| Objectives: Increasing evidence suggests that the therapeutic effect of implanted MSCs is not stem cells themselves but their secreted trophic factors and cytokine mediated.In the present study,We aimed to efficiently isolate and culture MSCs from degenerated nucleus pulposus and umbilical cord tissue,and the biological characteristics of the cells were identified and compared.And the effect of conditioned medium from human umbilical cord mesenchymal stem cells(UCMSCs-CM)on nucleus pulposus derived stem/progenitor cells from degenerated disc(D-NPMSCs)was further evaluated from cell morphology,cell phenotype,cell proliferation ability,three-line differentiation potential,cell pluripotency and nucleus pulposus specific marker gene e×pression.Methods: Nucleus pulposus derived stem/progenitor cells from degenerated IVD were isolated and purified by using enzymolysis tissue block method,human umbilical cord mesenchymal stem cells(UCMSCs)were isolated and purified by using explants culture method.The obtained cells were passaged for purification and cultured in vitro followed by morphological observation.The expression of surface markers was detected by flow cytometry and the positive cell rates were compared,and then its multilineage differentiation potential was identified and compared.The conditioned medium was collected from human UCMSCs to treat with the D-NPMSCs,and the morphological changes of the cells were observed.Flow cytometry was employed to detect the expression of MSCs and NP cell specific surface markers,cell apoptosis and cell cycle.Proliferative capacity and cell viability were evaluated with Edu,CCK8 and growth curve analysis.Multilineage potential was measured with specific cytochemical staining.The qRT-PCR was used to analyze the mRNA expressions of NP specific markers and pluripotency related genes.Finally,we investigated the effect of UCMSCs-CM on the phosphorylation of ERK in D-NPMSCs by western blot.Results: We added human UCMSCs conditioned media to cultured degenerate NPSCs in vitro.The results showed that the appearance of D-NPSCs changed significantly,and the proportion of spindle spindle cells increased significantly.Through flow cytometry,we found that the expression level of specific surface antigens of mesenchymal stem cells was significantly upregulated;the proliferative capacity was significantly increased,while the cells in the dividing S phase were significantly increased,and the apoptotic cells were significantly reduced.The osteogenic and chondrogenic differentiation potential was also significantly enhanced in D-NPMSCs.In addition,we found that the expression of pluripotency genes(Nanog,OCT4)and NP progenitor cell marker gene Tie2 in D-NPMSCs was significantly upregulated after UCMSCs-CM treatment,whereas NP cell specific markers(ACAN?COL2A1?SOX9?CD24)were significantly downregulated.Moreover,phosphorylation of ERK is significantly reduced.Conclusion: Our finding suggested that MSCs-derived conditioned medium was able to rejuvenate the degenerated NP stem/progenitor cells by improving the cell proliferation capacity,stemness and osteogenic and chondrogenic differentiation potential differentiation potential.This study provides insights and perspectives for development of stem cell-based therapeutic strategies for IVD degeneration. |
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