Extending the activities of human nucleus pulposus cells with recombinant adeno-associated virus vector-mediated human telomerase reverse transcriptase gene transfer.

Abstract

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.