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WO2017026878A1 - Composition de milieu de culture pour induire des cellules progénitrices musculo-squelettiques et composition pharmaceutique comprenant des cellules progénitrices musculo-squelettiques pour la prévention ou le traitement de maladies musculo-squelettiques - Google Patents

Composition de milieu de culture pour induire des cellules progénitrices musculo-squelettiques et composition pharmaceutique comprenant des cellules progénitrices musculo-squelettiques pour la prévention ou le traitement de maladies musculo-squelettiques Download PDF

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WO2017026878A1
WO2017026878A1 PCT/KR2016/008997 KR2016008997W WO2017026878A1 WO 2017026878 A1 WO2017026878 A1 WO 2017026878A1 KR 2016008997 W KR2016008997 W KR 2016008997W WO 2017026878 A1 WO2017026878 A1 WO 2017026878A1
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musculoskeletal
cell
hmspc
cells
bone
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Korean (ko)
Inventor
한명관
김정렬
허진주
송화령
서난희
이은혜
김승국
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Industry Academic Cooperation Foundation of Chonbuk National University
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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

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  • the present invention relates to a medium composition for inducing differentiation from stem cells to a musculoskeletal progenitor cell, a method for inducing differentiation from stem cells to musculoskeletal precursor cells using the medium composition, and a method for inducing differentiation into musculoskeletal progenitor cells, To a pharmaceutical composition and a cell treatment agent for preventing or treating musculoskeletal diseases.
  • Osteification is a process of bone formation, which is known to be due to two methods of intramedullary or intramedullary ossification.
  • Intramural ossification is a direct process that is converted into mesenchymal bone, which occurs within the skull bone.
  • ossification of cartilage occurs by the process of cartilage tissue being converted into bone after the process of cartilage tissue formation from agglomerated mesenchymal cells. This process of ossification is essential for most bone formation in vertebrate animals.
  • hESCs human embryonic stem cells
  • hESCs human embryonic stem cells
  • hiPSCs Human induced lpuripotent stem cells
  • pluripotent stem cells capable of differentiating into any cell type.
  • hiPSC is a cell that is useful for studying embryonic development at the cellular level and is attracting attention as a cell therapy agent. Since these cells can be differentiated into bone structures such as bone and cartilage by transplantation, they can be usefully used for repairing and repairing damaged skeletal tissues.
  • MSCs Mesenchymal stem cells
  • mice skeletal stem / progenitor cells are distinct from mesenchymal stem cells, and are highly likely to be specifically differentiated into bone and cartilage. Therefore, there is an increasing need for studies on cells capable of differentiating into bone and cartilage through osteoarthritis while overcoming the limitations of mesenchymal stem cells.
  • the present inventors have been able to induce musculoskeletal precursor cells from human embryonic stem cells or human induced pluripotent stem cells, and that the musculoskeletal precursor cells can be differentiated into bone through osteoarthritis and differentiated into cartilage,
  • the present invention has been completed.
  • the present invention provides a pharmaceutical composition comprising a FGF2 (Fibroblast Growth Factor 2) signaling activator, a TGF- ⁇ / activin / nodal signal transduction inhibitor, a Wnt signal activator, an extracellular signal-regulated kinase signaling inhibitor, and a leukemia inhibitory factor (LIF).
  • FGF2 Fibroblast Growth Factor 2
  • TGF- ⁇ / activin / nodal signal transduction inhibitor a Wnt signal activator
  • an extracellular signal-regulated kinase signaling inhibitor a leukemia inhibitory factor (LIF).
  • LIF leukemia inhibitory factor
  • the present invention provides a mucsloskeletal progenitor cell produced by the above method.
  • the present invention also provides a pharmaceutical composition for preventing or treating musculoskeletal diseases, which comprises the above musculoskeletal precursor cells.
  • the present invention also provides a cell therapy agent for treating musculoskeletal diseases, which comprises the above musculoskeletal precursor cells.
  • the culture medium composition of the present invention comprises LIF and is useful for stimulating stem cells by activating FGF2 signaling, inhibiting TGF-beta / activin / nodal signaling, activating Wnt signaling, and inhibiting ERK signaling
  • FGF2 signaling activating FGF2 signaling
  • TGF-beta / activin / nodal signaling activating Wnt signaling
  • ERK signaling Can induce the differentiation of musculoskeletal precursor cells efficiently, and the musculoskeletal progenitor cells obtained through this can differentiate into bone through ossification of cartilage and can be differentiated into cartilage, tendon, and muscle so that prevention of various musculoskeletal diseases Or may be useful for treatment.
  • Figure 1A shows the result of observing the morphology of hMSPC induced differentiation from hESC.
  • FIG. 1B is a graph showing the results of immunofluorescence detection of the expression of a fully differentiable marker in hMSPC.
  • FIG. 1C shows the results of RT-PCR analysis of the expression of the differentiation-ability marker in hMSPC.
  • FIG. 1D is a graph showing the results of immunofluorescence for expression of ectoderm, mesoderm, and endoderm markers in hMSPC.
  • FIG. 1E shows the results of RT-PCR for expression of ectoderm, mesoderm, and endoderm markers in hMSPC.
  • 2A shows the results of flow cytometry analysis of expression of mesenchymal stem cell markers in hMSC and hMSPC.
  • FIG. 2B is a graph showing the results of comparing bone formation, cartilage formation, and fat formation in hMSC and hMSPC.
  • FIG. 2C is a graph showing the change in bone cell marker expression during the bone formation process of hMSC and hMSPC for 9 days.
  • FIG. 2D shows the results of comparing changes in expression of cartilage markers before and after hESC-induced differentiation of hMSPC into chondrocytes.
  • Fig. 3A shows the results of immunohistochemical staining of expression of SM22a, a smooth muscle marker, in hMSPC.
  • FIG. 3B is a graph showing the expression of SM-MHC, a smooth muscle marker, in hMSPC by immunofluorescence.
  • FIG. 3C is a graph showing the results of immunofluorescence for the expression of CD31, an endothelial cell marker, in hMSPC.
  • FIG. 3E is a graph showing the expression of MAP2, a neuronal cell marker in hMSPC, by immunofluorescence.
  • FIG. 4A is a graph showing the results of immunofluorescence detection of the expression of the differentiation-ability markers in hMSPC derived from hiPSC.
  • FIG. 4B shows the results of RT-PCR analysis of the expression of the fully differentiable marker in hMSC derived from hiPSC.
  • 4C is a graph showing the results of immunofluorescence for expression of ectoderm, mesoderm, and endodermic markers in hMSC derived from hiPSC.
  • FIG. 4D shows the results of RT-PCR analysis of the expression of ectoderm, mesoderm, and endodermic markers in hMSC derived from hiPSC.
  • FIG. 4E shows the results of flow cytometry analysis of the expression of the mesenchymal stem cell marker in hMSC derived from hiPSC.
  • FIG. 4F shows the results of comparing bone formation, cartilage formation, and fat formation in hMSC derived from hiPSC.
  • FIG. 4G shows the expression of smooth muscle marker in hMSC derived from hiPSC by immunofluorescence.
  • FIG. 5A is a graph showing the result of transplanting hMSPC subcutaneously and confirming its differentiation.
  • FIG. 5B is a diagram showing the results of transplantation of hMSPC into the fascia and confirmation of its differentiation.
  • FIG. 5C is a diagram showing the results of transplantation of hMSPC under the fascia around the tendon and confirmation of its differentiation.
  • 6A shows the result of transplantation of hMSPCs under the kidney capsules of immunodeficient mice and histological examination by H & E staining 5 weeks later.
  • Figure 6B shows the results of transplantation of hMSPCs under the kidney capsules of immunodeficient mice and tissue validation by Movat ' s pentachrome staining 5 weeks later.
  • FIG. 6C is a graph showing the results of confirming the relationship between ossification of osteochondral and angiogenesis.
  • 7A is an experimental design diagram for a fracture study.
  • FIG. 7B is a photograph showing a fracture study.
  • FIG. 7C is a view showing results of fracture studies for hMSPC.
  • FIG. 7D is a view showing results of fracture studies on hMSPC.
  • FIG. 7E is a view showing a result of a fracture study on hMSPC.
  • FIG. 7F is a graph showing the result of confirming the bone formation effect of hMSPC.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an FGF2 (Fibroblast Growth Factor 2) signaling activator, a TGF-beta / activin / nodal signal transduction inhibitor, a Wnt signal activator, an extracellular signal- An inhibitor, and a leukemia inhibitory factor (LIF).
  • FGF2 Fibroblast Growth Factor 2
  • TGF-beta / activin / nodal signal transduction inhibitor e.g., a Wnt signal activator
  • LIF leukemia inhibitory factor
  • stem cell in the present invention is an undifferentiated cell having an ability to differentiate into various body tissues, including a totipotent stem cell, a pluripotent stem cell, a multipotent stem cell, cell.
  • the stem cells may be used in combination with terms such as precursor cells, progenitor cells, and the like.
  • the stem cell may be an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC). That is, the culture medium composition of the present invention can induce the differentiation of musculoskeletal precursor cells from embryonic stem cells or induced pluripotent stem cells.
  • ESC embryonic stem cell
  • iPSC induced pluripotent stem cell
  • the embryonic stem cell refers to a cell having a starch-like ability, and it is a cell of embryonic stem cell including the ability to develop into any cell derived from transplant without proliferation, infinite proliferation, autoregulation and all three embryonic stages , But is not limited thereto.
  • mucosloskeletal progenitor cell in the present invention refers, without limitation, to cells that can differentiate into bone, cartilage, tendons, and muscles.
  • the term "differentiation” refers to a phenomenon in which the structure or function of a cell is specialized during the growth of a cell by the proliferation and proliferation, that is, the cell or tissue of the organism is changed in shape or function to perform a task given to each.
  • a relatively simple system is separated into two or more qualitatively different systems.
  • the state in which a difference occurs or as a result is divided into qualitatively distinguishable parts or partial systems is called eruption.
  • the embryonic stem cell or induced pluripotent stem cell used in the present invention is derived from human, bovine, horse, goat, sheep, dog, cat, mouse, rat or alga, preferably human.
  • the stem cells used in the method of the present invention may be autologous or allogenic to the subject to be derived.
  • the FGF2 signaling activator may include, but is not limited to, bFGF (basic FGF).
  • the inhibitor of TGF-beta / activin / nodal signal transduction is E-616452 (2- [3- (6-methyl-2-pyridinyl) Yl) -1,5-naphthyridine), A-83-01 (3- (6-methyl-2-pyridinyl) -1-carbothioamide) or SB431542 (4- [4- (1,3-benzodioxol-5-yl) -5- (2- pyridinyl) But is not limited thereto.
  • the Wnt signal activator is SB216763 (3- (2,4-dichlorophenyl) -4- (1 -methyl-1 H-indol- (3-chloro-4-hydroxyphenyl) amino] -4- (2-nitrophenyl) -lH- pyrrole-2,5-dione), Kenpaullone Dihydro-pyrido [3,2-d] - [1] benzazepin-6 (5H) -one), CHIR99021 (9- , 2 ': 2,3] azepino [4,5-b] indol-6 (5H) -one), CP21R7 (3- Yl) -pyrrole-2,5-dione), SB203580 (4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -5- ), H-89 (5-isoquinolinesulfonamide), Purmorphamine (2- (1-naphthoxy) -6- (4-morpholin
  • the ERK signal inhibitor is selected from the group consisting of AS703026 (N - [(2S) -2,3-dihydroxypropyl] -3 - [(2-fluoro-4-iodophenyl) amino ]- isonicotinamide) (4-bromo-2-chloroanilino) -7-fluoro-N- (2-hydroxyethoxy) -3-methylbenzimidazole-5-carboxamide, PD0325901 - [(2-fluoro-4-iodophenyl) amino] -benzamide), ARRY-438162 (2R) -2,3-dihydroxypropoxy] -3,4-difluoro-2- Fluoro-N- (2-hydroxyethoxy) -1-methyl-1H-benzimidazole-6-carboxamide ), RDEA119 ((S) -N- (3,4-difluoro-2 - ((2- fluoro-4-iodophenyl)
  • the culture medium composition of the present invention may preferably include N2B27 medium and KOSR medium.
  • the N2B27 medium contains neural basal medium (Neurobasal, Gibco), DMEM / F12 (Gibco), N2 (Gibco, catalog number: 17502048), and B27 (Gibco, catalog number: 12587010)
  • the cells were cultured in RPMI 1640 medium supplemented with 48% DMEM / F12, 1% N2, 2% B27, 1 mM glutamine, 1% nonessential amino acid, 0.1 mM? -Mercaptoethanol, 0.1% penicillin-streptomycin, Serum albumin.
  • the KOSR medium can be prepared by replacing DMEM / F12 with Knockout DMEM (Life Technologies) in complete medium and may be adjusted accordingly.
  • the complete medium consisted of 20% KnockOut Serum Replacement (Invitrogen), 1 mM glutamine (Invitrogen), 1% nonessential amino acid (Invitrogen), 0.1 mM? -Mercaptoethanol (Invitrogen), and 0.1% penicillin / streptomycin , And DMEM / F12 (Invitrogen) supplemented with 15 ng / ml bFGF (R & D Systems).
  • the present invention also provides a method for inducing differentiation of stem cells into musculoskeletal progenitor cells, comprising culturing stem cells in the culture medium.
  • the stem cells that can be induced into musculoskeletal precursor cells by culturing in the medium composition are preferably embryonic stem cells or inducible pluripotent stem cells.
  • the present invention provides a mucsloskeletal progenitor cell produced by the above method.
  • the musculoskeletal precursor cells of the present invention can differentiate into bone, cartilage, muscle, or tendon.
  • the musculoskeletal precursor cells of the present invention can be differentiated into ectoderm or mesoderm.
  • the musculoskeletal precursor cells of the present invention are negative for Oct4, Nanog, Sox2 or Gdf3 among the differentiation-inducing markers and positive for Lin28.
  • the present invention also provides a pharmaceutical composition for preventing or treating musculoskeletal diseases, which comprises the above musculoskeletal precursor cells.
  • the present invention also provides a cell therapy agent for treating musculoskeletal diseases, which comprises the above musculoskeletal precursor cells.
  • the pharmaceutical composition and the cell treatment agent can be applied to all diseases to which stem cells can be applied, but most preferably they can be used for prevention or treatment of musculoskeletal diseases.
  • the pharmaceutical composition or cell treatment agent comprising the musculoskeletal precursor cells of the present invention can be used for osteoporosis, osteogenesis, osteogenesis imperfecta, osteopetrosis, osteosclerosis, Paget's disease, , Osteoarthritis, rickets, fracture, periodontal disease, segmental bone defect, osteolytic bone disease, primary and secondary hyperparathyroidism, hyperostosis, degenerative arthritis, degenerative arthritis, deformity arthropathy, deformity ankle arthritis, deformed arthropathy, deformed dog Arthritis, arthrosis of the patella, osteoarthritis of the patella, osteoarthritis of the simple humerus, osteochondroma of the humerus, lateral humeral condylar hyperhidrosis, humeral medial rectal hyperplasia, Hevadyn's nodule, Bushard's nodule, deformed moyamoya CM arthropathy, meniscus injury, disc disc degeneration, B
  • the pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carriers to be contained in the composition include those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, But are not limited to, cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • the pharmaceutical composition may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally.
  • parenteral administration it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and intrathecal administration.
  • the composition may be administered by any device capable of transferring the active agent to the target cell.
  • composition of the present invention may be prepared in unit dose form by incorporating into a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art, or may be prepared by inserting it into a multi-dose container .
  • the formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
  • the composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. It may also be administered once or, if necessary, further.
  • cell therapeutic agent used in the present invention refers to a medicament (US FDA regulation) used for the purpose of treatment, diagnosis and prevention of cells and tissues produced by separation, culture and special manipulation from human, refers to drugs used for therapeutic, diagnostic and prophylactic purposes through a series of actions, such as living, proliferating, screening, or otherwise altering the biological characteristics of a cell in vitro or in vitro.
  • " prevention " in the present invention means all the actions of inhibiting or delaying the progress of musculoskeletal diseases by administration of the composition or cell therapy of the present invention.
  • " treatment " used in the present invention means all the actions of improving or alleviating musculoskeletal diseases by administration of the composition or cell therapy of the present invention.
  • mice (20-24 g) in the background at 7-10 weeks of age were purchased from Orient bio (seongnam, Korea). All animal-related experiments were conducted in accordance with the Guidelines of the Animal Care and Use Committee of Chonbuk National University. The animals were maintained at a controlled temperature (21-24 ° C) and a 12:12 h light / dark cycle environment and allowed free access to water and food.
  • hESC human embryonic stem cells
  • hiPCS human induces lpuripotent stem cells
  • hMSPC human muscloskeletal progenitor cells
  • H9 hESCs were purchased from WiCell (Madison, MI, USA). hESC and hiPSC were transferred into a mitomycin C-treated CF1 monolayer on plates prepared 1 day before and cultured.
  • the culture medium (complete medium) contained 20% KnockOut Serum Replacement (Invitrogen), 1 mM glutamine (Invitrogen), 1% nonessential amino acid (Invitrogen), 0.1 mM? -Mercaptoethanol (Invitrogen), and 0.1% penicillin / streptomycin (Invitrogen) supplemented with 10 ng / ml bovine serum albumin (Invitrogen) and 15 ng / ml bFGF (R & D Systems).
  • the differentiation induction medium contained 20 ng / ml human LIF (Life Technologies), 15 ng / ml basic FGF, 3 ⁇ M CHIR99021 (Calbiochem), 1 ⁇ M PD0325901 (Calbiochem), 10 ⁇ M SB431542 (Sigma).
  • Differentiated cells were induced by culturing trypsinized hESCs or hiPSCs with TrypLE (Life technology) in induction medium on induction medium on bitronectin + gelatin (1 ng / ml, Sigma). Cells were transfected every 2-3 days with trypsinization without ROCK inhibitor and PKC inhibitor.
  • the digested samples were fixed overnight at 4 ° C in 2% paraformaldehyde (PFA) (Wako) and lime was removed with 0.4 M EDTA in PBS (pH 7.2) for 2 weeks at 4 ° C.
  • PFA paraformaldehyde
  • the samples were then dehydrated in alcohol or xylene, embedded in paraffin or embedded in OCT by cryoprotection in sucrose and cut. Representative sections were stained with H & E, and modified Movat pentachrome (Cosmobio).
  • the cells were then stained with secondary antibody Alexa Fluor 488-goat anti-mouse IgG, Alexa Fluor 594-donkey anti-rabbit IgG, Alexa Fluor 488-donkey anti-rabbit IgG, and Alexa Fluor 594-donkey anti-mouse IgG Invitrogen) .
  • the nuclei were stained with DAPI (4,6-diamidino-2-phenylindole). Images were acquired using an Olympus IX71 fluorescence microscope and MetaMorph software (Molecular Devices).
  • the primary antibodies treated at the cross-sections were: a mouse monoclonal antibody (Abcam) for HLA class I, a goat polyclonal antibody (Santacruz) for Collagen Type II, a rabbit polyclonal antibody (Santacruz) for osteocalcin, , Osterix (Abcam), p-myosin light chain (Abcam), Scleraxis (antibodies-online), Runx2 (Novus), and Sclerostin (Santacruz).
  • the secondary antibodies used are Alexa 555 (Invitrogen) and Alexa 488 (Invitrogen) IgG. Immunostained sections were stained with TO-PRO3 (Invitrogen) to stain nuclei. Fluorescence labeled tissue sections with a Leica DM 5000 microscope (Leica Microsystems) or a confocal microscope (LSM510; Carl Zeiss) were captured and validated with Zen software.
  • the cultured cells were treated with trypsin / EDTA to separate into a single cell suspension, blocked with 2% BSA in PBS, and then stained with CD73, CD90, and CD90 in buffer solution [1XPBS, 1% BSA, and 0.01% sodium azide] 0.0 > CD205, < / RTI > CD146, CD166 (BD Biosciences).
  • Cells were then incubated with Alexa Fluor 488 secondary mouse IgGs (Invitrogen, Carlsbad, Calif.) And analyzed using a flow cytometer (FACStar Plus Flow Cytometer, BD Biosciences). Normal mouse IgGs (BD Biosciences) were used as negative control.
  • MSC mesenchymal stem cells
  • hMSPC hMSPC into osteoblasts and adipocytes
  • cells were isolated with trypsin / EDTA, centrifuged at 100 x g for 5 min and seeded at 5 x 10 3 cells / cm 2 in a culture vessel Respectively.
  • Cells were cultured in induction medium at 37 ° C, 5% CO 2 for one day.
  • the medium was replaced with a pre-warmed complete osteogenesis differentiation kit (Life Technology) or a StemPro adipogenesis Differentiation Kit (Life Technology).
  • the cultures were re-fed every 3-4 days. After 14 days, the cells were stained with alkaline phosphatase staining (Roche) or alizarin red S (Sigma) for observation of osteogenesis, and oil red O) (Sigma).
  • the cells were separated with trypsin / EDTA, centrifuged at 100 ⁇ g for 5 minutes, and reconstituted with 1 ml of StemPro chondrogenesis differentiation kit (Life Technology) After cloudy, the cells were centrifuged again.
  • the pellet was resuspended in 1 x 105 viable cells / l in the differentiation medium and 5 ul of the cell solution was inoculated at the center of the unsubstantiated 96-well plate.
  • the cartilage formation medium heated in the culture vessel was added and cultured in a 5% CO 2 incubator at 37 ° C. The cultures were re-fed every 3-4 days. After 14 days, the cartilage-producing pellet was stained with Alcian blue, and gene expression analysis was performed.
  • hMSPC and HUVEC were differentiated into endothelial cells (ECs) or smooth muscle cells (SMCs).
  • the cells were treated with 50 ng / ml vascular endothelial growth factor (ProSpec, Rehovot, Israel) and 10 ng / ml bFGF (basic fibroblast growth factor) in EC differentiation medium (EGM) -2 (Lonza, Walkersville, MD) ml), 2.5 ng / ml TGF- ⁇ 1 (5 ⁇ g / ml) in SMC differentiation medium (SMCM: ScienCell Research Laboratories, Carlsbad, Calif. (transforming growth factor beta 1, ProSpec) for 6 days.
  • EMM EC differentiation medium
  • SMC differentiation medium SMC differentiation medium
  • hNSCs Human neural stem cells differentiated from ready-to-use H9 hESCs were purchased from GIBCO. hNSCs were maintained in knockout DMEM / F12 (GIBCO) containing 2 mM GlutaMAX, 20 ng / ml bFGF, 20 ng / ml EGF and 2% StemPro neural supplements. To differentiate into neurons, hMSPC and hNSC were plated in polyornithine and laminin-coated culture dishes. Two days later, the medium was replaced with neural differentiation medium (Neurobasal medium containing 2% B27, 2 mM GlutaMAX and antibiotics). On the 7th day of differentiation, 0.5 mM dibutyl cAMP (Sigma) was added daily for 3 days.
  • hMSPC (10 6 -10 7 cells in Matrigel) was transplanted into the subcutaneous and fascia of Balb / c nude mice to determine the differentiability of hMSPC.
  • hMSPC aggregates (4 x 10 5 ) were implanted under the kidney capsules of Balb / c nude mice. After 2-6 weeks of transplantation, the transplanted cells were removed and the tissue was analyzed.
  • Collagen cell carriers (CCC, 500042933, Viscofan-bioengineering, Weinheim, Germany) were placed in phosphate buffered saline (PBS) for 30 minutes to analyze the bone formation of hMSPCs in the long bone fracture model. After washing the PBS, the CCC was left overnight to dry to make it slightly opaque. SPC was inoculated into CCC and cultured. In one 6-week-old Balb / c-nude mouse, unilateral femoral osteotomy was pinned. The SPC supported by the CCC was then inserted into the fracture site of the mouse. The fractured bone was placed for 6 weeks. Images of the fracture site were obtained using an X-ray (Kodak DXS 4000 pro system, Rochester, USA).
  • hMSPC bone formation in a skull fracture model
  • a 7-week-old Balb / c-nude mouse was made with a 5-mm bony skull of the right parietal region.
  • SPC cells were primed for 7 days in StemPro Osteogenesis Differentiation Kit (Life technology). After priming SPC, 1x10 4 cells were inoculated into a scaffold made of hyaluronic acid-loaded poly (lactic-co-glycolic acid (HA-PLGA) for 24 hours and transplanted into empty areas.
  • HA-PLGA hyaluronic acid-loaded poly
  • CT computed tomography
  • hMSPCs The differentiation of hMSPCs was induced from hESCs as shown in Example 1.2 above, and the morphological changes of induced hMSPCs were observed, and the results are shown in Fig. 1A.
  • the expression of the differentiation marker was observed by immunofluorescence in the hMSPC after passage from the hESC to the passage of 10 or more passages, and the observation result is shown in Fig. 1B.
  • H9 hESC was positive for both OCT4, NANOG, SOX2, and LIN28, confirming the ability to differentiate.
  • hMSPC derived from H9 hESC showed negative for OCT4, NANOG and SOX2, but positive for LIN28.
  • 1C shows the results of RT-PCR of the expression of the differentiation-ability markers identified by immunofluorescence in the hMSPC of 5, 10, and 15 passages, respectively.
  • ectoderm, mesoderm, and endoderm markers in hMSPC derived from hESC of passage number 10 was confirmed by immunofluorescence, and the results are shown in Fig. 1D.
  • FIG. 1E shows the results.
  • Flow cytometry analysis was performed on the mesenchymal stem cell markers CD73, CD90, CD105, CD146 and CD166 in hMSC and the hMSPC of Example 2.1 above.
  • the results of flow cytometry analysis in which the mesenchymal stem cell markers are indicated by orange lines and the control group is indicated by blue lines are shown in Fig. 2A.
  • hMSPC has similar potential for differentiation to mesenchymal stem cells.
  • hMSCs were differentiated into bone, cartilage and fat, and hMSPC was differentiated into bone and cartilage but not into fat.
  • the bone cell markers ALP and RUNX2 were expressed in the bone formation process of hMSPC, and it was confirmed that the markers were expressed at a higher level in the bone formation process of hMSPC than hMSC. Therefore, it was confirmed that hMSPC could be differentiated into bone and cartilage.
  • FIG. 2D shows the results of comparing the expression of the cartilage markers AGC, SOX9, COL1A1, COL1A2 and catrigen2 before and after differentiation of hMSPC derived from hESC of 2.1 above into chondrocytes.
  • the cartilage markers AGC, catrigen2, COL1A1, and COL1A2 were expressed more in hMSPC than in hMSC, and the cartilage markers were expressed at a higher level after hMSPC was differentiated into cartilage cells.
  • hMSPC is more likely to differentiate into bone cells and cartilage cells than hMSC, and hMSPC is not differentiated into adipocytes.
  • SM22 ⁇ and SM-MHC smooth muscle myosin heavy chain
  • HASMC Human atrial smooth muscle cells
  • hMSPC had the potential to differentiate into smooth muscle.
  • FIGS. 3C and 3D The results of immunofluorescence for the endothelial cell markers CD31 and VE-cadherin are shown in FIGS. 3C and 3D.
  • HUVEC was used as a positive control for endothelial cell differentiation.
  • FIG. 3E shows the results of performing immunofluorescence on MAP2 as a neural cell differentiation marker.
  • NSC neurovascular stem cells
  • hMSPC is not capable of differentiating into endothelial cells.
  • hMSPC is likely to develop into ectoderm but not into neurons. Therefore, hMSPC can be differentiated into endoderm, more specifically bone, cartilage, and muscle.
  • hiPSC was prepared by reprogramming IMR90 fetal fibroblasts by overexpression of sendai virus-mediated OCT4, KLF4, SOX2, and MYC according to the protocol developed by Hasegawa et al. (Fusaki et al., 2009).
  • HMSPC was derived from hiPSC in the same manner as in Example 1.2 to obtain hMSPC.
  • the expression levels of Oct4, Nanog, Sox2, and Gdf3 as hmPSC-derived hMSPCs were confirmed by immunofluorescence and RT-PCR, respectively, and are shown in Figs. 4A and 4B, respectively.
  • iPS cells were positive for both OCT4, NANOG, SOX2, and LIN28, indicating that they were capable of differentiating.
  • hMSPC derived from hiPSC was negative for OCT4, NANOG and SOX2, but positive for LIN28.
  • Flow cytometric analysis of mesenchymal stem cell markers CD73, CD90, CD105, CD146 and CD166 was performed in hMSPC derived from hiPSC.
  • the results of flow cytometry analysis in which the mesenchymal stem cell markers are indicated by orange lines and the control group is indicated by blue lines are shown in Fig. 4E.
  • hMSCs derived from hiPSC had similar potential for differentiation to mesenchymal stem cells.
  • hMSC derived from hiPSC was differentiated into bone and cartilage but not into fat.
  • 4G shows the result of performing immunofluorescence on SM22 ⁇ , SM-MHC (smooth muscle myosin heavy chain) as markers of smooth muscle in hMSPC derived from hiPSC.
  • hMSPC derived from hiPSC had the potential to differentiate into smooth muscle.
  • hMSPC was transplanted into the subcutaneous and fascia of immunodeficient mice. Five weeks after transplantation of hMSPCs onto the site, the tissues were stained with H & E and Movat's pentachrome and stained for comparison with TO-PRO3. The results are shown in FIGS. 5A to 5C.
  • hMSPC formed mineralized cartilage tissue (green) and non-mineralized cartilage tissue (yellow) under the skin (FIGS. 5Aa and 5Ab).
  • a typical cartilage-like structure was formed by H & E staining, and thus it can be seen that hMSPC formed chondrocytes.
  • immunohistochemical analysis showed that collagen type II (Col2), a cartilage marker, and hLA (human leukocyte antigen), a human cell marker, were positive, indicating that the transplanted hMSPCs differentiated into chondrocytes. [Scale bar: 1 mm (a), 100 ⁇ m (b, c), 500 ⁇ m (i), 100 ⁇ m (ii, iii, iv)].
  • hMSPC was differentiated into a typical muscle form in the fascia as a result of Movat's Pentachrome staining (Fig. 5Ba and Fig. 5Bb) and H & E (Fig. 5Bc) staining.
  • p-MLC phosphorylated myosin light chain
  • hLA human leukocyte antigen
  • hMSPC was differentiated into a typical muscle form under the fascia around the tendon as a result of Movat's Pentachrome staining (Figs. 5Ca and 5Cb) and H & E (Fig. 5Cc) staining.
  • immunohistochemical analysis revealed that the transfected hMSPCs were differentiated into dry cells because it was confirmed that Scm (scleraxis), which is a key marker, and hLA (human leukocyte antigen), a human cell marker, were positive.
  • Scm scleraxis
  • hLA human leukocyte antigen
  • the hMSPC of the present invention can be differentiated into cartilage, muscle, tendon and endochondral bone depending on the transplantation site, and excellent in the differentiation ability.
  • Example 1.2 The same induction hMSPC (4 x 10 5 cells) and the rats are implanted under the kidney capsule of immunodeficient mice. After hMSPC was transplanted to the site, the transplanted cells were removed, and the tissues were stained with H & E and Movat's pentachrome, and stained with TO-PRO3. The results are shown in FIGS. 6A and 6B.
  • Fig. 6A it was confirmed that hard tissue, that is, bone was formed at the implantation site of hMSPC (Fig. 6Aa and Fig. 6Ab). It was confirmed that bone and cartilage were formed together with H & E staining and pentachrome staining. It was confirmed that the tissue was composed of mineralized cartilage tissue (green), non-mineralized cartilage tissue (yellow ) And mineralized bone tissue (red) (Fig. 6Ac and Fig. 6Ad). Therefore, it can be seen that the transplanted hMSPC is differentiated into cartilage and bone under the kidney capsule.
  • hLA human leukocyte antigen
  • Col II a cartilage marker
  • the bone marrow section was stained with anti-vWF (von Willebrand factor) antibody.
  • vWF von Willebrand factor
  • FIG. 6C vWF-positive cells were found at the junction of kidney and bone marrow. Since vWF-positive cells are derived from mice, it can be seen that angiogenesis is necessary for intra-cartilage differentiation.
  • the hMSPC When the hMSPC is implanted under the kidney capsule, the hMSPC is differentiated into bone and cartilage, and the process of forming the lecture is completed and the cartilage disappears. Thus, it can be understood that the hMSPC is differentiated into the bone through ossification of the cartilage.
  • Example 1.2 In order to confirm the effect of the hMSPC induced recovery in the same manner as in Example 1.2 on fracture healing, a fracture study was conducted as shown in Examples 1.11, 7A and 7B, and the results are shown in Figs. 7C to 7E .
  • hMSPC bone formation of hMSPC was confirmed in vivo by injecting HA-PLGA and cells into the infected skull of immunodeficient mice. After priming of hMSPC with osteogenic medium for 7 days, cells were inoculated into HA-PLGA graft. As shown in FIG. 7F, Runx2, a bone formation marker, was expressed in chondrocytes and osteocytes of tissues, and it was confirmed that transplantation of HA-PLGA-loaded hMSPC completely cures skull bone defects. Therefore, it was confirmed that hMSPC could be involved in intramural ossification.
  • the results of the above experiments show that the FGF2 (Fibroblast Growth Factor 2) signaling activator, TGF- ⁇ / activin / nodal signal transduction inhibitor, Wnt signal activator, ERK (extracellular such as embryonic stem cells or inducible pluripotent stem cells, by using a medium composition for inducing the differentiation of stem cells into mucosal bone marrow progenitor cells, which comprises a signal-regulated kinase signaling inhibitor and a leukemia inhibitory factor (LIF) It is possible to differentiate into hMSPC from cells, and since hMSPC having differentiation ability into bone, muscle, cartilage, and tendon in vivo can be used effectively for various musculoskeletal diseases.
  • FGF2 Fibroblast Growth Factor 2
  • TGF- ⁇ / activin / nodal signal transduction inhibitor Wnt signal activator
  • ERK extracellular such as embryonic stem cells or inducible pluripotent stem cells

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Abstract

La présente invention concerne : une composition de milieu de culture pour induire une différenciation d'une cellule progénitrice musculo-squelettique à partir d'une cellule souche ; un procédé pour induire la différenciation d'une cellule progénitrice musculo-squelettique à partir d'une cellule souche à l'aide de la composition de milieu de culture ; une cellule progénitrice musculo-squelettique produite par le procédé ; et une composition pharmaceutique et un agent de thérapie cellulaire contenant la cellule pour la prévention ou le traitement de maladies musculo-squelettiques. La composition de milieu de culture selon la présente invention contient un LIF, et elle est capable d'induire de façon efficace la différenciation d'une cellule progénitrice musculo-squelettique à partir d'une cellule souche par activation du transfert de signal FGF2, inhibition du transfert de signal TGF-ß/activine/nodal, activation d'un signal Wnt et inhibition d'un signal ERK. La cellule progénitrice musculo-squelettique ainsi obtenue peut être différenciée dans un os par ossification endochondrale, mais aussi dans du cartilage, du tendon et du muscle. Elle peut par conséquent être utile dans la prévention ou le traitement de diverses maladies musculo-squelettiques.
PCT/KR2016/008997 2015-08-13 2016-08-16 Composition de milieu de culture pour induire des cellules progénitrices musculo-squelettiques et composition pharmaceutique comprenant des cellules progénitrices musculo-squelettiques pour la prévention ou le traitement de maladies musculo-squelettiques Ceased WO2017026878A1 (fr)

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