Cell activity and stem cell-related genes expression of mesenchymal stromal cells from non-degenerative and degenerative nucleus pulposus: a comparative study

English Keywords:Intervertebral disc degeneration  Nucleus pulposus mesenchymal stem cells  Stem cell-related genes  Cell activity

English Abstract:

  【Abstract】 Objectives： To compare the cell activity and stem cell-related genes expression of mesenchymal stem cells(MSCs) in nucleus pulposus(NP) from non-degenerative and degenerative disc． Methods: The human nucleus pulposus mesenchymal stem cells(NPMSCs) were isolated by using standard MSC culture medium as suitable for MSCs culture by Cyagen from 6 non-degenerative patients of either scoliosis or burst fracture and 6 patients with degenerative intervertebral disc separately． One case of cells was selected from each group, respectively, and immunophenotype(CD90/CD105/CD73/CD45/CD34 and HLA-DR) and multilineage(Osteogenic, chondrogenic and adipogenic) differentiation potential were analyzed under the criteria to define MSCs, which was stated by the International Society for Cellular Therapry(ISCT). The cell activity of second passage from each group was analyzed by using cell counting kit-8(CCK-8). The total RNA of both non-degenerative and degenerative group were extracted, then the Real-Time PCR was used to detect the relative expressions of stem cell-related genes Oct4 and Nanog. Results: The original cells from both non-degenerative and degenerative group showed adherent growth, cell morphology of two groups showed no significant difference. The immune phenotype showed both normal and degenerative cells highly expressed the mesenchymal stem cell surface markers CD90, CD105, CD73(expression ratio as high as 96%, 98%, 95%, respectively). And hematopoietic markers CD45, CD34, HLA-DR were lower expressions(all less than 4%). Multilineage differentiation results showed cells obtained from both non-degenerative and degenerative NP developed red stained calcium salts, and adipocyte-like cells which were stained red by Oil red O, and chondrocyte-like cells stained blue by toluidine blue, after Osteogenic, chondrogenic and adipogenic differentiation for 28 days. Together with the immunophenotype, cells obtained from both non-degenerative and degenerative NP fulfilled the criteria to define MSCs stated by ISCT. Cell viability assay by CCK-8 showed that cell activity in normal group was stronger than that in the degeneration group at 5, 7, 9, 11, 13 days, there was significant difference between two groups(P<0.05). The Real-Time PCR results showed that Stemness maintenance related gene Oct4 and Nanog expression in normal group was 4.63±1.17, 7.36±1.19 times respectively than degeneration group. The Stemness maintenance related gene expression in normal group was significantly higher than that in the degeneration group(P<0.05). Conclusions: Both the non-degenerative and degenerative NP contain NPMSCs． Moreover, the non-degenerative NPMSCs show stronger ability of cell metabolic activity and stem cell-related genes expression.