WO2018101723A1 - Nouvelles cellules progénitrices mésenchymateuses fonctionnellement améliorées, composition d'agent thérapeutique cellulaire anti-inflammatoire les contenant, et procédé de préparation de cellules progénitrices mésenchymateuses - Google Patents

Nouvelles cellules progénitrices mésenchymateuses fonctionnellement améliorées, composition d'agent thérapeutique cellulaire anti-inflammatoire les contenant, et procédé de préparation de cellules progénitrices mésenchymateuses Download PDF

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WO2018101723A1
WO2018101723A1 PCT/KR2017/013766 KR2017013766W WO2018101723A1 WO 2018101723 A1 WO2018101723 A1 WO 2018101723A1 KR 2017013766 W KR2017013766 W KR 2017013766W WO 2018101723 A1 WO2018101723 A1 WO 2018101723A1
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progenitor cells
cells
mesenchymal
mesenchymal progenitor
stem cells
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Korean (ko)
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정형민
홍기성
신정민
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주식회사 미래셀바이오
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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
    • 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
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • 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
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • 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
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells

Definitions

  • the present invention provides a novel functionally enhanced mesenchymal progenitor (FEMP) that overexpresses the epidermal growth factor receptor (EGF-R) and low expresses CD95 (Cluster Differentiation 95). It relates to an anti-inflammatory cell therapy composition comprising and a method for producing mesenchymal progenitor cells.
  • FEMP functionally enhanced mesenchymal progenitor
  • Mesenchyme refers to the tissues of mesoderm corresponding to the mesenchyme formed by Epithelial to Mesenchyme Transition (EMT), which occurs during embryonic development.
  • EMT Epithelial to Mesenchyme Transition
  • Human embryonic stem cells can explain this early development in vitro. It can be used as the only source of beneficial cells.
  • mesenchymal stem cells or mesenchymal precursor cells derived from human embryonic stem cells as well as adults, suggesting that there are progenitor cells capable of forming bone in bone marrow like mesenchymal stem cells in adults.
  • progenitor cells capable of forming bone in bone marrow like mesenchymal stem cells in adults.
  • it has been reported for the first time that it can be separated from dense bone, peripheral blood, adipose tissue, and fetal tissues such as cord blood and amniotic membrane.
  • adult-derived mesenchymal stem cells have various characteristics that can be useful for cell therapy, myocardial regeneration, pulmonary fibrosis, and spinal cord injury in the musculoskeletal system and cardiovascular system such as bone, cartilage and tendon in actual clinical treatment Attempts have been made in cell therapy in various fields.
  • adult mesenchymal stem cells unlike embryonic stem cells, are known to exhibit irregular proliferative capacity when they are passaged in vitro for a long time, so that the division capacity of one cell does not exceed 40 times.
  • mesenchymal stem cells from human embryonic stem cells.
  • FACS flow activated analyzer
  • the method of processing is generally used.
  • the method using a flow cytometer does not only lower the viability of the cells because of the use of a laser, but also increases the incubation period due to the small number of cells obtained after separation, and in the case of cytokine treatment, Even after differentiation, there is a problem that it is difficult to effectively remove the pluripotency of embryonic stem cells.
  • the present invention is to solve the above-mentioned problems of the prior art, an object of the present invention is a novel mesenchymal progenitor cell having improved proliferative capacity and differentiation capacity and improved mobility, angiogenesis efficacy and body viability, cell therapeutic composition comprising the same And it provides a method for producing such mesenchymal progenitor cells.
  • EGF-R epidermal growth factor receptor
  • MSC mesenchymal Stem Cell
  • CD95 Cluster Differentiation
  • FEMPs Functionally Enhanced Mesenchymal Progenitors
  • the mesenchymal progenitor cells are embryonic stem cells (ESC, Embryonic Stem Cell), induced pluripotent stem cells (iPSC) or somatic cell nuclear transfer stem cells (SCNT, Somatic Cell Nuclear) Transfer cell).
  • ESC embryonic stem cells
  • iPSC induced pluripotent stem cells
  • SCNT somatic cell nuclear transfer stem cells
  • the embryonic stem cells may be derived from mammals.
  • the mesenchymal progenitor cells is compared to mesenchymal stem cells Latent-Transforming Growth Factor Beta-binding Protein (LTBP), Vascular Endothelial Growth Factor (VEGF), Kinase insert Domain Receptor (KDR), FZD
  • Latent-Transforming Growth Factor Beta-binding Protein LTBP
  • VEGF Vascular Endothelial Growth Factor
  • KDR Kinase insert Domain Receptor
  • FZD One or more angiogenesis factors selected from the group consisting of (Frizzled), Fibroblast Growth Factor (FGF), Angiopoietin (ANG), Platelet-Derived Growth Factor Receptor (PDGFR), and Endothelial Monocyte-Activating Polypeptide (EMAP).
  • FGF Fibroblast Growth Factor
  • ANG Angiopoietin
  • PDGFR Platelet-Derived Growth Factor Receptor
  • EEMAP Endothelial Monocyte-Activating Polypeptid
  • the mesenchymal progenitor cells may overexpress one or more migration factors of Matrix Metalloproteinase (MMP) and Hepatocyte growth factor (HGF) compared to mesenchymal stem cells.
  • MMP Matrix Metalloproteinase
  • HGF Hepatocyte growth factor
  • the mesenchymal progenitor cells may have differentiation ability into adipocytes, osteocytes, osteoblasts, chondrocytes or myocytes.
  • the mesenchymal progenitor cells may have a doubling time (20 hours to 35 hours).
  • the mesenchymal progenitor cells can inhibit the proliferation of Peripheral Blood Mononuclear Cell (PBMC).
  • PBMC Peripheral Blood Mononuclear Cell
  • the mesenchymal progenitor cells can induce the secretion of IL-10 (Interleukin 10).
  • an anti-inflammatory cell therapy composition comprising the mesenchymal progenitor cells (FEMP) as an active ingredient.
  • FEMP mesenchymal progenitor cells
  • the embryonic stem cells ESC
  • iPSC induced pluripotent stem cells
  • SCNT somatic cell nuclear transfer stem cells
  • the pore size of the porous membrane may be 1 ⁇ m to 20 ⁇ m.
  • the cell culture insert may comprise one or more additives selected from the group consisting of Fetal bovine serum (FBS), Serum replacement (SR) and Human platelet lysate (HPL).
  • FBS Fetal bovine serum
  • SR Serum replacement
  • HPL Human platelet lysate
  • Mesenchymal progenitor cells of the present invention may exhibit excellent proliferative, differentiating, migrating and angiogenic efficacy according to changes in specific marker expression patterns.
  • the cell therapeutic composition comprising such mesenchymal progenitor cells can implement an excellent anti-inflammatory effect and can be applied as an effective therapeutic agent for the site of inflammation caused by atopic dermatitis, cystitis and the like.
  • mesenchymal progenitor cells using a cell culture insert with a porous membrane, it is possible to improve cell viability and effectively remove the pluripotency of embryonic stem cells at low cost.
  • FEMPs mesenchymal progenitor cells
  • FIG. 2 is an image of each step of the method for producing mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.
  • FEMP mesenchymal progenitor cells
  • Figure 3 is a graph measuring the level of Oct4 and Nanog expression of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.
  • Figure 4 is an image of the cell morphology according to the culture conditions of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.
  • FEMP mesenchymal progenitor cells
  • Figure 5 shows the results of karyotype analysis of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.
  • Figure 6 is a graph measuring the EGF-R and CD95 expression rate of mesenchymal progenitor cells (FEMP) and bone marrow-derived mesenchymal stem cells (BM-MSC) according to an embodiment of the present invention.
  • FEMP mesenchymal progenitor cells
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • Figure 7 is an image of the adipocytes and osteocytes (Osteocyte) differentiated mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.
  • Figure 8 shows the effect of inhibiting apoptosis by Fas Ligand of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.
  • FEMP mesenchymal progenitor cells
  • FIG. 10 is a graph showing the expression level of angiogenesis factor and migration factor of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention compared with bone marrow-derived mesenchymal stem cells ( cutoff: 1.1).
  • FEMP mesenchymal progenitor cells
  • FIG. 11 is a graph showing PBMC proliferation inhibitory activity of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.
  • FEMP mesenchymal progenitor cells
  • FEMP mesenchymal progenitor cells
  • FEMP mesenchymal progenitor cells
  • FEMPs Functionally Enhanced Mesenchymal Progenitors
  • FEMPs mesenchymal progenitor cells
  • EGF-R epidermal growth factor receptor
  • MSC mesenchymal Stem Cell
  • FEMPs mesenchymal progenitor cells
  • MSC Mesenchymal stem cell
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • mesenchymal stem cells may be derived from mammals other than humans, fetuses or humans. Mammals other than humans include, but are not limited to, canines, felines, monkeys, cattle, sheep, pigs, horses, rats, mice, and guinea pigs.
  • the mesenchymal progenitor cells overexpress epidermal growth factor receptor (EGF-R) and low expression of CD95, compared to conventional mesenchymal stem cells (MSCs), and thus superior to the mesenchymal stem cells. It possesses proliferative capacity, differentiation capacity, and mobility, and can also exhibit excellent body viability. That is, the mesenchymal progenitor cells (FEMP) may refer to cells that are much more functional than the adult adult mesenchymal stem cells.
  • the epidermal growth factor receptor is a cell-surface receptor for the epidermal growth factor family of extracellular protein ligands, which promotes paracrine of stem cells and results in VEGF (Vascular Endothelial Growth Factor). ), And may promote the expression of angiogenesis factors such as Fibroblast Growth Factor (HGF), and increase the expression of various growth factors or cytokines such as Heparin-Binding EGF-like Growth Factor (HBEGF) and Amphiregulin (AREG). Can improve stem cell proliferation, differentiation ability.
  • HGF Fibroblast Growth Factor
  • HGF Heparin-Binding EGF-like Growth Factor
  • AVG Amphiregulin
  • EGF-R can act on stem cells with MAP kinase (Mitogen-Activated Protein kinase), PKC (Protein Kinase C) and the like to improve the mobility.
  • MAP kinase Mitogen-Activated Protein kinase
  • PKC Protein Kinase C
  • FEMP mesenchymal progenitor cells overexpressing EGF-R may exhibit superior proliferative, differentiating and migrating ability compared to conventional mesenchymal stem cells.
  • the EGF-R may promote anti-inflammatory activity and skin regeneration through interaction with epidermal growth factor (EGF).
  • EGF epidermal growth factor
  • the CD95 Cluster Differentiation 95
  • FasR Fes Receptor
  • APO-1 Apoptosis antigen 1
  • TNFRSF6 Tumor Necrosis Factor Receptor Superfamily Member 6
  • FasL Fas Ligand
  • CD95L CD95 Ligand
  • the mesenchymal progenitor cells can have excellent proliferative capacity compared to mesenchymal stem cells (MSC), in particular bone marrow-derived mesenchymal stem cells (BM-MSC). This property may mean that in stem cell grafts, it possesses excellent cell viability.
  • FEMP mesenchymal progenitor cells
  • ESC embryonic stem cells
  • iPSC induced pluripotent stem cells
  • SCNT somatic cell nuclear transfer stem cells
  • embryonic stem cell refers to the extraction of inner cell mass (ICM, Inner Cell Mass) from blastocyst embryos just before mammalian fertilized egg implants into the mother's womb and cultured in vitro. As one, it refers to a pluripotency stem cell that can differentiate into all the cells of the animal. In a broad sense, this concept includes an embryoid body derived from embryonic stem cells, an induced pluripotent stem cell (iPSC) and a somatic cell nuclear transfer stem cell (SCNT). It can be understood as.
  • ICM Inner Cell Mass
  • the embryonic stem cells include, but are not limited to, all embryonic stem cells derived from mammals including humans, primates, cattle, pigs, sheep, horses, dogs, mice, rats, and livestock. Stem cells.
  • human embryonic stem cells for example, H9 (James A. Thomson et. Al. Embryonic Stem Cell Lines Derived from Human Blastocysts. Science 1998 Dec 4; 282 (5395): 1827) and the like may be used.
  • human embryonic stem cells can be readily constructed by those skilled in the art.
  • FEMP mesenchymal progenitor cells
  • LTBP latent-transforming growth factor beta-binding protein
  • VEGF vascular endothelial growth factor
  • KDR Kinase insert Domain Receptor
  • FZD compared to mesenchymal stem cells
  • angiogenesis factors selected from the group consisting of (Frizzled), Fibroblast Growth Factor (FGF), Angiopoietin (ANG), Platelet-Derived Growth Factor Receptor (PDGFR), and Endothelial Monocyte-Activating Polypeptide (EMAP). Can overexpress.
  • FEMP mesenchymal progenitor cells
  • MMP matrix metalloproteinase
  • HGF hepatocyte growth factor
  • the mesenchymal progenitor cells When the mesenchymal progenitor cells (FEMP) are present around the wounded tissue, anti-inflammatory activity, cell migration capacity and vascular regeneration efficacy may be important factors for the treatment thereof. That is, the stem cells may be moved to tissues or organs that are inflamed by a wound, thereby alleviating inflammation and sequentially regenerating damaged blood vessels. At this time, the effect of wound treatment may be determined according to the level of each anti-inflammatory activity, mobility and angiogenesis efficacy.
  • the mobility may be understood as a concept including a homing ability to move to a target organ or tissue.
  • FEMPs mesenchymal progenitor cells
  • MSCs mesenchymal stem cells
  • FGF Fibroblast Growth Factor
  • EGF-R epidermal growth factor receptor
  • the mesenchymal progenitor cells as described above may have an excellent differentiation ability, for example, differentiation into adipocytes (adipocytes), osteocytes (osteocytes), chondrocytes (myocytes) or myocytes (myocytes).
  • the mesenchymal progenitor cells can be used as a cell therapy suitable for the treatment of diseases, symptoms, such as diabetic retinopathy, interstitial cystitis, irritable cystitis, and may exhibit an excellent effect in the treatment of inflammatory diseases.
  • the mesenchymal progenitor cells can implement an enhanced proliferative capacity compared to conventional mesenchymal stem cells by overexpressing the epidermal growth factor receptor (EGF-R).
  • EGF-R epidermal growth factor receptor
  • the doubling time of doubling the number of cells from the time of the stem cell culture may be 20 hours to 35 hours, preferably 24 hours to 32 hours.
  • Such cell proliferation ability means that the effect of FEMP can be further improved as a cell therapy agent.
  • the mesenchymal progenitor cells may exhibit anti-inflammatory activity by inhibiting proliferation of Peripheral Blood Mononuclear Cells (PBMCs) or inducing secretion of Interleukin 10 (IL-10). Accordingly, the cell therapy composition may be applied as an effective therapeutic agent for atopic dermatitis, rheumatoid arthritis, interstitial cystitis and the like.
  • the PBMC may include T-cells, B-cells, natural killer (NK) cells, monocytes, macrophages, and the like.
  • the mesenchymal progenitor cells can inhibit the expression of Major Histocompatibility Complex (MHC) in macrophages and inhibit T-cell activity by inhibiting the interaction of CD28, CD80, CD86 and the like.
  • MHC Major Histocompatibility Complex
  • IL-10 secreted by mesenchymal progenitor cells inhibits the expression of inflammation-related enzymes such as inducible Nitric Oxide Synthase (iNOS) and Inducible Cyclooxyganas (COX-2) in macrophages.
  • inflammation-related enzymes such as inducible Nitric Oxide Synthase (iNOS) and Inducible Cyclooxyganas (COX-2) in macrophages.
  • iNOS inducible Nitric Oxide Synthase
  • COX-2 Inducible Cyclooxyganas
  • the mesenchymal progenitor cells can be used as an anti-inflammatory cell therapy composition.
  • the cell therapy composition may further include a support for receiving the mesenchymal progenitor cells (FEMP), preferably a biodegradable support.
  • FEMP mesenchymal progenitor cells
  • the biodegradable support may be a hydrogel such as fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose, pectin, chitin, polyglycolic acid, or polylactic acid, but is not limited thereto.
  • the cell therapy composition may further include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, HSA (Human serum albumin) and one or more of these components. If desired, other conventional additives such as antioxidants, buffers and bacteriostatic agents can be added.
  • the cell therapy composition may be formulated into an injectable formulation such as an aqueous solution, suspension, emulsion, or the like by additionally adding a diluent, a dispersant, a surfactant, a binder, and a lubricant, but is not limited thereto.
  • an injectable formulation such as an aqueous solution, suspension, emulsion, or the like by additionally adding a diluent, a dispersant, a surfactant, a binder, and a lubricant, but is not limited thereto.
  • ESC embryonic stem cells
  • iPSC induced pluripotent stem cells
  • SCNT somatic cell nuclear transfer stem cells
  • Embryonic stem cells, induced pluripotent stem cells and somatic cell nuclear transfer stem cells of step (a) are as described above.
  • embryoid body refers to a cell aggregate formed by attachment between embryonic stem cells, and when inoculated into a hydrophobic culture vessel, the embryonic stem cells are removed from the elements that maintain the undifferentiated state. Embryonic stem cells do not adhere to the bottom of the culture vessel and aggregate between cells to form embryoid bodies having a size of 1 mm or less.
  • the embryoid body may have a trioderm (endoderm, mesoderm, ectoderm) differentiating ability constituting the body composition of the mammal.
  • the term "cell culture insert” is a device having a porous membrane, epithelium that occurs during embryonic development when cultured embryonic stem cells, induced pluripotent stem cells or somatic cell nuclear transfer stem cells derived embryonic- Epithelial-Mesenchymal Transition (EMT) refers to the mechanism by which naturally occurs.
  • EMT embryonic- Epithelial-Mesenchymal Transition
  • the pore size of the porous membrane may be 1 ⁇ m to 20 ⁇ m, preferably 6 ⁇ m to 12 ⁇ m, more preferably 8 ⁇ m.
  • EGM2-MV, DMEM, MEM- ⁇ , STEMPRO-MSC, MesenCult-MSC medium can be used as the medium of the cell culture insert, preferably EGM2-MV, DMEM or MEM- ⁇ , It is not limited to this.
  • the cell culture insert may include one or more additives selected from the group consisting of Fetal bovine serum (FBS), Serum replacement (SR) and Human platelet lysate (HPL).
  • FBS Fetal bovine serum
  • SR Serum replacement
  • HPL Human platelet lysate
  • the additive may be 1-10% FBS, 1-10% SR or 1-10% HPL, preferably 5% FBS or 2.5-5% HPL, but is not limited thereto. no.
  • the progenitor cells specifically mesenchymal progenitor cells (FEMP)
  • FEMP mesenchymal progenitor cells
  • FEMP multifunctional mesenchymal progenitor cells
  • zymatic separation refers to a method for separating cell aggregates through enzymatic treatment, specifically collagenase (Collagense), accutase including collagenase I, II, III, IV (Accutase), Dispase, trypLE or trypsin-EDTA can be isolated by treatment, preferably may be a hypoallergenic enzyme trypLE, but is not limited thereto.
  • a method of separating and culturing in the state of maintaining the original shape of the epithelial cell sheet using a scraper can be used.
  • Step (c) compares the expression level of specific markers of progenitor cells isolated in step (b) with the expression levels of mesenchymal stem cells (MSC), specifically bone marrow-derived mesenchymal stem cells (BM-MSC).
  • the markers may be epidermal growth factor receptor (EGF-R) and CD95.
  • step (c) when the progenitor cells isolated in step (c) overexpress epidermal growth factor receptor (EGF-R) and low CD95 expression compared to the mesenchymal stem cells, multifunctional mesenchymal progenitor cells (FEMP) It can be judged as Specific marker characteristics are as described above.
  • EGF-R epidermal growth factor receptor
  • FEMP multifunctional mesenchymal progenitor cells
  • the mesenchymal progenitor cells prepared according to the above method are particularly excellent in anti-inflammatory activity, they can be selected and formulated by mixing with a support, a carrier, a diluent, and other additives according to marker expression levels such as EGF-R and CD95.
  • the support, carrier and the like are as described above.
  • Example 1 Isolation of Human Embryonic Stem Cells (hESC) -derived Mesenchymal Progenitor Cells (FEMP)
  • Figure 2 is an image of each step of the method for producing mesenchymal progenitor cells according to an embodiment of the present invention.
  • human embryonic stem cells in the normal oxygen state (37 °C, 5% CO 2 cell incubator) after initial differentiation through the formation of embryoid body (B), cells having a pore size of 8 ⁇ m Smear on culture inserts (C).
  • Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F12) containing 20% Serum Replacement (SR), 1% Non-essential amino acid (NEAA), 0.1% ⁇ -mercaptoethanol, and 1% anti-biotic Medium was used.
  • Embryos were cultured on the cell culture inserts for 1 to 10 days, thereby confirming that progenitor cells migrated below the inserts to form epithelial cell-like sheets (D).
  • RT-PCR analysis confirmed the transition pattern of each cell and the results are shown in FIG. 3.
  • Specific conditions such as marker gene, primer sequencing and the like are shown in Table 1 below.
  • the human embryonic stem cells (hESC) remaining on the cell culture insert express a certain level of pluripotency markers Oct4 and Nanog, while the mesenchymal cells migrated to the cell culture insert below.
  • Progenitor cells (FEMP) were found to express little Oct4 and Nanog similarly to human embryonic fibroblasts (hEF).
  • FEMPs mesenchymal progenitor cells migrated to the lower cell culture inserts through epithelial-mesenchymal transition can be easily separated, and the mesenchymal progenitor cells (FEMPs) are human embryonic stem cells (hESCs). It can be expected to show a different characteristic from).
  • Figure 2 is an image of each step of the method for producing a stem cell according to an embodiment of the present invention.
  • the mesenchymal progenitor cells (FEMP) sheet formed in the lower part of the cell culture was scraped off using a scraper (E) and then passaged to a new culture dish and cultured.
  • the culture medium was EGM-2MV medium containing 5% fetal bovine serum (FBS, Fetal Bovine Serum).
  • the epithelial cell-shaped sheet is rolled out, and passaged again to obtain stem cells continuously. Remaining stem cells were removed and passaged using trypLE.
  • FEMP mesenchymal progenitor cells
  • FEMP mesenchymal progenitor cells
  • the procedure of leaving for 10 minutes was repeated twice.
  • the cells were suspended in 0.5 ml of fixed solution and cell suspension was added dropwise onto a slide glass immersed in 70% ethanol. Ethanol on the slide was dried using an alcohol lamp, and water was removed from the air, and then the coverslip was covered and observed under a microscope (FIG. 5).
  • FEMP mesenchymal progenitor cells
  • FEMP mesenchymal progenitor cells
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • mesenchymal progenitor cells compared to BM-MSC overexpressing EGF-R about 40% to 70%, it can be seen that CD95 is about 80% low expression.
  • FEMP mesenchymal progenitor cells
  • markers such as CD73 were strongly expressed, and hematopoietic stem cell markers such as CD34 and CD45 were not expressed, and thus, the mesenchymal progenitor cells (FEMP) were analyzed to include the characteristics and multipotency of conventional mesenchymal stem cells. In fact, it was confirmed that the differentiation into adipocytes, bone cells, myocytes and the like (FIG. 7).
  • FEMP mesenchymal progenitor cells
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • A549 was used as a positive control group.
  • seeding of BM-MSC, mesenchymal progenitor cells (FEMP), and A549 cells at 1 ⁇ 10 4 cells / well at 100 ⁇ l was performed on each 96 well culture plates, and cultured for 37 to 24 hours, followed by apoptosis.
  • apoptotic cells increased in a concentration-dependent manner when Fas Ligand was treated in BM-MSC.
  • Fas Ligand was treated in BM-MSC.
  • apoptotic cells increased by 40.64 ⁇ 12.02% on day 1 after treatment with 500ng / ml Fas Ligand.
  • FEMP mesenchymal progenitor cells
  • FEMP mesenchymal progenitor cells
  • FEMP mesenchymal progenitor cells
  • Each stem cell was plated with 3 wells of each passage (16, 17, 18, 19 passages) at a concentration of 2.0x10 4 cells / plate in a 6 well plate dish. Each stem cell was cultured at normal oxygen partial pressure, and then separated from the culture dish using 0.25% trypsin. The average cell number was confirmed by counting the cell number three times using a hemocytometer, and the results are shown in Table 3 below.
  • FEMP mesenchymal progenitor cells
  • BM-MSCs showed a doubling time of more than 39 hours
  • mesenchymal progenitor cells FEMPs
  • FEMPs mesenchymal progenitor cells
  • FEMP mesenchymal progenitor cells
  • FEMP mesenchymal progenitor cells
  • BM-MSC mesenchymal progenitor cells
  • FEMP mesenchymal progenitor cells
  • the results of the analysis are expressed as a fold change of FEMP / BM-MSC, and the cutoff value is set to 1.1 and is shown in FIG. 10.
  • FEMP mesenchymal progenitor cells
  • LTBP-1 mesenchymal progenitor cells
  • VEGF-B mesenchymal progenitor cells
  • KDR mesenchymal progenitor cells
  • FEMP mesenchymal progenitor cells
  • Example 9 Analysis of PBMC proliferation inhibitory effect of mesenchymal progenitor cells (FEMP)
  • PBMC Peripheral Blood Mononuclear Cell
  • FEMP mesenchymal progenitor cells
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • the mesenchymal progenitor cells showed improved PBMC inhibition rate compared to BM-MSC in the same cell number, and in particular, in the experimental group having 10,000 cell numbers, the inhibition rate was approximately 10-fold. .
  • FEMP mesenchymal progenitor cells
  • FEMP mesenchymal progenitor cells
  • IL-10 Interleukin 10
  • Peripheral Blood Mononuclear at a concentration of 1x10 6 cells / well Cells were plated in a 96 well plate and induced an inflammatory response at 100 ng / ml LPS.
  • the concentration of IL-10 in each experimental group was measured with or without LPS treatment and cells (FEMP or BM-MSC) treatment, and the results are shown in Table 5 and FIG. 12.
  • IL-10 IL-10 was secreted from PBMCs that induce an inflammatory response with LPS compared to before adding LPS.
  • IL-10 was further increased when PBMCs, which caused inflammatory responses with LPS, were co-cultured with BM-MSC or FEMP.
  • BM-MSCs bone marrow-derived mesenchymal stem cells
  • FEMPs mesenchymal progenitor cells
  • FEMP mesenchymal progenitor cells
  • Tissues were prepared by extracting the tissues at 4 and 8 weeks after cell transplantation, and H & E staining was performed.
  • FEMP mesenchymal progenitor cells

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Abstract

Un mode de réalisation de la présente invention concerne de nouvelles cellules progénitrices mésenchymateuses fonctionnellement améliorées surexprimant le récepteur du facteur de croissance épidermique (EGF-R) par comparaison avec des cellules souches mésenchymateuses (CSM), et sous-exprimant le cluster de différenciation 95 (CD95) par comparaison avec les CSM.
PCT/KR2017/013766 2016-12-02 2017-11-29 Nouvelles cellules progénitrices mésenchymateuses fonctionnellement améliorées, composition d'agent thérapeutique cellulaire anti-inflammatoire les contenant, et procédé de préparation de cellules progénitrices mésenchymateuses WO2018101723A1 (fr)

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KR20160163584 2016-12-02
KR10-2016-0163586 2016-12-02
KR20160163586 2016-12-02
KR10-2016-0163584 2016-12-02
KR10-2017-0106994 2017-08-24
KR1020170106994A KR101900199B1 (ko) 2016-12-02 2017-08-24 기능성이 향상된 신규 간엽성 전구세포, 이를 포함하는 항염증용 세포치료제 조성물 및 간엽성 전구세포의 제조방법

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CN112522189A (zh) * 2020-12-01 2021-03-19 深圳先进技术研究院 一种调控间充质干细胞分化和旁分泌功能的方法

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KR20160002248A (ko) * 2014-06-30 2016-01-07 건국대학교 산학협력단 다공성 막을 가진 세포배양 삽입체를 이용한 배아줄기세포로부터 다분화능 신경능 줄기세포의 분리 방법
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US20160130556A1 (en) * 2013-01-18 2016-05-12 Escape Therapeutics, Inc. Enhanced differentiation of mesenchymal stem cells
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US20160130556A1 (en) * 2013-01-18 2016-05-12 Escape Therapeutics, Inc. Enhanced differentiation of mesenchymal stem cells
KR20160002248A (ko) * 2014-06-30 2016-01-07 건국대학교 산학협력단 다공성 막을 가진 세포배양 삽입체를 이용한 배아줄기세포로부터 다분화능 신경능 줄기세포의 분리 방법
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112522189A (zh) * 2020-12-01 2021-03-19 深圳先进技术研究院 一种调控间充质干细胞分化和旁分泌功能的方法

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