Abstract
Stem cells are undifferentiated primitive cells that are capable of self-renewal and multipotency of differentiation, which can be applied in cell or gene therapy. Human bone marrow mesenchymal stem cell (hMSC) is a type of multipotent stem cell. It has the characteristics of being cultured and expanded in vitro easily, and multipotency of osteogenic, chondrogenic, and adipogenic differentiation. However, the source of bone marrow is not easy to obtain and the life span of hMSC is limited due to senescence. In recent studies, the attempt of the transformation of SV40T antigen (SV40T) or human telomere reverse transcriptase (hTERT) genes into MSCs to delay senescence have been failed to prevent cells from entering crisis stage and losing multipotency. We had constructed three vectors carrying combination of SV40T and hTERT genes by using a bicistronic retroviral plasmid construct with Cre-loxP recombination system for reversible immortalization in previous studies. In this study, three reversible immortalized MSC lines, including MSC-SV40T, MSC-hTERT, and MSC-SV40T-hTERT, were successfully established by using these three vectors, and their characteristics were investigated. The transformed hMSCs expressed either SV40T or hTERT, or both, on mRNA and protein levels. Telomerase activity was detected in MSC-hTERT and MSC-SV40T-hTERT. Untransformed hMSCs appeared senescenced at PD 36 during in vitro culture. MSC-SV40T showed decreased growth rate, morphological changes and shortened telomere length at PD 75. MSC-hTERT showed morphological changes and low multipotency beyond PD 55. No apparent difference in growth rate, cell morphology, telomere length and differentiation potential of adipocyte, osteocyte and chondrocyte of MSC-SV40T-hTERT at PD150 was observed, nor tumorogenicity. The study suggested that transfection of both SV40T and hTERT genes into hMSCs could overcome M1 and M2 crises, which allowed cells being immortalized and also maintaining multipotency of differentiation.