Stem Cells Differentiation Into Nucleus Pulposus Like-cells Based On The New Phenotype Of Young Healthy Nucleus Pulposus Cells For Intervertebral Disc Regeneration

Part 1: The Expression of Newly Defined Phenotype of Young Healthy Nucleus Pulposus Cells in Rat Intervertebral DiscObjective: The main purpose of this study is to confirm the expression of healthy nucleus pulposus(NP)phenotypic markers recently recommended by the Spine Research Interest Group in rat nucleus pulposus cells(NPCs).Methods: Immunofluorescence was performed to evaluate the expression of NP markers.The expression of these NP markers was also quantified by quantitative polymerase chain reaction(qPCR)and western blotting analysis under normoxic and hypoxic conditions.Results: The results by qPCR,western blot and immunofluorescence microscopy results showed that the recommended newly defined young healthy NPCs phenotypes were expressed both in the normoxic and hypoxic culture conditions.The expression of aggrecan,brachyury,carbonic anhydrase 3,carbonic anhydrase 12,CD24,collagen II,Glut-1,Hif-1α,Hif-2α,cytokeratin 8,cytokeratin 18,cytokeratin 19 and shh were significantly higher in the hypoxic culture condition compared to the normoxic culture condition.Conclusion: These findings suggested that NP lives in hypoxic microenvironment and thus,we propose that these recommended newly defined young healthy NP phenotypic markers can be used as a phenotypic marker of NPCs in further researches.Part 2: Conditioned Medium Regulates Mesenchymal Stem Cells Differentiation into Nucleus Pulposus-like CellsObjective: The main purpose of this study is to evaluate the effectiveness of nucleus pulposus cell(NPC)conditioned medium for promoting the expression of mesenchymal stem cells(MSCs).Methods: The expression of MSCs treated with 50% or 100% NPC conditioned medium was investigated using quantitative polymerase chain reaction(qPCR)and western blotting under normoxic and hypoxic conditions.Results: qPCR and western blotting demonstrated significant upregulation of NP marker expression in MSCs cultured under hypoxic conditions and treated with 50% or 100% NPC conditioned medium,compared with those cultured under normoxic conditions.Upregulation was highest in the presence of 100% NPC conditioned medium compared with the control group(aggrecan,p < 0.01;brachyury,p < 0.05;collagen II,p < 0.001;KRT8,p < 0.01;KRT19,p < 0.001;and Shh,p < 0.01).The expression levels of genes in MSCs treated with 50% NPC conditioned medium also showed upregulation compared with the control group(collagen II,p < 0.05;KRT8,p < 0.05;and KRT19,p < 0.01).Conclusion: These findings suggested that the NPC conditioned medium stimulated MSC differentiation into an NP-like phenotype with distinct characteristics.The results could inform strategies for IVD regeneration.