Effect of serum concentration on the biological activity of human nucleus pulposus mesenchymal stem cells

English Keywords:Intervertebral disc degeneration  Nucleus pulposus mesenchymal stem cell  Fetal bovine serum  Biological activity

English Abstract:

  【Abstract】 Objectives: To observe and research the effects of different serum concentrations on the biological activity of human normal nucleus pulposus mesenchymal stem cells (NPMSCs) in vitro. Methods: The nucleus pulposus tissue of patients with congenital scoliosis correction(Pfirrmann grade Ⅰ or Ⅱ) was selected and NPMSCs were isolated and cultured in vitro. First, Flow cytometry was used to detect cells phenotypes(CD105, CD90, CD73, CD45, CD34, and HLA-DR). The ability to induce differentiation into osteogenic, adipogenic and chondrogenic cells was then observed. After that, third passage NPMSCs were cultured in DMEM medium containing different concentrations of fetal bovine serum(FBS) (0%, 5%, and 10%) to determine their cell proliferation capacities, cell cycles, apoptosis rates, and stemness genes (Oct4, Nanog, Jag1, and Notch1) and functional genes (collagen Ⅰ, collagen Ⅱ, aggrecan, and SOX-9) expressions were detected by Quantitative Real-time PCR(QRT-PCR), respectively. Results: The cells isolated and cultured from normal nucleus pulposus tissues grew in a spindle shape, adhered to the wall, and could be passaged in vitro. The immunophenotype results showed the cells highly expressed CD73 (98.9%), CD90 (97.9%), and CD105 (98.9%), and lowly expressed HLA-DR (3.11%), CD34 (1.83%), and CD45 (1.19%). The isolated cells could be differentiated into osteoblasts, adipocytes and chondrocytes. According to the criteria of the International Society for Cellular Therapy(ISCT), the isolated and cultured cells were NPMSCs. With the decrease of FBS concentration, the cell proliferation rate decreased, cell apoptosis rate increased significantly, and the G1 phase length prolonged (P<0.05). The QRT-PCR results showed that with the decrease of FBS concentration, the relative expression level of stemness genes (Oct4, Nanog, Jag1, and Notch1) decreased significantly, the relative expression level of collagen Ⅰ increased, and the relative expression levels of collagen Ⅱ, aggrecan and SOX-9 decreased. There were statistically significant differences(P<0.05). Conclusions: Decreased serum concentration can significantly suppress the proliferation of NPMSCs, and inhibit the expression of stemness genes and related functional genes.