Figure 1.
Extending the replicative lifespan of hES-derived epithelial cells by evading the p16INK4A senescence mechanism. (A): p16 immunocytochemical staining of hESderK cells in their third and final passage before senescence, growing in Gibco K-sfm medium (phase contrast left; bright-field, right). Note that small proliferative, colony-forming cells are p16-negative and large, nondividing cells are p16-positive. The large, very flat cells, p16-negative in these fields are irradiated 3T3 fibroblast feeder cells that had been carried over from the previous passage. Bar in top left panel is 200 μm long. (B): Western blot analysis of bmi1 and p16 expression. Lanes: (1) early passage strain N epidermal keratinocyte; (2) late passage N/bmi1; (3) hESderK/bmi1; (4) hESderK/E6E7 clone K; (5) late passage HBl-10U; (6) late passage HBL-10U/bmi1. Note that constitutive bmi1 expression is associated with reduced p16 expression in normal keratinocytes, hESderK cells, and normal urothelial cells and that hESderK/E6E7 cells express high levels of p16 but are still able to proliferate, consistent with Rb-inactivation by E7. (C–E): Replicative lifespans of primary cell lines and engineered derivatives; arrows indicate timing of transductions during serial passage of this line; asterisks indicate the normal replicative lifespan limit (senescence) of the primary cell line. (C): Lifespan extension and immortalization of hESderK cells. Note that hESderK cells senesced by 10 population doublings (PD) and were not immortalized by TERT. Expression of bmi1 in the parent line conferred a long extension of lifespan to ∼60 PD. Transduction of the parent line to express E6 and E7 resulted in immortalization and also a substantial increase in growth rate to the ∼1 PD/day rate exhibited by early passage normal keratinocytes and their engineered derivatives. (D): Lifespan extension and immortalization of normal primary corneal keratinocyte line KL5. Note that KL5 had a very short lifespan of ∼23 PD, which was not altered by TERT expression. Transduction of KL5 to express bmi1 resulted in extension of lifespan to ∼37 PD and subsequent transduction of KL5/bmi1 cells to express TERT resulted in immortalization. (E): Lifespan extension and immortalization of normal primary human epidermal keratinocyte strain N. Note that transduction of strain N to express bmi1 resulted in extension of replicative lifespan from 70 to 90 PD, and subsequent transduction of N/bmi1 to express TERT resulted in immortalization. Transduction of strain N to express HPV16E6 and E7 also resulted in immortalization. Abbreviations: TERT, telomerase catalytic subunit.