WO2020122405A1 - Composition pour la prévention ou le traitement de maladies inflammatoires, comprenant des cellules souches mésenchymateuses dérivées de cellules souches dédifférenciées - Google Patents

Composition pour la prévention ou le traitement de maladies inflammatoires, comprenant des cellules souches mésenchymateuses dérivées de cellules souches dédifférenciées Download PDF

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WO2020122405A1
WO2020122405A1 PCT/KR2019/013764 KR2019013764W WO2020122405A1 WO 2020122405 A1 WO2020122405 A1 WO 2020122405A1 KR 2019013764 W KR2019013764 W KR 2019013764W WO 2020122405 A1 WO2020122405 A1 WO 2020122405A1
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msc
ips
inflammatory
stem cells
cells
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양철우
정병하
김경운
김보미
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가톨릭대학교 산학협력단
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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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

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  • the present invention relates to mesenchymal stem cells having improved immunomodulatory ability and uses thereof, specifically, dedifferentiated stem cell-derived mesenchymal stem cells (iPS-MSC) having superior immune modulating ability compared to mesenchymal stem cells isolated directly from tissues. ) And pharmaceutical compositions for the prevention or treatment of immune-related diseases including the same.
  • iPS-MSC dedifferentiated stem cell-derived mesenchymal stem cells
  • the immune system protects the body from harmful foreign substances, antigens.
  • antigens include bacteria, viruses, toxins, cancer cells, blood and tissues of other people or animals.
  • the immune system responds to these harmful substances and produces antibodies to eliminate them.
  • an autoimmune disorder occurs, the immune system cannot distinguish its healthy body organs from harmful antigens, destroying normal tissues, and autoimmune disease caused by this reaction occurs.
  • a reaction occurs to normal tissues of the body. The cause of autoimmune abnormalities is not clear, but there is a hypothesis that microorganisms or drugs, such as bacteria, cause disease in people born with genes prone to autoimmune diseases.
  • a number of chemical and biological immunotherapy methods have been developed for autoimmune treatment.
  • a typical treatment method is to weaken the body's immune response as a whole by using chemical agents such as steroids or anti-immune cell antibodies. For example, by administering an antibody that binds to CD22, CD20, CD19, CD74, or HLA-DR antigen, it weakens the body's overall immune response, including the normal function of B cells.
  • Republic of Korea Patent Publication No. 2008-0109705 relates to this increased mesenchymal stem cell-mediated autologous dendritic cells with immunosuppression, and discloses dendritic cells with improved T-cell suppression ability and their use of autoimmune diseases.
  • U.S. Patent No. 7,074,403 relates to a method of treating autoimmune diseases using antibodies targeting B cells, and discloses antibodies that specifically bind to B-cell antigens.
  • U.S. Patent No. 7,735,592 relates to a cell composition for the treatment of immune disease using cells from other subjects, and discloses a T cell composition coated with an anti-CD3, anti-CD28 antibody.
  • Conventional autoimmune disease treatment is to weaken the overall immune system of the body, causing the side effects of other diseases by dropping the body's immune function that can respond to external invasion.
  • mesenchymal stem cells are stem cells present in the whole body including bone marrow capable of differentiating into various cell systems such as adipocytes, bone cells and chondrocytes.
  • Mesenchymal stem cells have been isolated from a number of species including humans, mice, mice, dogs, goats, rabbits and cats. Recently, it has been reported that mesenchymal stem cells exert immunoregulatory ability by inhibiting several T lymphocyte activities in vitro and in vivo, and induced pluripotent stem cells (iPS) for smooth supply of mesenchymal stem cells. Although a method of using has been suggested, mesenchymal stem cells derived directly from mesenchymal stem cells derived from induced pluripotent stem cells have not been sufficiently studied for the difference in immune modulating ability.
  • the present inventors studied the immune modulating ability of dedifferentiated stem cell-derived mesenchymal stem cells (iPS-MSC) derived from cells isolated from healthy humans, iPS- compared to bone marrow-derived mesenchymal stem cells (BM-MSC) It has been found that the immunomodulatory ability of MSC is excellent, and the invention of the composition for the treatment of autoimmune diseases or inflammatory diseases using iPS-MSC was completed.
  • iPS-MSC dedifferentiated stem cell-derived mesenchymal stem cells derived from cells isolated from healthy humans, iPS- compared to bone marrow-derived mesenchymal stem cells (BM-MSC)
  • iPS-MSC differentiated stem cell-derived mesenchymal stem cell
  • Another object of the present invention is a pharmaceutical composition for the prevention or treatment of autoimmune diseases or inflammatory diseases, including dedifferentiated stem cell-derived mesenchymal stem cells (iPS-MSC) with improved immune regulation compared to directly isolated MSC. Is to provide.
  • iPS-MSC dedifferentiated stem cell-derived mesenchymal stem cells
  • a further object of the present invention relates to a method for the prevention or treatment of inflammatory diseases, comprising the step of administering mesenchymal stem cells derived from dedifferentiated stem cells to a subject in need thereof.
  • mesenchymal stem cells iPS-MSC
  • iPS-MSC cultures derived from dedifferentiated stem cells which have improved immune regulation compared to directly isolated MSCs
  • iPS-MSC cultures from which cells have been removed are used as active ingredients. It relates to a pharmaceutical composition for the prevention or treatment of an autoimmune disease or an inflammatory disease.
  • dedifferentiated stem cells may be used interchangeably with “derived stem cells”, “derived pluripotent stem cells” or “induced pluripotent stem cells” (iPS).
  • derived stem cells derived pluripotent stem cells
  • iPS induced pluripotent stem cells
  • cell therapy refers to the proliferation and selection of living autologous, allogenic, and xenogenic cells in vitro to restore the function of cells and tissues, or the biological properties of cells by other methods. It refers to a medicine used for the purpose of treatment, diagnosis, and prevention through a series of actions such as changing.
  • the US has been managing cell therapy products as pharmaceuticals since 1993, and Korea since 2002.
  • These cell therapy products can be broadly classified into two fields, the first is “stem cell therapy” for tissue regeneration or long-term function recovery, and the second is the suppression of immune responses in vivo or the regulation of immune responses such as the improvement of immune responses. It can be classified as a therapeutic agent for immune cells.
  • An example of the present invention is to provide an induced pluripotent stem cell-derived mesenchymal stem cell (iPS-MSC) derived from normal cells, which has improved immune regulation, for example, increased anti-inflammatory factors and reduced inflammatory factors.
  • iPS-MSC induced pluripotent stem cell-derived mesenchymal stem cell
  • a further example of the present invention relates to a cell therapeutic agent comprising dedifferentiated stem cell-derived mesenchymal stem cells (iPS-MSC) derived from normal cells with improved immune regulation.
  • iPS-MSC dedifferentiated stem cell-derived mesenchymal stem cells
  • Another example of the present invention relates to a composition for the prevention or treatment of autoimmune diseases or inflammatory diseases, including dedifferentiated stem cell-derived mesenchymal stem cells (iPS-MSC) derived from normal cells with improved immune regulation.
  • iPS-MSC dedifferentiated stem cell-derived mesenchymal stem cells
  • the mesenchymal stem cells (iPS-MSC) derived from dedifferentiated stem cells are derived from dedifferentiated stem cells derived from cells isolated from mammals, and thus MSCs isolated directly from tissues, such as bone marrow. It is distinguished from a derived mesenchymal stem cell (BM-MSC) or an embryonic stem cell-derived mesenchymal stem cell driven from Embryonic stem cell (ES-MSC).
  • BM-MSC derived mesenchymal stem cell
  • ES-MSC embryonic stem cell-derived mesenchymal stem cell driven from Embryonic stem cell
  • the iPS-MSC may be derived from cells isolated from normal humans.
  • MSC Mesenchymal Stem Cell
  • a stem cell obtained by being directly separated from bone marrow, blood, dermis and periosteum and is composed of various cells such as adipocytes, chondrocytes, and bone cells.
  • mesenchymal stem cells obtained directly from tissue are separated from mesenchymal stem cells from mammalian, preferably human, mesenchymal stem cell sources, such as blood or bone marrow (the bone marrow is tibia, femur, spinal cord).
  • the tissue-derived mesenchymal stem cells are umbilical cord mesenchymal stem cells, cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, fat-derived mesenchymal stem cells, muscle-derived mesenchymal stem cells, nerve-derived mesenchymal stem cells, It may be one or more selected from the group consisting of skin-derived mesenchymal stem cells, amniotic-derived mesenchymal stem cells, and placental-derived mesenchymal stem cells.
  • IPS-MSC according to the present invention has an improved immunomodulatory ability compared to MSC isolated directly from tissues, for example, has an increased anti-inflammatory factor and a reduced inflammatory factor.
  • the degree of improvement of the immunomodulatory ability can be evaluated by an inflammatory index (R), and the inflammatory index (R) is expressed as an inflammatory cytokine expression level compared to an anti-inflammatory cytokine expression level (Equation 1 below). Specifically, the lower the value of the inflammatory index, it is evaluated that the immunomodulatory ability increased.
  • the inflammatory index of iPS-MSC according to the present invention may be expressed as 40% or less based on 100% of the inflammatory index of mesenchymal stem cells isolated directly from tissues.
  • the MSC directly separated from the iPS-MSC is in contact with synovial cells and co-cultured with synovial cells. It can be divided into cells and cultured alone, and can be expressed as a multiple ratio between iPS-MSC and directly separated MSC in the case of two cultures.
  • the inflammatory index of iPS-MSC has a lower value than the inflammatory index of MSC isolated directly from tissue, which has an excellent anti-inflammatory effect compared to MSC obtained by iPS-MSC directly isolated from tissue. Indicates.
  • the expression level of the cytokine can be used in the cytokine expression level measurement by a person skilled in the art freely selecting a method known in the art according to the purpose, for example, Enzyme linked immunosorbent assay (ELISA). ), flow cytometry, real-time polymerase chain reaction (real-time PCR), and confocal microscopy (confocal microscopy).
  • ELISA Enzyme linked immunosorbent assay
  • flow cytometry flow cytometry
  • real-time polymerase chain reaction real-time PCR
  • confocal microscopy confocal microscopy
  • the inflammatory cytokine may be selected without limitation as long as it is an inflammatory cytokine known in the art.
  • the inflammatory cytokine is one selected from the group consisting of tumor necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b), IL-2, IL-6, IL-8, and IL-17.
  • TNF-a tumor necrosis factor alpha
  • IL-1b interleukin 1 beta
  • IL-2 interleukin 1 beta
  • IL-6 interleukin 1 beta
  • IL-8 interleukin 17
  • the above may be, and may be one or more selected from the group consisting of IL-6, IL-8 and IL-17, but is not limited thereto.
  • the anti-inflammatory cytokine may be selected without limitation as long as it is an anti-inflammatory cytokine known in the art.
  • it may be one or more selected from the group consisting of tumor necrosis factor beta (TNF-b), IL-4, IL-10, and IL-13, preferably TNF-b or IL-10, more preferably May be IL-10, but is not limited thereto.
  • the inflammatory index (R) of the iPS-MSC is 0.00001 to 15, 0.0001 to 15, 0.001 to 15, 0.01 to 15, 0.00001 to 10, 0.0001 to It may be 10, 0.001 to 10, 0.00001 to 8, 0.0001 to 8, 0.001 to 8, 0.00001 to 6, 0.0001 to 6, or 0.001 to 6, but is not limited thereto.
  • the inflammatory index (R) of the iPS-MSC is based on 100% of the inflammatory index of the MSC (eg, BM-MSC) isolated directly from the tissue, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, 0.5% or less, 0.3% or less, 0.2% or less, 0.1% Hereinafter, it may be 0.01% or less or 0.001% or less.
  • the inflammatory index of the iPS-MSC is based on 100% of the inflammatory index of the MSC (eg, BM-MSC) isolated directly from the tissue.
  • IL-6/IL-10 expression ratio (R) is 0.3% or less, 0.2% or less, 0.15% or less, 0.10% or less, 0.01% or less, 0.07% or less, 0.05% or less, 0.03% or less, 0.01% or less , Or a value in the range of 0.001% or less, and the expression ratio (R) of IL-8/IL-10 is 30% or less, 25% or less, 20% or less, 17% or less, 15% or less, 13% or less, or 10 Values in the range of% or less may be indicated.
  • the inflammatory index of iPS-MSC is IL-6/IL-10 (p ⁇ 0.01) and IL-8/IL-10
  • the expression level ratio of (p ⁇ 0.05) was statistically significantly lower than that of bone marrow-derived stem cells (BM-MSC).
  • BM-MSC bone marrow-derived stem cells
  • iPS-MSC The inflammatory index, such as the IL-6/IL-10 ratio showed a value of about 0.053% when the BM-MSC value was set at 100%, and the IL-8/IL-10 ratio was the value of the BM-MSC. When the value was set at 100%, it was about 15%.
  • the iPS-MSC has an excellent anti-inflammatory effect compared to the BM-MSC.
  • the ratio of IL-6/IL-10 of iPS-MSC has a p value of 0.01 or less and an inflammatory index value of 1 or less and/or IL
  • the ratio of -8/IL-10 may have a p value of 0.05 or less and an inflammatory index value of 10 or less.
  • the expression of the cytokine of iPS-MSC Ratio is 0.001 to 1.5, 0.01 to 1.5, 0.1 to 1.5, 0.2 to 1.5, 0.3 to 1.5, 0.001 to 1.0, 0.01 to 1.0, 0.1 to 1.0, 0.2 to 1.0, 0.3 to 1.0, 0.001 to 0.7, 0.01 to 0.7, 0.1 to 0.7, 0.2 to 0.7, or 0.3 to 0.7.
  • the ratio of the anti-inflammatory cytokine to the inflammatory cytokine is when mesenchymal stem cells (MSC) come into contact with synovial cells, for example, co-culture of synovial cells and mesenchymal stem cells or mesenchymal stem cells are injected into synovial cells.
  • MSC mesenchymal stem cells
  • R cytokine expression ratio
  • iPS-MSC cytokine expression ratio of iPS-MSC is 75% or less, 70% or less, 65% or less, 60% or less , 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 5% or more and 15% or less.
  • human monocyte cells were stimulated with Th17 differentiation conditions and co-cultured with iPS-MSC or BM-MSC in order to confirm the immunomodulatory ability of iPS-MSC.
  • IL-17 production in cells decreased more than when co-cultured with BM-MSC, while the amount of Treg marker was not significantly different. That is, it was confirmed that iPS-MSC according to the present invention further suppresses inflammatory Th17 cells (IL-17 production) than bone marrow-derived stem cells (BM-MSC), while maintaining immunoregulatory T cells (Treg).
  • IL-17 production which is an inflammatory cytokine
  • IL-10 an anti-inflammatory cytokine
  • the iPS-MSC according to the present invention is derived from dedifferentiated stem cells, and first, cells, for example, monocytes are obtained from normal human blood, and coated with monocytes in 24-well plates with vitronectin (BD BioCoat TM ) and Dedifferentiated stem cells (iPS) were prepared through Sendai virus (SeV) (CytoTune®-iPS 2.0 Reprogramming kit, Life Technologies, Invitrogen).
  • the dedifferentiated stem cells are separated from colony-shaped cells to produce an EB (embryo body) and differentiated through b-FGF, rh TGF-b cytokines in ⁇ -Minimum Eagle's Medium culture medium containing 20% fetal calf serum.
  • iPS-MSC is obtained.
  • Methods for preparing dedifferentiated stem cells from the monocytes and obtaining iPS-MSC from dedifferentiated stem cells are well known.
  • the method of manufacturing iPS-MSC according to the present invention (1) isolates mononuclear cells from the blood of a normal person, and uses Sendai virus (SeV), CytoTune®-iPS 2.0 Reprogramming kit to reverse-differentiate stem cells ( iPS) and (2) b-FGF, rh TGF- in ⁇ -Minimum Eagle's Medium culture medium containing 20% fetal calf serum by tearing cells in colony form to produce an EB (embryo body).
  • b Differentiation through cytokines may include the step of preparing an iPS-MSC.
  • the dedifferentiated stem cell is selected from the group consisting of monocytes, umbilical cord blood, bone marrow, fat, muscle, nerves, skin, amniotic membranes and placenta 1 It may be from a species or higher.
  • normal or normal person means an individual or a person who is not an autoimmune disease or an inflammatory disease patient, and includes a state prior to the onset or diagnosis time point of an autoimmune disease or an inflammatory disease.
  • the individual may be a human, including a human, a primate such as a monkey, a mammal including a rodent such as a rat, a mouse, and preferably human, but is not limited thereto.
  • the inflammatory disease according to the present invention is a cytokine-mediated inflammatory disease
  • examples of the cytokine-mediated inflammatory disease include systemic lupus erythematosus, Hashimoto's thyroiditis, type 1 diabetes, hypothyroidism, hyperthyroidism, and Sjogren's syndrome ( Sjogren's syndrome), psoriasis, Crohn's disease, thyroiditis, and ulcerative colitis may include one or more selected from the group consisting of rheumatoid arthritis.
  • the pharmaceutical composition provided herein is an oral dosage form, such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, formulated according to a conventional method, or suspension, emulsion, lyophilized preparation, It can be formulated and used as a parenteral formulation such as an external preparation, a suppository, a sterile injection solution, and an implantable formulation.
  • the pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto. It is not.
  • the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.
  • a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, a talct, a talct, a talct, a stevia, glycerin, a stevia, glycerin, glycerin, g
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally.
  • the pharmaceutical composition of the present invention can be administered by intravenous infusion, subcutaneous infusion, intramuscular infusion and intraperitoneal infusion.
  • the pharmaceutical composition of the present invention it is preferable that the route of administration is determined according to the type of disease to be applied. For example, when applied to type I diabetes, intraperitoneal administration is most preferred, because the administered dendritic cells can effectively migrate to the pancreas without dilution.
  • the pharmaceutical composition of the present invention when applied to a patient with arthritis, it may be administered by intravenous injection, but most preferably, it is administered by intra-articular injection.
  • Suitable dosages of the pharmaceutical compositions of the present invention may be variously prescribed by factors such as formulation method, mode of administration, recipient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response sensitivity. Can be.
  • the dosage of the pharmaceutical composition of the present invention is preferably 1 x 10 3 to 1 x 10 12 cells/kg per day.
  • the pharmaceutical composition of the present invention is prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person skilled in the art to which the present invention pertains, or It can be manufactured by incorporating into a multi-dose container.
  • the present invention relates to a pharmaceutical composition for the prevention or treatment of autoimmune diseases or inflammatory diseases, including iPS-MSC, which has excellent immune-modulating ability compared to BM-MSC, and the supply of MSC compared to MSC directly isolated. It is easy and shows a significantly superior immunomodulatory effect compared to directly isolated MSC, and thus can be used for the prevention or treatment of autoimmune diseases or inflammatory diseases including rheumatoid arthritis.
  • Figure 1a is a photomicrograph showing the change in cell appearance in the process of preparing mesenchymal stem cells derived from pluripotent stem cells (hiPS) derived from normal humans (denoted as iMSC or iPS-MSC).
  • hiPS pluripotent stem cells
  • 1B is a graph confirming that iPS-MSC normally expresses MSC-specific markers in addition to external features.
  • Figure 1c is observed in the iPS-MSC formed by the method of Example 1 in the differentiation conditions to determine whether it has the ability to differentiate into chondrocytes, osteoblasts and adipocytes, as in the directly separated MSC, observed by staining the differentiated cells It is a picture.
  • 2A and 2B are graphs comparing the production of inflammatory cytokines and anti-inflammatory cytokines of iPS-MSC and BM-MSC.
  • Figures 3a and 3b is a specific marker for IL-17 and Treg cells produced by Th17 cells after co-culture with BM-MSC or iPS-MSC after inducing differentiation under Th17 differentiation conditions by separating human monocytes. It is a graph comparing the ratio of Foxp3 + CD4 + cells.
  • Figure 3c is a graph comparing the amount of IL-17 and IL-10 in the culture when human monocytes and BM-MSC or human monocytes and iPS-MSC are co-cultured under Th17 differentiation conditions.
  • Figures 4a and 4b is a graph analyzing the ratio of Th17 cells or Treg cells by co-culture with synovial cells isolated from patients with rheumatoid arthritis under i17-differentiation conditions with iPS-MSC.
  • FIG. 5 is a graph showing the results of analyzing the amount of IL-17 and IL-10 involved in the inflammatory response by co-cultivating synovial cells and BM-MSC or iPS-MSC isolated from rheumatoid arthritis patients under Th17 differentiation conditions.
  • Example 1 Preparation of mesenchymal stem cells derived from dedifferentiated stem cells (iPS_MSC)
  • Monocytes were isolated from the blood of a healthy person (hereinafter referred to as a'normal person') who was not diagnosed with an immune disease, and used to produce dedifferentiated stem cells. Specifically, normal human blood was obtained from a normal donor at clergy Mall, and 10 mL of blood was centrifuged using Ficoll Paque TM PLUS (GE Healthcare) for 2,000 rpm for 30 minutes to separate mononuclear cells.
  • the isolated monocytes were cultured for 4 days in StemSpan TM ACF (Stem Cell Technologies) supplemented with StemSpan cc110. Subsequently, mononuclear cells were coated on 24-well plates with vitronectin (BD BioCoat ⁇ ) and Sendai virus (SeV) (CytoTune®-iPS 2.0 Reprogramming kit, Life Technologies, Invitrogen) to prepare dedifferentiated stem cells (iPS) Did. Medium was changed daily until iPS colonies formed. After manual picking, iPS lines were maintained in plates coated with vitronectin (Life Technologies, Invitrogen) in TeSR-E8 medium (Stem Cell Technologies). On day 12 after transduction, new iPS colonies were picked individually and expanded for characterization. After transfection, cells were cultured at 37°C and 5% CO 2 conditions from 3 to 21 days to obtain dedifferentiated stem cells.
  • StemSpan TM ACF StemSpan TM ACF (Stem Cell Technologies) supplemented with StemSpan cc110.
  • iPS-MSC mesenchymal stem cells
  • Dedifferentiated stem cells obtained by the method of Example 1-1 were PBS washed with iPS cells grown with 100% confluence in one 100mm dish to differentiate again with iMSC, and then washed with 1mg/ml type IV Collagenase solution in 100mm dish Into 3 ml, and reacted at 37°C for 10 minutes, and after adding 6 ml of media, centrifugation was performed at 1100 rpm for 3 minutes.
  • ⁇ -Minimum Eagle's Medium 0.1 mM nonessential amino acid (100x) gibco, 1 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 1% penicillin streptomycin
  • a change in cell morphology in the process of changing from iPS to iPS-MSC was observed using a microscope.
  • Cell samples for observation were prepared with cells with passage 5 or higher after growing iPS-MSC for about 4 weeks.
  • Figure 1a is a photomicrograph showing the change in cell appearance in the process of preparing mesenchymal stem cells derived from pluripotent stem cells (hiPS) derived from normal humans (denoted as iMSC or iPS-MSC). Colony formation was prominent in hiPS, but the appearance of these colonies was not observed in iMSC, so external changes were observed.
  • hiPS pluripotent stem cells
  • CD34, CD31, CD19, CD11, HALDR (Negative Marker), CD44, CD73, CD105, and CD90 (Positive Marker), known as MSC markers was examined for these iPS-MSCs. Specifically, after growing iMSC for about 4 weeks, it was confirmed by flow cytomery in cells with passage 5 or higher. 1B is a graph confirming that iPS-MSC normally expresses MSC-specific markers in addition to external features.
  • iPS-MSC induced by the method of Example 1-2 exhibits characteristics as mesenchymal stem cells.
  • the iPS-MSC of the present invention has a multipotential ability unique to mesenchymal stem cells and can differentiate into chondrocytes, osteoblasts, and adipocytes. Specifically, after performing safranin-O, alkaline phosphatase (ALP) substrate, and Oil Red O staining, it was performed by observing and visualizing with a Zeiss microscope (LSM 510 Meta, Carl Zeiss, Oberkochen, Germany). The results are shown in Figure 1c. As can be seen in Figure 1c, it was confirmed that the iPS-MSC of the present invention has a multi-lineage differentiation that can differentiate into chondrocytes, osteoblasts, and adipocytes.
  • ALP alkaline phosphatase
  • FIG. 1c stains the differentiated cells after induction with each differentiation condition to confirm that the iPS-MSC formed by the method of Example 1 has differentiation ability into chondrocytes, osteoblasts, and adipocytes, as in the directly isolated MSC. It is a picture observed. Differentiation into all of chondrocytes, osteoblasts, and adipocytes was confirmed, indicating that iPS-MSC has the same function as MSC directly isolated.
  • the inflammatory cytokine or anti-inflammatory cytokine expression level was confirmed by ELISA.
  • iPS-MSC prepared in Example 1-2 was used as a control group after bone marrow-derived stem cells (BM-MSC) as a control group, and after growing stem cells with 90% confluence in a 100 mm dish. Place the ELISA microplates according to the manufacturer's instructions according to the inflammatory cytokines IL-6, IL-8, IL-2 and anti-inflammatory cytokines IL-10 and TGF-b (R&D Systems, Minneapolis, MN, USA). ELISA was performed using a absorbance reader (Molecular Devices, Sunnyvale, CA, USA) measured at 405 nm.
  • BM-MSC was purchased from the clergyman and used.
  • Mesenchymal stem cells were cultured in a 37°C, 5% carbon dioxide, humidity 95% incubator by adding 10% FBS and antibiotics to DMEM/F12 (Gibco).
  • 2A and 2B are graphs comparing the production of inflammatory cytokines and anti-inflammatory cytokines of iPS-MSC and BM-MSC.
  • IL-6 and IL-8 are inflammatory cytokines, IL-10, TGF-b corresponds to a non-inflammatory cytokine, it can be confirmed that the reduction of inflammatory cytokines is particularly pronounced.
  • Table 1 below shows the ratio of inflammatory cytokines and anti-inflammatory cytokines of iPS-MSC and BM-MSC.
  • the immune system maintains a balance like the seesaw, both immune tolerance and immune response, and is properly regulated.
  • the immunological homeostasis collapses, and the collapse of the immunological homeostasis causes various forms of immunologically related diseases.
  • This immunological balance can be controlled by inflammatory and anti-inflammatory cytokines.
  • the expression level of anti-inflammatory cytokines in iPS-MSC and BM-MSC is not different, but the expression level of inflammatory cytokines, i.e., IL-6/IL-10 and IL-8/IL-10, is iPS -Since it is statistically significantly reduced in MSC compared to BM-MSC, it can be predicted that the immunological control ability is excellent.
  • IL-6/IL-10 p ⁇ 0.01
  • IL-8/IL-10 The expression level ratio of p ⁇ 0.05
  • Th17 cells are known to be involved in various autoimmune diseases, such as arthritis, multiple sclerosis, psoriasis, and inflammatory bowel disease, by producing IL-17 that disrupts the cytokine immune system.
  • Th17 cells which produce a lot of specific cytokines IL-17, have been found to have a direct role in the autoimmune disease rheumatoid arthritis etiology through animal models and human cell research, and to host defense against pathogens or induce adverse immune responses.
  • immunoregulatory T cells suppress the immune response, and serve to prevent excessive immune response in the body.
  • monocyte cells of normal humans were subjected to Th17 cell differentiation conditions (anti-CD3 (1 ⁇ g/ml; BD Biosciences), anti-CD28 (1 ⁇ g/ml; BD Biosciences), IL-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), neutralizing antibodies 10% fetal calf in iPS-MSC or BM-MSC and RPMI 1640 medium medium by stimulation with against interferon-gamma (IFN- ⁇ ; 2 ⁇ g/ml; R&D Systems), IL-4 (2 ⁇ g/ml; R&D Systems) Serum (FCS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine were added, and air-cultured at 37°C and Th17 cell differentiation conditions (anti-CD3 (1 ⁇ g/ml; BD Biosciences
  • IL-17 (FIGS. 3A and 3B) and CD25 + Foxp3 + expression generated in immunoregulatory T cells were examined, and the contents of IL-17 and IL-10 in each culture were analyzed using ELISA (FIG. 3C). ).
  • FACS method is a cell surface staining method, monoclonal antibody anti-CD4-PE / Cy7 (RPA-T4, IgG1, BioLegend, San Diego, CA, USA) and anti-CD25-APC (M-A251, IgG1 , ⁇ , BD Biosciences), washed with cells by intracellular staining, made permeable, and monoclonal antibody anti-IL-17-PE (eBio64dec17, IgG1, ⁇ , eBioscience, San Diego, CA, USA) , Incubated with anti-IFN- ⁇ -FITC (4S.B3, IgG1, ⁇ ; eBioscience) and anti-Foxp3-FITC (PCH101, IgG2a, ⁇ ; eBioscience) antibodies. In addition, an appropriate isotype antibody was prepared and stained. Intracellular cytokines were analyzed with a flow cytometer (FACS Calibur; Becton Dickinson, San Diego, CA).
  • sandwich ELISA method monoclonal IL-6, IL-8, IL-2, IL-10, TGF-b, IL-17 (R&D systems, USA) in a 96 well plate (NUNC, Denmark) for Sandwich ELISA 50 ⁇ l/well at 4 ug/mL, and reacted overnight at 4° C., followed by blocking solution (1% (w/v) BSA/ PBS (Phosphate-Buffered Saline), 0.05% (v/v) tween 20/ PBS) was added at 200 uL/well and reacted at room temperature for 2 hours.
  • BSA/ PBS Phosphate-Buffered Saline
  • Recombinant IL-6, IL-8, IL-2, IL-10, TGF-b, IL-17 were used as a control group to measure concentrations of 5 ng/mL to 78 pg/mL, respectively.
  • the separated patient serum to be measured together with the standard sample was added at 50 uL/well and reacted for 2 hours at room temperature.
  • the reaction vessel was washed 4 times with a washing solution (0.05% (v/v)Tween 20/PBS) and the biotinylated IL-6, IL-8, IL-2, IL-10, TGF-b, and IL-17 antibodies were 200.
  • FIGS. 3A to 3C The analysis results are shown in FIGS. 3A to 3C.
  • Figures 3a and 3b is a specific marker for IL-17 and Treg cells produced by Th17 cells after co-culture with BM-MSC or iPS-MSC after inducing differentiation under Th17 differentiation conditions by separating human monocytes. This graph compares the percentage of Foxp3+CD4+ cells.
  • iPS-MSC further suppresses inflammatory Th17 cells (IL-17 production) than bone marrow-derived stem cells (BM-MSC), whereas immunoregulatory T cells (Treg) Confirmed that it was maintained.
  • IL-17 production which is an inflammatory cytokine, decreased and IL-10, an anti-inflammatory cytokine, increased.
  • FIG. 3c is a graph comparing the amount of IL-17 and IL-10 in the culture when human monocytes and BM-MSC or human monocytes and iPS-MSC are co-cultured under Th17 differentiation conditions.
  • the amount of inflammatory cytokine IL-17 was significantly reduced, and the anti-inflammatory cytokine IL-10 was significantly increased.
  • Table 17 shows the ratio of IL-17 to IL-10 as a result of Th17 differentiation conditions and co-culture with each MSC. Specifically, in the Th17 differentiation condition, the IL-17/IL-10 ratio was 2.3, but when co-cultured with BM-MSC, it fell to 1.6, and significantly decreased to 0.5 in iPS-MSC.
  • Rheumatoid arthritis is known to activate the disease by increasing the inflammatory cytokines as cells in the synovial membrane become active and infiltrate into joint tissue. It was intended to confirm whether the iPS-MSC of the present invention has a disease control effect against rheumatoid arthritis.
  • SFMC synovial fluid mononuclear cells isolated from synovial fluid of patients with rheumatoid arthritis
  • SFMC of rheumatoid arthritis patients with Th17 cell differentiation condition After stimulation, when co-cultured with iPS-MSC, IL-17, a cytokine produced in Th17 cells, and CD25+Foxp3+ expression, a marker produced in immunoregulatory T cells (Treg), were examined by FACs.
  • synovial fluid from patients with rheumatoid arthritis was obtained from the Department of Internal Medicine, clergy University of Rheumatology.
  • Mononuclear cells were obtained from synovial fluid isolated from a patient with rheumatoid arthritis by the method described in Example 1 (RA SFMC; synovial fluid mononuclear cells from RA patients), and Th17 differentiation conditions were formed as described in Example 3 to form iPS-MSC and 37.
  • Air conditioning was performed for 48 hours at 5° C. and 5% CO 2 conditions.
  • the amount of IL-17 formed in Th17 from the culture and CD25+Foxp3+ expression generated in immunoregulatory T cells (Treg) were analyzed in substantially the same manner as FACS in Example 3, and the results were shown in FIGS. 4A and 4B. Shown.
  • Figures 4a and 4b is a graph analyzing the ratio of Th17 cells or Treg cells by co-culture with synovial cells isolated from patients with rheumatoid arthritis under i17-differentiation conditions with iPS-MSC.
  • the iPS-MSC derived from normal humans also has immunomodulatory ability against SFMC, which is an actual pathogen of rheumatoid arthritis, and confirmed the therapeutic effect on rheumatoid arthritis disease at the patient cell level.
  • Example 5 Evaluation of anti-inflammatory efficacy of rheumatoid arthritis of iPS-MSC
  • the ratio of inflammatory cytokines IL-17 and anti-inflammatory cytokines IL-10 was examined to confirm that the iPS-MSC of the present invention has superior inflammation control effect of rheumatoid arthritis compared to bone marrow-derived MSC (BM-MSC).
  • mononuclear cells were isolated by obtaining synovial fluid from a patient with rheumatoid arthritis in the same manner as in Example 4-1.
  • the isolated mononuclear cells were co-cultured with iPS-MSC or BM-MSC, and ELISA analysis was performed in substantially the same manner as in Example 3 ELISA to measure the content of cytokines in the cells, and the results are shown in FIG. 5.
  • Table 2 Table 2.
  • FIG. 5 is a graph showing the results of analyzing the amount of IL-17 and IL-10 involved in the inflammatory response by co-cultivating synovial cells and BM-MSC or iPS-MSC isolated from rheumatoid arthritis patients under Th17 differentiation conditions.
  • the amount of inflammatory cytokine IL-17 in iPS-MSC is significantly reduced, and the amount of anti-inflammatory cytokine IL-10 is significantly increased compared to BM-MSC, resulting in iPS-MSC of the present invention. It can be seen that it exhibits an excellent anti-inflammatory effect compared to BM-MSC.

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Abstract

La présente invention concerne une composition pharmaceutique pour la prévention ou le traitement de maladies auto-immunes ou de maladies inflammatoires, comprenant des cellules souches mésenchymateuses (iPS-MSC) dérivées de cellules souches pluripotentes produites à partir de cellules somatiques, ou un milieu conditionné associé. La composition pharmaceutique selon la présente invention présente des effets immunorégulateurs et anti-inflammatoires supérieurs par comparaison avec des cellules souches mésenchymateuses directement isolées à partir de tissus, et peut être facilement fournie, et est donc efficace pour prévenir et traiter des maladies auto-immunes telles que la polyarthrite rhumatoïde.
PCT/KR2019/013764 2018-12-14 2019-10-18 Composition pour la prévention ou le traitement de maladies inflammatoires, comprenant des cellules souches mésenchymateuses dérivées de cellules souches dédifférenciées WO2020122405A1 (fr)

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