Induction of mesenchymal stem cells to nucleus pulposus phenotype by indirect co-culture in vitro

Objectives: To isolate and co-culture notochordal cells(NC) and mesenchymal stem cells(MSCs) from immature nucleus pulposus(NP) of New Zealand rabbit,and to investigate the induction of notochordal cells to mesenchymal stem cells.Methods: Notochordal cells were harvested from immature NPs of 8 New Zealand rabbits(4-6 week-old) and purified by discontinuous gradient density centrifugation.MSCs were released from femur bone marrow of New Zealand rabbits and purified by discontinuous gradient density centrifugation.MSCs were cultured alone and non-contact co-cultured with NCs(1 ∶1) respectively,the expressions of collagenⅡand proteoglycan were determined by toluidine blue and immunocytochemistry staining.The expressions of collagenⅠ/Ⅱand proteoglycan in gene and protein level were detected.Results: NCs and MSCs were isolated and purified.NCs with diameter of 20-30μm had abundant intracytoplasmic vesicles and poor proliferation.MSCs showed as fusiform,arrayed with whirlpool.After indirect co-culture of NCs and MSCs for 3 days,the cells of experiment group demonstrated a robust cellular and extracellular matrix,with well-de fined cells that demonstrated intense metachromatic staining when stained with toluidine blue,which was in sharp contrast to the control and more obvious by continued co-culture.The indirect co-cultured cells also demonstrated positive immune reactivity to collagen Type II and brown particles were scattered in intracellular.After indirect co-culture for 5 days,The mRNA level of collagenⅠdecreased to a lower level in NC/MSC sample compared with the higher level in MSC sample.Collagen II and aggrecan gene expression increased significantly in NC/MSC sample,almost 1.61 and 2.35 fold than that in the control.After indirect co-cultured,MSCs from experiment group were observed expression of collagen Ⅱand proteoglycan compared with negative expression in the group of MSCs cultured alone.Conclusions: By co-culture,NCs can stimulate MSCs′ proliferation and differentiation toward nucleus pulposus cell,which may provide a new choice to cell therapy for intervertebral disc regeneration.