[Extension of life-span of normal human fibroblasts by reconstitution of telomerase activity].

Abstract:

Most of normal human somatic cells can divide only a finite number of times and inevitably become senescent. Telomerase is an enzyme that imparts replicative immortality by maintaining the length of the telomeres when expressed in reproductive and cancer cells. Cells that are mortal do not express the telomerase. Recently it was reported that the life-span of the normal human cells could be successfully extended by introduction of telomerase into these cells. We have found, in the previous work, that fibroblasts exhibited an osteogentic potential, and therefore, can be considered as a type of "seed cells" in tissue engineering for bone repairing and reconstruction. But this potential was impaired by the limitation in life-span and proliferative capacity of the normal fibroblasts. In the present work, plasmid pGRN145 bearing a cDNA insert of human telomerase reverse transcriptase (hTERT) was introduced into the fibroblasts with osteogenic potential by electroporation. The stable hTERT+ fibroblast clones was established and cultured for long-term in a medium containing hygromycin-B. The exogenous hTERT mRNA expression and telomerase activity were detected. The hTERT+ fibroblasts showed shorter population doubling time and no beta-galactosidase stain, which indicated a stronger proliferative capacity and fewer signs of cell senescence, compared to their hTERT- counterpart. These evidenced that the life-span of hTERT+ fibroblasts was extended. The assays for DNA euploidy by flow cytometry and chromosome karyotype by cytogenetic technique showed no signs of heteroploidy, providing the data for cell carcinogenesis and utilization safety. The results of the present study suggested that the introduction of hTERT could make the life-span of normal fibroblast extended without causing their malignant transformation, and such type of "longevous" fibroblasts might be clinically useful in tissue engineering for bone repairing and reconstruction.

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