猪髓核基质成分诱导骨髓间充质干细胞分化为脊索样细胞
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摘要
背景:脊索细胞对椎间盘内严酷的内环境有良好的抵抗性,或许是治疗椎间盘退变性疾病合适的种子细胞来源。猪髓核终生含有大量脊索细胞,其基质内含有多种细胞因子可以促进骨髓间充质干细胞向脊索样细胞分化。目的:观察猪髓核基质对骨髓间充质干细胞分化的影响。方法:体外分离、培养、鉴定猪骨髓间充质干细胞;制备猪髓核粉末,并用配置好的DMEM完全培养基重悬粉末制备成含猪髓核组织的DMEM培养基,对骨髓间充质干细胞进行诱导3周,RT-PCR检测脊索细胞标记性基因鼠短尾突变体表型(T)、细胞角蛋白8及细胞角蛋白18的表达。该实验经复旦大学附属金山医院动物实验伦理委员会批准。结果与结论:(1)原代培养的骨髓间充质干细胞具有贴壁生长能力,在特定诱导条件下具有成骨、成脂分化能力,且能表达间充质干细胞表面标记物CD90、CD105;(2)在猪髓核基质成分诱导下骨髓间充质干细胞形态发生变化,表达鼠短尾突变体表型、细胞角蛋白8、细胞角蛋白18;(3)结果显示,猪髓核基质成分能诱导骨髓间充质干细胞分化为具有脊索细胞表型的脊索样细胞,为椎间盘退行性疾病治疗提供可靠的种子细胞。
BACKGROUND: As notochord cells can develop resistance to a harsh microenvironment in the intervertebral disc, it may be a suitable cell source for the treatment of degenerative diseases of the intervertebral disc. Porcine nucleus pulposus contains a large number of notochord cells throughout the life, and various cytokines in the cell matrix can induce the differentiation of bone marrow mesenchymal stem cells into notochord-like cells. OBJECTIVE: To explore the effect of porcine nucleus pulposus matrix on the differentiation of bone marrow mesenchymal stem cells. METHODS: Porcine bone marrow mesenchymal stem cells were isolated, cultured and identified in vitro. Porcine nucleus pulposus powder was prepared, and suspended in the DMEM complete medium to prepare the DMEM medium containing porcine nucleus pulposus matrix. Then, the bone marrow mesenchymal stem cells were induced in the prepared medium. After 3 weeks of induction, Notochord cell marker genes, including brachyury, cytokeratin 8, and cytokeratin 18, were evaluated by RT-PCR. The study protocol was approved by the Animal Ethics Committee of Jinshan Hospital affiliated to Fudan University. RESULTS AND CONCLUSION:(1) Primary cultured bone marrow mesenchymal stem cells exhibited adherent growth. These cells had the ability of osteogenic and adipogenic differentiation under special conditions, and expressed CD90 and CD105.(2) Under the induction of porcine nucleus pulposus matrix, the cell morphology was changed, and the expression of brachyury, cytokeratin 8, and cytokeratin 18 was enhanced. To conclude, porcine nucleus pulposus matrix can induce the differentiation of bone marrow mesenchymal stem cells into notochord-like cells with notochord cell phenotype, which may provide a reliable cell source for the cell therapy of degenerative diseases of the intervertebral disc.
BACKGROUND: As notochord cells can develop resistance to a harsh microenvironment in the intervertebral disc, it may be a suitable cell source for the treatment of degenerative diseases of the intervertebral disc. Porcine nucleus pulposus contains a large number of notochord cells throughout the life, and various cytokines in the cell matrix can induce the differentiation of bone marrow mesenchymal stem cells into notochord-like cells. OBJECTIVE: To explore the effect of porcine nucleus pulposus matrix on the differentiation of bone marrow mesenchymal stem cells. METHODS: Porcine bone marrow mesenchymal stem cells were isolated, cultured and identified in vitro. Porcine nucleus pulposus powder was prepared, and suspended in the DMEM complete medium to prepare the DMEM medium containing porcine nucleus pulposus matrix. Then, the bone marrow mesenchymal stem cells were induced in the prepared medium. After 3 weeks of induction, Notochord cell marker genes, including brachyury, cytokeratin 8, and cytokeratin 18, were evaluated by RT-PCR. The study protocol was approved by the Animal Ethics Committee of Jinshan Hospital affiliated to Fudan University. RESULTS AND CONCLUSION:(1) Primary cultured bone marrow mesenchymal stem cells exhibited adherent growth. These cells had the ability of osteogenic and adipogenic differentiation under special conditions, and expressed CD90 and CD105.(2) Under the induction of porcine nucleus pulposus matrix, the cell morphology was changed, and the expression of brachyury, cytokeratin 8, and cytokeratin 18 was enhanced. To conclude, porcine nucleus pulposus matrix can induce the differentiation of bone marrow mesenchymal stem cells into notochord-like cells with notochord cell phenotype, which may provide a reliable cell source for the cell therapy of degenerative diseases of the intervertebral disc.
引文
[1]Luo J,Wang H,Peng J,et al.Rate of Adjacent Segment Degeneration of Cervical Disc Arthroplasty Versus Fusion Meta-Analysis of Randomized Controlled Trials.World Neurosurg.2018;113:225-231.
[2]Crevensten G,Walsh AJ,Ananthakrishnan D,et al.Intervertebral disc cell therapy for regeneration:mesenchymal stem cell implantation in rat intervertebral discs.Ann Biomed Eng.2004;32(3):430-434.
[3]Tang R,Jing L,Willard VP,et al.Differentiation of human induced pluripotent stem cells into nucleus pulposus-like cells.Stem Cell Res Ther.2018;9(1):61.
[4]Steck E,Bertram H,Abel R,et al.Induction of intervertebral disc-like cells from adult mesenchymal stem cells.Stem Cells.2005;23(3):403-411.
[5]Hu J,Deng G,Tian Y,et al.An in vitro investigation into the role of bone marrow-derived mesenchymal stem cells in the control of disc degeneration.Mol Med Rep.2015;12(4):5701-5708.
[6]Lee EH,Hui JH.The potential of stem cells in orthopaedic surgery.J Bone Joint Surg Br.2006;88(7):841-851.
[7]VadalàG,Studer RK,Sowa G,et al.Coculture of bone marrow mesenchymal stem cells and nucleus pulposus cells modulate gene expression profile without cell fusion.Spine(Phila Pa 1976).2008;33(8):870-876.
[8]Peroglio M,Douma LS,Caprez TS,et al.Intervertebral disc response to stem cell treatment is conditioned by disc state and cell carrier:An ex vivo study.J Orthop Translat.2017;9:43-51.
[9]Ishiguro H,Kaito T,Yarimitsu S,et al.Intervertebral disc regeneration with an adipose mesenchymal stem cell-derived tissue-engineered construct in a rat nucleotomy model.Acta Biomater.2019;87:118-129.
[10]Bach FC,Tellegen AR,Beukers M,et al.Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix:from bench towards bedside.Oncotarget.2018;9(41):26507-26526.
[11]de Vries S,Doeselaar MV,Meij B,et al.Notochordal Cell Matrix As a Therapeutic Agent for Intervertebral Disc Regeneration.Tissue Eng Part A.2019;25(11-12):830-841.
[12]Lin X,Fang X,Wang Q,et al.Decellularized allogeneic intervertebral disc:natural biomaterials for regenerating disc degeneration.Oncotarget.2016;7(11):12121-12136.
[13]Saggese T,Thambyah A,Wade K,et al.Differential Response of Bovine Mature Nucleus Pulposus and Notochordal Cells to Hydrostatic Pressure and Glucose Restriction.Cartilage.2018:1947603518775795.
[14]Li G,Yu F,Lei T,et al.Bone marrow mesenchymal stem cell therapy in ischemic stroke:mechanisms of action and treatment optimization strategies.Neural Regen Res.2016;11(6):1015-1024.
[15]Prockop DJ.Marrow stromal cells as stem cells for nonhematopoietic tissues.Science.1997;276(5309):71-74.
[16]VadalàG,Russo F,Ambrosio L,et al.Stem cells sources for intervertebral disc regeneration.World J Stem Cells.2016;8(5):185-201.
[17]Steffen F,Smolders LA,Roentgen AM,et al.Bone Marrow-Derived Mesenchymal Stem Cells as Autologous Therapy in Dogs with Naturally Occurring Intervertebral Disc Disease:Feasibility,Safety,and Preliminary Results.Tissue Eng Part C Methods.2017;23(11):643-651.
[18]Marcucio RS,Nauth A,Giannoudis PV,et al.Stem Cell Therapies in Orthopaedic Trauma.J Orthop Trauma.2015;29Suppl 12:S24-27.
[19]Deng P,Torrest A,Pollock K,et al.Clinical trial perspective for adult and juvenile Huntington's disease using geneticallyengineered mesenchymal stem cells.Neural Regen Res.2016;11(5):702-705.
[20]Zeng Y,Feng S,Liu W,et al.Preconditioning of mesenchymal stromal cells toward nucleus pulposus-like cells by microcryogels-based 3D cell culture and syringe-based pressure loading system.J Biomed Mater Res B Appl Biomater.2017;105(3):507-520.
[21]Han XB,Zhang YL,Li HY,et al.Differentiation of Human Ligamentum Flavum Stem Cells Toward Nucleus Pulposus-Like Cells Induced by Coculture System and Hypoxia.Spine(Phila Pa 1976).2015;40(12):E665-674.
[22]VadalàG,Sowa G,Hubert M,et al.Mesenchymal stem cells injection in degenerated intervertebral disc:cell leakage may induce osteophyte formation.J Tissue Eng Regen Med.2012;6(5):348-355.
[23]Smolders LA,Bergknut N,Grinwis GC,et al.Intervertebral disc degeneration in the dog.Part 2:chondrodystrophic and non-chondrodystrophic breeds.Vet J.2013;195(3):292-299.
[24]Bergknut N,Rutges JP,Kranenburg HJ,et al.The dog as an animal model for intervertebral disc degeneration.Spine(Phila Pa 1976).2012;37(5):351-358.
[25]Dominici M,Le Blanc K,Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement.Cytotherapy.2006;8(4):315-317.
[26]Salzig D,Schmiermund A,Gebauer E,et al.Influence of porcine intervertebral disc matrix on stem cell differentiation.JFunct Biomater.2011;2(3):155-172.
[27]Potier E,de Vries S,van Doeselaar M,et al.Potential application of notochordal cells for intervertebral disc regeneration:an in vitro assessment.Eur Cell Mater.2014;28:68-80.
[28]Vujovic S,Henderson S,Presneau N,et al.Brachyury,a crucial regulator of notochordal development,is a novel biomarker for chordomas.J Pathol.2006;209(2):157-165.
[2]Crevensten G,Walsh AJ,Ananthakrishnan D,et al.Intervertebral disc cell therapy for regeneration:mesenchymal stem cell implantation in rat intervertebral discs.Ann Biomed Eng.2004;32(3):430-434.
[3]Tang R,Jing L,Willard VP,et al.Differentiation of human induced pluripotent stem cells into nucleus pulposus-like cells.Stem Cell Res Ther.2018;9(1):61.
[4]Steck E,Bertram H,Abel R,et al.Induction of intervertebral disc-like cells from adult mesenchymal stem cells.Stem Cells.2005;23(3):403-411.
[5]Hu J,Deng G,Tian Y,et al.An in vitro investigation into the role of bone marrow-derived mesenchymal stem cells in the control of disc degeneration.Mol Med Rep.2015;12(4):5701-5708.
[6]Lee EH,Hui JH.The potential of stem cells in orthopaedic surgery.J Bone Joint Surg Br.2006;88(7):841-851.
[7]VadalàG,Studer RK,Sowa G,et al.Coculture of bone marrow mesenchymal stem cells and nucleus pulposus cells modulate gene expression profile without cell fusion.Spine(Phila Pa 1976).2008;33(8):870-876.
[8]Peroglio M,Douma LS,Caprez TS,et al.Intervertebral disc response to stem cell treatment is conditioned by disc state and cell carrier:An ex vivo study.J Orthop Translat.2017;9:43-51.
[9]Ishiguro H,Kaito T,Yarimitsu S,et al.Intervertebral disc regeneration with an adipose mesenchymal stem cell-derived tissue-engineered construct in a rat nucleotomy model.Acta Biomater.2019;87:118-129.
[10]Bach FC,Tellegen AR,Beukers M,et al.Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix:from bench towards bedside.Oncotarget.2018;9(41):26507-26526.
[11]de Vries S,Doeselaar MV,Meij B,et al.Notochordal Cell Matrix As a Therapeutic Agent for Intervertebral Disc Regeneration.Tissue Eng Part A.2019;25(11-12):830-841.
[12]Lin X,Fang X,Wang Q,et al.Decellularized allogeneic intervertebral disc:natural biomaterials for regenerating disc degeneration.Oncotarget.2016;7(11):12121-12136.
[13]Saggese T,Thambyah A,Wade K,et al.Differential Response of Bovine Mature Nucleus Pulposus and Notochordal Cells to Hydrostatic Pressure and Glucose Restriction.Cartilage.2018:1947603518775795.
[14]Li G,Yu F,Lei T,et al.Bone marrow mesenchymal stem cell therapy in ischemic stroke:mechanisms of action and treatment optimization strategies.Neural Regen Res.2016;11(6):1015-1024.
[15]Prockop DJ.Marrow stromal cells as stem cells for nonhematopoietic tissues.Science.1997;276(5309):71-74.
[16]VadalàG,Russo F,Ambrosio L,et al.Stem cells sources for intervertebral disc regeneration.World J Stem Cells.2016;8(5):185-201.
[17]Steffen F,Smolders LA,Roentgen AM,et al.Bone Marrow-Derived Mesenchymal Stem Cells as Autologous Therapy in Dogs with Naturally Occurring Intervertebral Disc Disease:Feasibility,Safety,and Preliminary Results.Tissue Eng Part C Methods.2017;23(11):643-651.
[18]Marcucio RS,Nauth A,Giannoudis PV,et al.Stem Cell Therapies in Orthopaedic Trauma.J Orthop Trauma.2015;29Suppl 12:S24-27.
[19]Deng P,Torrest A,Pollock K,et al.Clinical trial perspective for adult and juvenile Huntington's disease using geneticallyengineered mesenchymal stem cells.Neural Regen Res.2016;11(5):702-705.
[20]Zeng Y,Feng S,Liu W,et al.Preconditioning of mesenchymal stromal cells toward nucleus pulposus-like cells by microcryogels-based 3D cell culture and syringe-based pressure loading system.J Biomed Mater Res B Appl Biomater.2017;105(3):507-520.
[21]Han XB,Zhang YL,Li HY,et al.Differentiation of Human Ligamentum Flavum Stem Cells Toward Nucleus Pulposus-Like Cells Induced by Coculture System and Hypoxia.Spine(Phila Pa 1976).2015;40(12):E665-674.
[22]VadalàG,Sowa G,Hubert M,et al.Mesenchymal stem cells injection in degenerated intervertebral disc:cell leakage may induce osteophyte formation.J Tissue Eng Regen Med.2012;6(5):348-355.
[23]Smolders LA,Bergknut N,Grinwis GC,et al.Intervertebral disc degeneration in the dog.Part 2:chondrodystrophic and non-chondrodystrophic breeds.Vet J.2013;195(3):292-299.
[24]Bergknut N,Rutges JP,Kranenburg HJ,et al.The dog as an animal model for intervertebral disc degeneration.Spine(Phila Pa 1976).2012;37(5):351-358.
[25]Dominici M,Le Blanc K,Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement.Cytotherapy.2006;8(4):315-317.
[26]Salzig D,Schmiermund A,Gebauer E,et al.Influence of porcine intervertebral disc matrix on stem cell differentiation.JFunct Biomater.2011;2(3):155-172.
[27]Potier E,de Vries S,van Doeselaar M,et al.Potential application of notochordal cells for intervertebral disc regeneration:an in vitro assessment.Eur Cell Mater.2014;28:68-80.
[28]Vujovic S,Henderson S,Presneau N,et al.Brachyury,a crucial regulator of notochordal development,is a novel biomarker for chordomas.J Pathol.2006;209(2):157-165.