The aim of this study was to evaluate whether human Wharton's jelly cells (WJCs) could be differentiated into nucleus pulposus (NP)-like cells by coculturing with NP cells (NPCs) in vitro. WJCs were isolated from the human umbilical cord, and NPCs were isolated from healthy human intervertebral disc. After coculturing WJCs with NPCs in a monolayer environment with or without cell-cell contact for 7 days, the real-time polymerase chain reaction showed the relative gene expressions of NP-marker genes (aggrecan, type II collagens, and SRY-type HMG box-9) were significantly increased (p<0.05) in all groups, and the increase in the group of 25:75/WJCs:NPCs was the largest (p<0.05). The increases of relative gene expression in WJCs cocultured with cell-cell contact were larger than those cocultured without contact in all ratios (p<0.05). WJCs were positive for telomerase expression. Flow cytometry analyses showed that WJCs expressed CD73, CD105, CD90, CD29, CD166, and human leukocyte antigen (HLA)-ABC while being negative for the expression of CD34, CD45, and HLA-DR. The results of this study indicated that the WJCs had the feature of the mesenchymal stem cell and might be induced to differentiate to NP-like cells by coculturing with NPCs.
Even though human nucleus pulposus (HNP) cells have a limited replicative lifespan in vitro culture, the lifespan of ovine nucleus pulposus cells transferred by lipofectamine with human telomerase reverse transcriptase (hTERT) gene could be prolonged. However, the lipofectamine is a transient expression system with an expressed duration of hTERT for less than 73 days. Here we present a viral vector system to investigate whether recombinant adenoassociated virus vector-mediated hTERT gene (rAAV-hTERT) could safely prolong the activities of HNP cells. Our hypothesis was that the activities of HNP cells could be extended with rAAV-hTERT gene transfer. Therefore, we designed an in vitro and in vivo evaluation of a HNP cells gene transfectant. Second-generation HNP cells were transfected with rAAV-hTERT and the expression of hTERT determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Cumulative population doubling, senescence assay, real-time RT-PCR, and enzyme-linked immunosorbent assay were used to determine cellular activities. Genetic phenotype was validated by karyotypic analysis and ontogenicity by nude mice injected in vivo. The continuing expression of hTERT gene and production of extra cellular matrix for 120 days were found in the transfected HNP cells, and karyotypic instability was detected, but there was no evidence of polyploidy or oncogenicity in nude mouse. The results showed that this transfection model might provide a resource cells necessary for the biological treatment of degenerative disc disease.