An in vitro study on the interaction of human cartilage endplate stem cells with nucleus pulposus cells in co-culture system

English Keywords:Cartilage endplate stem cells  Degenerative nucleus pulposus cells  Co-colture

English Abstract:

  【Abstract】 Objectives: To investigate the biological effects of Cartilage endplate stem cells(CESCs) and degenerative nucleus pulposus cells(NPCs) when co-cultured under a transwell system. Methods: The Cartilage endplate organization and degenerative nucleus pulposus tissue were obtained from the patients undergoing discectomy and fusion for lumbar degenerative disease. The cells were subcultured in the agarose culture and the cell clones were selected and expanded in vitro for the assays of stem cell markers by using immunofluorescence and flow cytometry assay. The third generation of CESCs and the primary NPCs were co-cultured in a transwell plate. The three groups were assigned as follows: CESCs group, NPCs group and co-colture group with CESCs and NPCs. The expressions of type Ⅱ collagen, aggrecan and Sox-9 of CESCs and NPCs were detected by RT-PCR at 3 days, 5 days and 7 days respectively. On the 7th day after co-culture, the Western-blot assay was performed to detect the expression of the protein. Results: Cultured cells in vitro exhibited a positive expression of the stem cell markers(the percentage of stem cell markers tested by flow cytometry showed CD73, CD90 and CD105 were detected and their positive rate was 97.5%, 98.7% and 98.7% respectively). The expressions of Agg, Coll Ⅱ and SOX-9 were not detected in CESCs group by RT-PCR. Higher levels of aggrecan, type Ⅱ collagen and Sox-9 expressions from RT-PCR were detected in NPCs from the co-cultured group ( their value was 1.32, 1.25, 0.92 respectively) than those in control group from the 5th day（P<0.05）, similarly, compared with the CESCs that not cultured with NPCs, the CESCs from the co-culture group showed statistically significant expressions of aggrecan, type Ⅱ collagen and Sox-9 from the 5th day(P<0.05)(the value in co-culture group was 0.10, 0.11, 0.15 respectively). On the 7th day after co-culture, the Western-blot showed that the CESCs from the co-culture group had a significantly higher expression of collagen protein Ⅱ, proteoglycan and SOX-9. Conclusions: During the co-colture process, the existence of NPCs can assist the CESCs diffierentiation into NPCs-like cells; furthermore, CESCs have the ability to enhance the expression of NPCs′s specific molecules.