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

English Keywords:Notochordal cells  Mesenchymal stem cells  Nucleus pulposus  Intervertebral disc  Co-culture

English Abstract:

  【Abstract】 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-defined 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 collagenⅡand 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.