Transformation Of Human Telomerase Gene Bone Marrow Mesenchymal Stem Cells And Its Cellular Characteristics Analysis

Telomerase is a ribonucleoprotein consisting of RNA and proteins.The RNA components are templates of telomere replication.The protein components consist of telomerase catalysis subunits and other associated proteins.The telomerase catalysis subunit is also named telomerase reverse transcriptase(TERT)that plays an important role in the replication of telomere repeat sequences and the extension of telomeres.In telomerase-negative cells,the cellular senescence and the life-span depend on the loss rate of telomeres during each cell division and on the primary length of telomere.In telomerase-positive cells,the telomerase Can extend the telomere length,prolong the life-span of cells and even cause the immortalization of cells.The human MSCs play important roles in cell transplantation,gene therapy and tissue engineering.The proliferation capacity of hMSCs in vitro is limitted.Therefore,it is important to prolong the life-span of human MSCs to make up the shortage of cell source.In the present study, we constructed a retrovirus expressing hTERT gene,established a line of hMSCs transduced with exogenous hTERT(hTERT-hMSCs)and investigated its sustaining cellular properties in a long-term culture for over 290 population doublings(PDs).Object:A line of hMSCs overexpressing hTERT is constructed by transducting hTERT genes into hMSCs with a recombined retrovirus.The cell proliferation and the life-span of the tranduced cells are observed during culture and passage.An in vitro assay of cell growth in soft agar and an in vivo assay of tumorigenicity in NOD-SCID mice are used to test the tumorigenicity of hTERT-hMSCs.The objective of the present study is to illuminate whether the hTERT gene can prolong the life-span of human MSCs,to investigate the surface antigens,karyotypic characteristics,tumorigenicity,to investigate the multipotentials of differentiation into adipocytes,chondrocytes and osteocytes,and to illuminate the differentially expressed proteins between the primary hMSCs and the hTERT-hMSCs.We hope the present_study can be useful to construct clinically applied cell lines of hMSCs with a prolonged life-span and multipotentials of differentiation into mesenchymal lineages in future.Method and Results:hMSCs were infected by the recombinant retrovirus expressing hTERT.TRAP-PCR assay proved that the activity of telomerase in the hMSCs had been reconstituted.We applied an in vitro assay of cell growth in soft agar and an in vivo assay of tumorigenicity in NOD-SCID to confirm the transforming activity of hTERT-hMSCs cells.The sign of transformation was not detected in hTERT-hMSCs, which showed that hTERT-hMSCs have no tumorigenicity potential.After exposure to adipogenic,chondrogenic and osteogenic differentiation media,hTERT-hMSCs could differentiate into adipocytes,chondrocytes and osteocytes respectively,which showed that hTERT-hMSCs have the multipotentials of differention into adipocytes, chondrocytes and osteocytes.By two-dimensional gel electrophoresis and peptide mass fingerprinting(PMF)using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS),we identified 100 proteins including 20 differently expressed proteins in hTERT-hMSCs.Conclusion:The telomerase activity of hMSCs was reconstituted by recombinant retrovirus expressing hTERT.The life-span of hMSCs was extended and the replicative senescence was overcome.The in vitro assay of cell growth in soft agar and in vivo assay of tumorigenicity in NOD-SCID confirmed no tumorigenicity potential of hTERTs-hMSCs,hTERTs-hMSCs have the multipotentials of differentiation into adipocytes,chondrocytes and osteocytes after exposure to adipogenic,chondrogenic and osteogenic differentiation media respectively.The present study shows that hTERT has the capacity of overcoming replicative senescence and prolonging the life-span of hMSCs.There were 11 proteins up-regulated and 9 proteins down-regulated in hTERTs-hMSC in comparison with primary hMSCs,hTERTs-hMSCs have an interesting perspective in tissue engineering,cell engineering and gene engineering.