Human Schwann cell senescence is not prevented by ectopic expression of human telomerase reverse transcriptase

BLANCHE KUO; PRIYAMVADA RAI; GONZALO PIÑERO; PAULA MONJE; NATALIA ANDERSEN; KRISTINE RAVELO

San Diego

Congreso; Society for Neuroscience 46th Annual Meeting; 2016

Society for Neuroscience

Isolated human Schwann cells (hSCs) typically become senescent and unresponsive to mitogenic factors with continued in vitro expansion. Data from our lab has shown that adult nerve-derived hSCs, in contrast to rodent (rat) SCs, inevitably stop proliferating and acquire a senescent phenotype characterized by high levels of senescence-associated (SA)-β galactosidase activity and morphological changes that include cell enlargement and appearance of multi-nucleated cells. As opposed to rodent SCs, hSC cultures consist of mixed populations of proliferating cells, senescent cells and cells at different stages of differentiation regardless of the nerve of origin and other donor-specific factors. RNA-seq analysis of representative cultures of hSCs did not reveal the presence of telomerase reverse transcriptase (TERT) mRNA while other TERT-related genes (e.g. TERF1, telomeric repeat binding factor, and TEP1, telomerase-associated protein) were well-represented in the hSC transcriptome. In an attempt to overcome senescence, we used retroviral vectors and antibiotic selection to generate hSC lines ectopically expressing human (h)-TERT. For these experiments, highly proliferative, non-senescent, early passage hSC cultures were stably transduced with the retroviruses h-TERT-hygro or h-TERT-puro, each encoding the h-TERT gene along with hygromycin or puromycin resistance genes, respectively. Transduced hSC cultures from three different donors were selected and subjected to three rounds of expansion in medium containing chemical mitogens. Subsequently, the cultures were analyzed for their rate of proliferation by means of EdU incorporation assays and the acquisition of senescence by means of SA-β galactosidase activity assays in each round. We found that whereas ectopic h-TERT expression extended the lifespan of cultured hSCs when compared to non-infected or GFP-expressing cells, it was not sufficient to confer immortalization and overcome senescence. In sum, our results suggest that progression of the hSCs to a senescent state likely is stress-induced rather than dependent on replication-associated telomere shortening.