Mesenchymal stem cells from Chinese Guizhou minipig by hTERT gene transfection.

INTRODUCTION: To provide standard implemental cells for biomedical research, an immortalized mesenchymal stem cell (MSC) lineage was established from the bone marrow of Chinese Guizhou minipigs. We investigated the characteristics of the MSC transfected with hTERT.

METHODS: MSCs isolated from bone marrow (BM) were steadily transfected using a plasmid containing human telomerase reverse transcriptase gene (pCI-neo-hTERT) at population doubling (PD) time of 3. The expression of hTERT was analyzed by reverse transcriptase polymerase chain reaction; the morphology of the cells, by phase contrast microscope; cell growth curve, by MTS assay, and phenotype, flow cytometry and immunofluorescence. Karyotype and osteogenic differentiation of transfected cells were compared with normal MSCs.

RESULTS: After transfection with the hTERT gene, the MSCs showed vigorous proliferation activity undergoing more than 60 PDs. Expression of the hTERT gene was detected in these cells. Transfected MSCs showed a prolonged life span, maintaining similar morphology and karyotype compared with normal MSCs. The apoptotic rate of transfected MSCs at PD60 was far below that of the normal MSCs at PD15. More than 99% of transfected MSCs were positive for stem cell markers, including SH-2, SH-3, SB-21, and CD29, and negative for CD34, CD45, and SLA-II. Transfected MSCs possessed the ability to differentiate into osteogenic as same as that of normal MSCs.

CONCLUSION: The hTERT gene-induced immortalization prolonged the life span of MSCs, maintaining the overall properties identical to the original cells, findings that will be useful for basic research.