD14, CD34, CD45, CD14, CD79 and HLA-DR.
  • exosomes refers to endoplasmic reticulum having a size of several tens to hundreds of nanometers (about 30 to 200 nm) composed of a double phospholipid membrane identical to the structure of a cell membrane.
  • the exosome may include a protein, a nucleic acid (mRNA, miRNA, etc.) called a exosome cargo.
  • the exosome cargo may include a wide range of signaling factors, and the signaling factors may be specific to a cell type and may be differently regulated according to the environment of the secretory cell.
  • the exosomes are intercellular signaling mediators secreted by cells, and various cellular signals delivered through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells.
  • the exosome may include both a nano-sized vesicle secreted from mesenchymal stem cells and released into the extracellular space or a vesicle having a composition similar to that of the exosome (eg, exosome-like vesicle). have.
  • the "inducing step” may include the following steps:
  • the "inducing step” may further include washing the dedifferentiated stem cells with PBS before step (a).
  • the collagenase refers to an enzyme capable of hydrolyzing collagen or gelatin, which is a kind of hard protein.
  • the collagenase may be any one of types 1, 2, 3 and 4.
  • the collagenase solution may contain collagenase at a concentration of 0.5 to 2 mg/mL.
  • the collagenase solution may contain collagenase type 4 (type IV Collagenase) at a concentration of 1 mg/mL.
  • Step (a) may be to react for 5 to 15 minutes.
  • step (a) may be to react the prepared retrodifferentiated stem cells with a collagenase type 4 solution for 10 minutes.
  • the step (b) may be centrifugation at 1000 to 1200 rpm, preferably centrifugation at 1100 rpm.
  • Step (b) may be centrifuged for 2 to 4 minutes.
  • step (b) may be centrifuging the dedifferentiated stem cells for 3 minutes.
  • the b-FGF (basic fibroblast growth factor), also known as FGF2 or FGF- ⁇ , refers to a growth factor encoded by the FGF2 gene.
  • the b-FGF may be stimulated at 0.5 to 1.5 ng/mL.
  • step (d) may be to stimulate the suspended dedifferentiated stem cells with 1 ng/mL of b-FGF.
  • the "inducing step” may further include stimulation with recombinant human TGF- ⁇ after stimulation with b-FGF.
  • the recombinant human TGF- ⁇ is also referred to as rh TGF- ⁇ , and may be rh TGF- ⁇ 1, rh TGF- ⁇ 2 or rh TGF- ⁇ 3.
  • step of isolating exosomes may include the following steps:
  • step (c) centrifuging the supernatant of the culture solution centrifuged in step (b) at 1500 to 2500 g at 0 to 5° C. for 15 to 30 minutes;
  • step (d) centrifuging the supernatant of the culture medium centrifuged in step (c) at 8,000 to 12,000 g at 0 to 5° C. for 20 to 40 minutes;
  • step (e) filtering the supernatant of the culture medium centrifuged in step (d) with a filter paper;
  • step (f) centrifuging the culture solution filtered in step (e) at 80,000 to 120,000 g for 100 to 140 minutes.
  • Step (a) may be culturing for 48 hours.
  • the “medium” may include essential components necessary for cell growth, survival, and proliferation in vitro .
  • the medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), ⁇ -MEM ( ⁇ -Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), Knockout DMEM, E8 (Essential 8 Medium), SF-DMEM (serum free-Dulbecco Modified Eagle Medium), such as commercially prepared medium or artificially synthesized medium may be used, but is not limited thereto.
  • DMEM Dulbecco's
  • the medium may be ⁇ -Minimum Eagle's Medium.
  • the ⁇ -Minimum Eagle’s Medium is also referred to as ⁇ -MEM, and refers to a medium in which amino acids and vitamins are additionally added to the components of MEM.
  • the ⁇ -MEM may include non-essential amino acids, glutamine, and 2-mercaptoethanol.
  • the ⁇ -MEM may include antibiotics such as penicillin and streptomycin.
  • the non-essential amino acids may refer to one or more selected from the group consisting of isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine and arginine.
  • the centrifugation can be carried out for any length of time at any speed and temperature suitable for the purpose as is known in the art.
  • the centrifugation is 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g, 2000 g, 3000 g, 4000 g, 5000 g, 6000 g, 7000 g, 8000 g, 9000 g, 10000 g, 20000 g, 30000 g, 40000 g, 50000 g, 60000 g, 70000 g, 80000 g, 90000 g, 100000 g, 200 to 400 g, 250 to 350 g, 1500-2500 g, 1800-2200 g, 1900 g-2100 g, 8,000-12,000 g, 9,000-11,000 g, or 80,000-120,000 g, 90,000-110,000 g.
  • the centrifugation may be performed at 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, 1-5 minutes, 5-10 minutes, 1-10 minutes, 5-15 minutes, 10-20 minutes, 15-20 minutes, 15-30 minutes, 20-30 minutes minutes, 20 to 40 minutes, 25 to 35 minutes, 20 to 60 minutes, 20 to 80 minutes, 20 to 100 minutes, 80 to 160 minutes, 100 to 140 minutes, 110 to 130 minutes.
  • the centrifugation is 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 40 It may be carried out at °C, 50 °C, 0 to 5 °C, 0 to 10 °C, 0 to 20 °C or 0 to 30 °C, but is not limited thereto.
  • the filter paper may have a pore size of 0.22 ⁇ m, but is not limited thereto.
  • Another aspect provides a pharmaceutical composition for preventing or treating autoimmune diseases comprising the exosomes prepared by the above manufacturing method.
  • exosome and the like are the same as described above.
  • prevention means any action that suppresses or delays the onset of the obesity or autoimmune disease
  • treatment means any action that improves or beneficially changes the symptoms of the autoimmune disease.
  • the "autoimmune disease” refers to a disease in which immune function is abnormal and immune cells attack organs or tissues of the body.
  • the autoimmune disease can be divided into organ-specific autoantibody-related diseases and organ non-specific (systemic) diseases.
  • the autoimmune disease is Hemophagocytic lymphohistiocytosis, systemic lupus erythematosus, Kikuchi's disease, vasculitis, Adult onset Still's disease, rheumatoid arthritis (rheumatoid arthritis) arthritis), Inflammatory Myositis, Behcet disease, IgG4-associated disease, Sjogren syndrome, Giant cell arteritis, Temporal arteritis, Type 1 diabetes, It may be selected from the group consisting of atopic dermatitis, Crohn's disease, systemic sclerosis, psoriasis, multiple sclerosis, and Graves hyperthyroidism, but is not limited thereto.
  • the pharmaceutical composition may include a pharmaceutically acceptable carrier.
  • the carrier is used in the sense of including excipients, diluents or adjuvants.
  • the carrier may be, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pi It may be selected from the group consisting of rolidone, water, physiological saline, buffers such as PBS, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil.
  • the composition may include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, or a combination thereof
  • the pharmaceutical composition may be provided to the subject in any formulation according to a conventional method, respectively.
  • the formulation may be an oral or parenteral formulation.
  • the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule.
  • the solid formulation may further include an excipient.
  • the excipient may be starch, calcium carbonate, sucrose, lactose, or gelatin.
  • the solid formulation may further include a lubricant such as magnesium stearate or talc.
  • the oral liquid formulation may be a suspension, an oral solution, an emulsion, or a syrup.
  • the liquid formulation may contain water or liquid paraffin.
  • the liquid formulation may include a wetting agent, a sweetening agent, a flavoring agent, or a preservative.
  • Formulations for parenteral administration may be sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilizations, or suppositories.
  • the non-aqueous solvent or suspending agent may include vegetable oil or ester.
  • the vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil.
  • the ester is, for example, ethyl oleate.
  • the base of the suppository may be witepsol, macrogol, tween 61, cacao butter, laurin, or glycerogelatin.
  • the pharmaceutical composition may include the exosome in a pharmaceutically effective amount.
  • effective amount refers to an amount sufficient to exhibit the effect of prophylaxis or treatment when administered to a subject in need thereof.
  • the effective amount can be appropriately selected by those skilled in the art depending on the individual.
  • the effective amount of the pharmaceutical composition is that the pharmaceutical composition contains the exosomes at least about 1 ⁇ g, at least about 2 ⁇ g, at least about 5 ⁇ g, at least about 10 ⁇ g, at least about 20 ⁇ g, at least about 50 ⁇ g, at least about 100 ⁇ g.
  • the effective amount may mean the amount of the exosome contained per 0.01 mL, 0.1 mL, 1 mL, 10 mL, or 100 mL of the pharmaceutical composition.
  • the pharmaceutical composition may be administered in a conventional manner via oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, or intradermal routes.
  • the pharmaceutical composition may be administered in the form of an injection, for example, subcutaneously, intravenously or intramuscularly.
  • the pharmaceutical composition may be administered systemically or locally, alone or in combination with other pharmaceutically active compounds.
  • the dosage of the pharmaceutical composition is, for example, about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 10 mg/kg, based on an adult.
  • about 0.1 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 5 mg/kg, or about 1 mg/kg to about 3 mg/kg may be within the range.
  • the administration is once a day, twice a day, three times a day, four times a day, once a week, once in two weeks, once in three weeks, once in four weeks, once in a month. It may be administered once, once every three months or once a year.
  • Another aspect provides a method for preventing or treating an autoimmune disease comprising administering the exosome prepared by the method for producing the exosome.
  • a pharmaceutical composition comprising an exosome prepared according to a manufacturing method according to an aspect significantly inhibits the production of inflammatory cytokines IL-2, IL-17, IFN- ⁇ in T h 0 and T h 17 cells, and , It significantly increases the production of IL-10, an anti-inflammatory cytokine, and does not affect the activity of immunoregulatory T cells (T reg ), so it shows a very good effect in the treatment of autoimmune diseases.
  • FIGS. 1A to 1C are diagrams analyzing cell shape and markers of dedifferentiated stem cell-derived mesenchymal stem cells according to an embodiment of the present invention.
  • 1a shows the expression of NANOG, SSEA-4, TRA-181, which are markers of iPSCs confirmed by immunofluorescence
  • FIG. 1b shows iPSC-derived mesenchymal stems. It shows the results of observing the cell morphology of the cells (iPSC-iMSC) for each stage of differentiation
  • FIG. 1c shows CD34, CD31, which are reported as MSC markers in iPSC-derived mesenchymal stem cells (hiPSC-MSCs).
  • hiPSC-MSCs shows the results of confirming the expression of CD19, CD11, HALDR (negative marker), CD44, CD73, CD105, and CD90 (positive marker).
  • FIGS. 2A to 2C are diagrams analyzing physical properties and markers of exosomes isolated from dedifferentiated stem cell-derived mesenchymal stem cells according to an embodiment of the present invention.
  • Figure 2a shows the size distribution of the exosomes measured using the NANOSIGHT instrument after the separation of the dedifferentiated stem cell-derived mesenchymal stem cell exo (iPSC-iMSC-Exo)
  • Figure 2b is a transmission electron microscopy (Transmission electron microscopy)
  • TEM shows the morphology of dedifferentiated stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo)
  • FIG. -Exo shows the expression of TGS101, CD9, CD63 (positive marker), which are reported as markers of exosomes confirmed by Western blotting.
  • Figures 3a to 3c show the results of confirming the immunomodulatory ability of the dedifferentiated stem cell-derived mesenchymal stem cell exosomes to T h 0 cells according to an embodiment of the present invention.
  • Figure 3a shows IL-17, IFN-g and immunity generated in Th0 cells after co-culture of mononuclear cells cultured in Th0 cell differentiation conditions with dedifferentiated stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo).
  • FIG. 3b is a graph showing the FACS result
  • Figure 3c is IL-17, IFN-g in the culture medium confirmed by ELISA and IL-10.
  • Figures 4a to 4c show the results of confirming the immune modulating ability of the dedifferentiated stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo) to T h 17 cells according to an embodiment of the present invention.
  • Figure 4a shows IL-17, IFN-g, and immunity generated in Th17 cells after co-culture of mononuclear cells cultured in Th17 cell differentiation conditions with dedifferentiated stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo). It is a result of FACS analysis of expression of CD25+Foxp3+, a marker generated in regulatory T cells (Treg), FIG. 4b is a graph showing the FACS result, and FIG. 4c is IL-17, IL-10 in the culture medium confirmed by ELISA. , and IL-2.
  • FIG. 5a to 5d show the results of comparing the disease control efficacy of the dedifferentiated stem cell-derived mesenchymal stem cell exosomes and the mesenchymal stem cell exosomes in an arthritis animal model according to an embodiment of the present invention.
  • Figure 5a shows the arthritis evaluation index of the group administered with the dedifferentiated stem cell-derived mesenchymal stem cell exo (iPSC-iMSC-Exo) and the mesenchymal stem cell exo (MSC-Exo), respectively, in the induction of the rheumatoid arthritis animal model.
  • Figure 5b shows changes in CD4+T intracellular cytokines (CD4+IL-17A+, CD4+IL-10, etc.) in splenocytes of an animal model of rheumatoid arthritis confirmed by FACS
  • Figure 5c shows rheumatoid arthritis Shows the histological evaluation results by H & E, toluidine blue, and safranin O staining of the arthritis animal model
  • Figure 5d is the inflammation index (inflammation score) and cartilage erosion score (Cartilage damage score) calculated in each group through (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 ).
  • FIG. 6a to 6d show the results of confirming the disease control efficacy in the arthritis animal model according to the concentration of the dedifferentiated stem cell-derived mesenchymal stem cell exosomes according to an embodiment of the present invention.
  • Figure 6a shows the arthritis evaluation index of the group administered with each concentration of immunized stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo) in the induction of an animal model of rheumatoid arthritis
  • Figure 6b is confirmed by FACS shows changes in cytokines (CD4+IL-17A+, CD4+IL-10, etc.) in splenocytes in an animal model of rheumatoid arthritis
  • FIG. 6c shows H&E, toluidine blue, and Shows the results of histological evaluation by safranin O staining
  • FIG. 6d shows the inflammation index and cartilage erosion score calculated in each group (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 ).
  • iPSC induced Pluripotent Stem Cells
  • Blood was obtained from a normal donor without disease with the approval of the institutional IRB and used.
  • To isolate mononuclear cells blood was centrifuged at 2,000 rpm for 30 minutes using a Ficoll Paque-PLUS (GE Healthcare).
  • the isolated mononuclear cells were cultured in StemSpan-ACF (Stem Cell Technologies) supplemented with StemSpan cc110 at 37° C. and 5% CO 2 conditions for 4 days. Thereafter, the cultured mononuclear cells were inoculated in a 24-well plate coated with vitronectin (Life Technologies, Invitrogen), and Sendai of the CytoTune®-iPS 2.0 Reprogramming Kit (Life Technologies, Invitrogen) according to the manufacturer's instructions.
  • IPSCs were prepared using a virus (Sendai virus). Specifically, from 3 to 21 days after transduction, cells were cultured at 37° C. and 5% CO 2 conditions, and on day 12, new iPSC colonies were individually picked and expanded for characterization. The medium was changed daily until iPSC colonies were formed. After manually picking iPSC colonies, they were cultured in TeSR-E8 medium (Stem Cell Technologies) in vitronectin-coated plates. Cultured cells were analyzed by flow cytometry for the expression of NANOG, SSEA-4, and TRA-181, which are markers of iPSCs, and identified as iPSCs.
  • a virus Sendai virus
  • Example 2 Preparation and identification of mesenchymal stem cells (iPSC-iMSC) derived from dedifferentiated stem cells
  • the retrodifferentiated stem cells cultured on a plate to 90% confluency (90% confluency) were washed once with PBS (1 x Phosphate buffered saline). did After that, 3 mL of a 1 mg/mL type IV collagenase solution was added to the plate and reacted for 10 minutes. After adding 6 mL of TeSR-E8 medium (Stem Cell Technologies), it was centrifuged at 1100 rpm for 3 minutes.
  • EB embryoid body
  • rh recombinant human TGF- ⁇ 1
  • 100 mm dishes coated with 1% gelatin for outgrowth recombinant human TGF- ⁇ 1
  • iPSC-MSCs were cultured until 90% confluency (90% confluency) in a gelatin-coated 100 mm dish while changing the culture medium every other day.
  • Example 2-1 it was confirmed through a marker whether dedifferentiated stem cell-derived mesenchymal stem cells (iPSC-MSC) were prepared.
  • iPSC-MSC dedifferentiated stem cell-derived mesenchymal stem cells
  • NANOG, SSEA-4 and TRA-181 which are markers of dedifferentiated stem cells, was confirmed by the immunofluorescence method, and the cell shape of dedifferentiated stem cell-derived mesenchymal stem cells was confirmed at each stage of differentiation.
  • CD34, CD31, CD19, CD11 and HALDR (negative markers), and CD44, CD73, CD105 and CD90 (positive markers), known as MSC markers were investigated for iPSC-MSCs. After culturing iPSC-MSCs for about 4 weeks, cells at passage 5 or higher were confirmed by flow cytometry.
  • Example 1C shows the expression of MSC markers by iPSC-MSCs confirmed by flow cytometry.
  • the retrodifferentiated stem cell-derived mesenchymal stem cells prepared in Example 2-1 expressed CD34, CD31, CD19, CD11 and HALDR, which are negative markers of MSC, in similar amounts as the isotype control, but CD44, a positive marker , CD73, CD105 and CD90 all expressed significantly higher amounts compared to the isotype control, confirming that dedifferentiated stem cell-derived mesenchymal stem cells were formed in Example 2-1.
  • Example 3 Isolation and identification of mesenchymal stem cell exo-some (MSC-Exo) and dedifferentiated stem cell-derived mesenchymal stem cell exo-some (iPSC-MSC-Exo)
  • Cord blood-derived MSCs and iPSC-MSCs were each cultured in a 100 mm dish to 100% confluence, and 5 mL of SF-DMEM (serum free-Dulbecco Modified Eagle Medium) was added thereto at 37°C and 5% CO 2 conditions for 48 hours. cultured. The culture medium was centrifuged for 10 minutes at 4 °C and 300 g conditions. The obtained supernatant was again centrifuged at 4°C and 2000 g conditions for 20 minutes. The obtained supernatant was centrifuged for 30 minutes at 10,000 g with a high-speed centrifuge (Hitachi KoKi #S306352A, Japan) to remove cell debris.
  • SF-DMEM serum free-Dulbecco Modified Eagle Medium
  • the supernatant was filtered through a 0.22 ⁇ m filter and then centrifuged for 120 minutes at 100,000 g in an ultra-high speed centrifuge (Thermo Scientific Fiberlite F50L-8x39 Rotor, Thermo Fisher Scientific). After taking out the tube from the centrifuge, the pellet part, which is the location of the exosomes, was marked in advance, all the rest except the pellet was removed to the opposite side, and the remaining pellet was suspended in PBS and stored at -80°C until use.
  • Thermo Scientific Fiberlite F50L-8x39 Rotor Thermo Fisher Scientific
  • the exosomes separated by Example 3-1 were diluted in PBS and measured six times consecutively with a NanoSight (Malvern instruments Ltd, Malvern, UK) instrument. .
  • the isolated exosomes were photographed with a transmission electron microscope (TEM). Then, in order to confirm whether the isolated exosome is correct, TSG101 (Santa Cruz Biotechnology), CD9 (Santa Cruz Biotechnology) and CD63 (Santa Cruz Biotechnology), known as exosome markers, were identified using a Western blot method.
  • Figure 2c shows that the exosomes (hiPSC-MSCs-Exo) isolated from dedifferentiated stem cell-derived mesenchymal stem cells are a control (Control: SF-DMEM medium) and dedifferentiated stem cell-derived mesenchymal stem cells themselves (hiPSCs).
  • -MSC it was confirmed that TSG101, CD9 and CD63, which are known as markers of exosomes, are expressed significantly more. Through this, it was confirmed that the material isolated in Example 3-1 was an exosome (iPSC-iMSC-Exo).
  • Example 4 Immune regulation ability of exosomes (iPSC-MSC-Exo) isolated from dedifferentiated stem cell-derived mesenchymal stem cells from T h 0 cells
  • Mononuclear cells obtained from normal human peripheral blood were differentiated into T h 0 cells as follows. Specifically, the mononuclear cells to be differentiated into T h 0 cells were transformed into RPMI 1640 supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine.
  • iPSC-iMSC-Exo prepared in Example 3 (0, 0.1, 1) inoculated into medium, treated with anti-CD3 (1 ⁇ g/mL; BD Biosciences) and anti-CD28 (1 ⁇ g/mL; BD Biosciences) , and 10 ⁇ g) and 37° C.
  • IL-17 and IFN- ⁇ which are pro-inflammatory cytokines produced in T h 0 cells, and CD25+Foxp3+ expression, a marker of immunoregulatory T cells (T reg ).
  • T reg a marker of immunoregulatory T cells
  • the contents of the inflammation-inducing cytokine IL-17 and the anti-inflammatory cytokine IL-10 in each culture were analyzed.
  • cytokine content and marker expression were analyzed using FACS and sandwich ELISA.
  • Monoclonal antibodies anti-CD4-PE/Cy7 (RPA-T4, IgG1, BioLegend, San Diego, CA, USA) and anti-CD25-APC (M-A251, IgG1, ⁇ isotype, BD Biosciences) were administered for FACS.
  • control group was stained with the same type of fluorescence-labeled immunoglobulin of the same type without antigen specificity with an isotype antibody.
  • Intracellular cytokines were analyzed by flow cytometry (FACS Calibur; Becton Dickinson, San Diego, CA).
  • IL-17, IFN- ⁇ , IL-10 and IL-2 antibodies were first added to a 96-well plate (NUNC, Denmark) for sandwich ELISA at 4 ⁇ g/mL, respectively. 50 ⁇ l per well of the furnace was added and reacted at 4° C. overnight, and then 200 ⁇ l of blocking solution (1% (w/v) BSA/PBS (Phosphate-Buffered Saline) and 0.05% (v/v) tween 20/PBS) was added per well. and reacted at room temperature for 2 hours.
  • BSA/PBS Phosphate-Buffered Saline
  • tween 20/PBS 0.05%
  • recombinant IL-17, IFN- ⁇ , IL-10 and IL-2 were used to 78 pg/mL, 156 pg/mL, 312 pg/mL, 625 pg/mL, and 1250 pg, respectively. /mL, 2500 pg/mL, 5000 pg/mL concentrations were measured.
  • Figures 3a to 3c show the results of FACS analysis of the amounts of IL-17 and IFN- ⁇ produced in T h 0 cells and CD25+Foxp3+ expression produced in immunoregulatory T cells (Tregs), respectively, and Figure 3c is in the culture medium.
  • the results of measuring IL-17, IFN- ⁇ and IL-10 by ELISA are shown.
  • Immunomodulatory stem cell-derived mesenchymal stem cell exosomes iPSC-iMSC-Exo
  • iPSC-iMSC-Exo inhibited the production of IL-17 and IFN- ⁇ , which are pro-inflammatory cytokines, while further increasing the production of IL-10, an anti-inflammatory cytokine, in cell culture medium.
  • Example 3 The immunomodulatory ability of iPSC-iMSC-Exo and MSC-Exo obtained in Example 3 was confirmed, respectively. Specifically, in order to differentiate mononuclear cells obtained from normal human peripheral blood into T h 17 cells, anti-CD3 (1 ⁇ g/mL; BD Biosciences), anti-CD28 (1 ⁇ g/mL; BD Biosciences), IL- Neutralizing antibody against 1 ⁇ (20 ng/mL; R&D Systems, Minneapolis, MN, USA), IL-6 (20 ng/mL; R&D Systems), IL-23 (20 ng/mL; R&D Systems), IFN- ⁇ (IFN- ⁇ ; 2 ⁇ g/mL; R&D Systems) and a neutralizing antibody against IL-4 (2 ⁇ g/mL; R&D Systems), and Example 4 except that T h 17 was used instead of T h 0 cells The same experiment was performed.
  • FIG. 4a and 4b show the results of FACS analysis of the expression of IL-17 generated in Th17 cells and CD25+Foxp3+ generated in immunoregulatory T cells (Tregs), respectively, and FIG. 4c shows IL-17 and IL-10 in the culture medium. , and IL-2 measured by ELISA are shown.
  • iPSC-iMSC-Exo Inversely differentiated stem cell-derived mesenchymal stem cell exosomes (iPSC-iMSC-Exo) inhibit the production of IL-17, an inflammation-inducing cytokine, but do not affect the activity of immunoregulatory T cells (T reg ) was able to confirm In addition, it was found that iPSC-iMSC-Exo decreased the production of IL-17 and IL-2, which are pro-inflammatory cytokines, and further increased the production of IL-10, an anti-inflammatory cytokine, in cell culture medium.
  • Example 6 Comparison of disease control effects in rheumatoid arthritis animal model of exosomes isolated from retrodifferentiated stem cell-derived mesenchymal stem cells (iPSC-iMSC-Exo) and mesenchymal stem cell exosomes (MSC-Exo)
  • Example 3 In order to confirm whether the exosomes (iPSC-iMSC-Exo) isolated from the dedifferentiated stem cell-derived mesenchymal stem cells obtained in Example 3 have an effect on rheumatoid arthritis, an animal model experiment was performed.
  • CII type II collagen
  • CFA Complete Freund's Adjuvant
  • Score 1 Mild swelling and redness localized to the foot or ankle joint.
  • Score 2 Mild swelling and redness from the ankle joint to the metatarsal.
  • Score 3 Moderate swelling and redness from the ankle joint to the tarsal bone.
  • cytokines IL-17, TNF-a, IL-1b, IL-10, IL-4, etc.
  • 5B and 6B show changes in CD4+T intracellular cytokines (CD4+IL-17A+, CD4+IL-10, etc.) in splenocytes of rheumatoid arthritis animal model mice measured by FACS.
  • CD4+IL-17A+, CD4+IL-10, etc. intracellular cytokines
  • the results in the rheumatoid arthritis animal model showed that the iPSC-iMSC-Exo administration group suppressed the production of inflammation-inducing cytokines (CD4+IL-17+), and the anti-inflammatory cytokines (CD4) compared to the MSC-Exo administration group. +IL-10+), and even when comparing the two groups iPSC-iMSC-Exo 50 ⁇ g, iPSC-iMSC-Exo100 ⁇ g, it was statistically significantly It was confirmed that it inhibits the production of IL-17+) and maintains the production of anti-inflammatory cytokines (CD4+IL-10+).
  • mice were sacrificed 5 weeks after the second boosting for further evaluation of the disease, and the leg joints of each mouse were excised and fixed in 10% formalin for 6 days, Calci -Decalcification was performed in a clear rapid solution for 7 hours. After fixation in 10% formalin for 2 days or in 10% formalin for 6 days, demineralized tissue was processed in calci-clear rapid for 7 hours by dehydration, clearing and paraffin infiltration in the following order.
  • the tissue was embedded in paraffin and manufactured as a block.
  • the tissue was sectioned into 4 ⁇ m using a microtome. Each slide was prepared so that there was one tissue per slide.
  • For hematoxylin & eosin staining paraffin-fixed tissue slides are placed in an oven at 65°C to melt paraffin for 1 hour, and then in NeoClear solution and different concentrations of ethanol for tissue deparaffinization and hydration. They were sequentially immersed for 5 minutes. After hematoxylin staining was performed for 10 seconds, the hematoxylin residue used for staining was removed by immersing the slide in tap water for 10 minutes to avoid direct contact with the slide.
  • the slide was immersed in the Eosin channel and stained for 10 minutes.
  • For dehydration sequentially immersed in ethanol and NeoClear solution of different concentrations, mounting was performed, and after drying in a cool place for more than a day, the tissue was checked through an optical microscope.
  • toluidine blue staining 0.1% toluidine blue solution was dispensed on the slides and stained for 10 minutes. For dehydration, they were sequentially immersed in different concentrations of ethanol and NeoClear solution. After mounting and drying in a cool place for more than one day, the tissue was confirmed through an optical microscope.
  • Histological evaluation of the rheumatoid arthritis animal model was performed according to the following evaluation criteria.
  • 1 point Slight thickening of the inner layer or some infiltrating cells in the lower layer.
  • 5c and 5d show the histological evaluation results of CIA, iPSC-iMSC-Exo and MSC-Exo.
  • the average joint index in the joint tissue was significantly lower in the iPSC-iMSC-Exo group compared to the MSC-Exo group or the CIA group through the inflammation score and the cartilage damage score. confirmed that.
  • 6c and 6d show the histological evaluation results of CIA, iPSC-iMSC-Exo 50 ⁇ g and 100 ⁇ g administration groups. Even when iPSC-iMSC-Exo 50 ⁇ g and iPSC-iMSC-Exo 100 ⁇ g administration group were compared, it was confirmed that the iPSC-iMSC-Exo concentration-dependent mean inflammation index and cartilage erosion score were low. That is, it was found that iPSC-iMSC-Exo had an excellent effect in treating rheumatoid arthritis in an animal model.

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Abstract

La présente invention concerne un procédé de production d'exosomes isolés à partir de cellules souches mésenchymateuses dérivées de cellules souches pluripotentes induites, et une utilisation associée, et les exosomes selon un mode de réalisation augmentent sensiblement la génération de IL-10, qui est une cytokine anti-inflammatoire, et suppriment sensiblement la génération de cytokines inflammatoires IL-2, IL-17 et IFN-γ dans les cellules Th0 et Th17, et n'ont donc pas d'effet sur l'activité des lymphocytes T régulateurs Treg), et présentent par conséquent d'excellents avantages pour le traitement de maladies auto-immunes.
PCT/KR2021/015286 2020-11-18 2021-10-28 Procédé de production d'exosomes isolés à partir de cellules souches mésenchymateuses dérivées de cellules souches pluripotentes induites, et utilisation associée Ceased WO2022108165A1 (fr)

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