Figure 1.

Generation of mTERT-MSC cell line. (a) Single cell cloning (limiting dilution) of mTERT-MSC cell line. Following cell transfection and G418 selection, single cell cloning was adopted to obtain a purified Sca-1^pos mTERT-MSC. Different time points along the culture are shown (bar: 50 μm). (b) Western blot analysis of mTERT protein expression tested on extracts from mTERT-MSC and control cells at different culture time points (p1, p10), and nuclear localization of mTERT protein; (c) Telomerase activity (TRAP Assay) of Sca-1^pos MSC transduced with mTERT, compared to controls, evaluated at different culture time points. Telomerase activity was tested on extracts corresponding to viable 5 × 10³ cells. The pattern of TRAP reaction represents a ladder of bands with a periodicity corresponding to multiples of TTAGGG telomeric repeats. Band intensity was quantified by bi-dimensional densitometry; (d) comparison between MSC and mTERT-MSC proliferation, expressed as population doublings (PD), determined as described in Section 2.