Comparison of biological characteristics of nucleus pulposus mesenchymal stem cells from non-degenerative and degenerative nucleus pulposus in rats

Abstract

Objective To compare the biological characteristics of nucleus pulposus mesenchymal stem cells (NPMSCs) derived from non-degenerative and degenerative intervertebral disc (IVD). Methods NPMSCs were isolated from non-degenerative (N-NPMSCs) and degenerative IVD (D-NPMSCs). Flow cytometry was performed to detect the expressions of cell surface markers. The expression of the stemness genes was detected by reverse-transcription quantitative polymerase chain reaction (RT-PCR). The biological characteristics of NPMSCs, including cell colony formation, cell proliferation, cell apoptosis, cysteinyl aspartate-specific protease (Caspase)-3 activity, mRNA and protein expressions of hypoxia inducible factor-1α (HIF-1α), glucose transporter 1(GLUT-1), vascular endothelial growth factor (VEGF), silent information regulator protein 1 (SIRT1) and SIRT6 were compared. Results NPMSCs from both groups highly expressed CD105, CD90 and CD73 and low expressed CD45 and CD11b/c. D-NPMSCs showed lower expression of stemness genes [sex determination region related high mobility group box protein-2 (Sox-2): 1.52±0.21 vs. 3.41±0.94, Nanog: 2.15±0.16 vs. 4.72±1.12, octamer-binding transcription factor 4 (Oct-4): 1.73±0.11 vs. 3.22±1.03) (t=6.145, 6.556, 7.806, P=0.000, 0.000, 0.000). D-NPMSCs showed lower clonogenic [(23.5±10.1)% vs. (39.4±12.2)%, t=5.304, P=0.001] and proliferative activity, while increased rate of cell apoptosis (10.43% vs. 4.69%) (t=7.689, P=0.000) and Caspase-3 activity (0.77 vs. 0.72) (t=2.743, P=0.016). D-NPMSCs also showed lower expression of HIF-1α, GLUT-1, VEGF, SIRT1 and SIRT6 in the mRNA and protein levels (The results of each experiment were 0.35±0.23, 0.51±0.22; 0.94±0.14, 0.90±0.13; 0.02±0.01, 0.41±0.02; 0.04±0.02, 0.73±0.34 and 0.51±0.22, 0.84±0.05; respecticely) (The t value was 6.486, 5.015; 2.271, 2.572; 4.921, 6.275; 7.898, 6.754 and 7.016, 7.956; respectively; The P value was 0.000, 0.001; 0.044, 0.023; 0.001, 0.000; 0.000, 0.000 and 0.000, 0.000; respectively). Conclusion D-NPMSCs showed different biological behaviors including lower proliferative activity, lower expression of stemness gene and higher cell apoptosis rate. The HIF-1α-mediated pathway may be involved in the mechanism of cell proliferation. Key words: Nucleus pulposus mesenchymal stem cells; Intervertebral disc degeneration; Proliferation; Apoptosis; Hypoxia inducible factor-1α