[An in vitro study on human bone marrow mesenchymal stem cells protecting nucleus pulposus cells from oxidative stress-induced apoptosis in a co-culture system of no direct cellular interaction]

Abstract

Objective: Bone marrow mesenchymal stem cells (BMSCs) transplantation can potentially regenerate the degenerated intervertebral disc, with the underlying regenerating mechanism remaining largely unknown. To investigate the potential of human BMSCs protecting nucleus pulposus cells (NPCs) from oxidative stress-induced apoptosis in a co-culture system, and to illustrate the possible mechanisms of BMSCs transplantation for intervertebral disc regeneration.

Methods: BMSCs collected by density gradient centrifugation in Percoll solution were cultured and sub-cultured till passage 3, and the surface molecules of CD34, CD45, and CD13 were identified. NPCs were isolated by collagenase digestion and the chondrocyte like phenotype was confirmed by morphologic observation after HE staining, inverted phase contrast microscope, proteoglycan, and collagen type II expression after toluidine blue and immunocytochemistry staining. The 3rd passage BMSCs and the 1st passage NPCs were divided into four groups: group A, NPCs (1 x 10(6) cells) were cultured alone without apoptosis inducing (negative control); group B, NPCs (1 x 10(6) cells) were co-cultured with BMSCs (1 x 10(6) cells) with apoptosis inducing; group C, NPCs (1 x 10(6) cells) were co-cultured with BMSCs (3 x 10(5) cells) with apoptosis inducing; group D, NPCs (1 x 10(6) cells) were cultured alone with apoptosis inducing (positive control). After 3 or 7 days of culture or co-culture, the NPCs in groups B, C, and D were exposed to 0.1 mmol hydrogen peroxide for 20 minutes to induce apoptosis. With DAPI staining cellular nucleus, Annexin-V/propidium iodide staining cellular membrane for flow cytometry analysis, the apoptosis of NPCs in each group was studied both qualitatively and quantitatively. Besides, the changes in Bax/Bcl-2 gene transcription and Caspase-3 protein content, were analyzed with semi-quantitative RT-PCR and Western blot.

Results: BMSCs were successfully isolated and CD34-, CD45-, and CD13+ were demonstrated; after isolated from degenerated intervertebral discs and sub-cultured, the spindle-shaped 1st passage NPCs maintained chondrocyte phenotype with the constructive expressions of proteoglycan and collagen type II in cytoplasm. DAPI staining showed the nucleus shrinkage of apoptosis NPCs. Co-cultured with BMSCs for 3 days and 7 days, the apoptosis rates of NPCs in groups B (29.26% +/- 8.90% and 18.03% +/- 2.25%) and C (37.10% +/- 3.28% and 13.93% +/- 1.25%) were lower than that in group D (54.90% +/- 5.97% and 26.97% +/- 3.10%), but higher than that of group A (15.67% +/- 1.74% and 8.87% +/- 0.15%); all showing significant differences (P < 0.05). Besides, semi-quantitative RT-PCR showed Bcl-2 gene transcription up-regulated (P < 0.05) and no significant change of Bax (P > 0.05); Western blot result showed that the Caspase-3 protein expression of groups B and C was lower than that of group D, and was higher than that of group A; all showing significant differences (P < 0.05).

Conclusion: In a co-culture system without direct cellular interactions, the oxidative stress-induced apoptosis of human NPCs was ameliorated by BMSCs. The enhanced anti-apoptosis ability of NPCs preconditioned by co-culturing with BMSCs might come from the decreased Bax/Bcl-2 gene transcription ratio.