Notochordala cells stimulate proliferation and differentiation of mesenchymal stem cells toward nucleus pulposus phenotype

Abstract

Objective The notochordal cells ( NC ) and bone mesenchymal stem cells ( MSCs )were isolated co-cultured from New Zealand rabbit immature nucleus pulposus (NP),and the contribution of NC to proliferation and differentiation of MSCs was assessed.Methods NC were got from immature NP of 4 New Zealand rabbits (4-6 week-old) and purified by discontinuous gradient density centrifugation,and MSCs were released from femur bone marrow and purified by discontinuous gradient density centrifugation.MSCs were cultured alone and co-cultured with NC ( 1:1/1:2/2:1 ).Cell proliferation was evaluated by cell counting Kit-8 ( CCK-8 ) -kit and expression levels of collagen Ⅱ and proteoglycan detected by toluidine blue and immunocytochemistry staining.The expression of collagen Ⅱ and proteoglycan mRNA was assayed.Results NCs and MSCs were isolated and purified.After co-culture,proliferation ability in the group of cell ratio at 1:1 ( NC:MSC) was increased significantly as compared with other groups.In the coculture group of MSCs and NC,the expression of collagen Ⅱ and proteoglycan was 2.0 and 1.35 times higher than in NC cultured alone.Conclusion By co-culture of 1:1,NC can stimulate proliferation of MSCs,and induce differentiation toward NP cells. Key words: Notochordal cells; Bone mesenchymal stem cells; Nucleus pulposus; Intervertebral disc; Co-culture