We next asked what the impact of culture induced
We next asked what the impact of culture-induced senescence of the fibroblasts was on the reprogramming efficiency. Four representative fibroblast lines from young (ages and 1year) and old (ages 81 and 83year) donors were propagated for up to 25 passages (Fig. 2A). The pluripotent reprogramming efficiency of early and late passage fibroblasts was studied by counting AP-positive iPSC colonies with hESC-like morphology 21days after the induction by non-integrating Sendai viral vectors. Regardless of donor age, early passage fibroblasts yielded significantly more iPSC colonies than late passage sodium calcium exchanger (Fig. 2B–D and Supplementary Fig. 2A). Similar to retroviral inductions, we observed the difference in reprogramming efficiencies of young (age 0, 1) and old fibroblasts (age 81, 83) when using Sendai viruses (Fig. 2C and D and Supplementary Fig. 2A). Although the older fibroblasts gave only few iPSC colonies, the iPSC lines (HEL24.3 and HEL47.2) generated from neonatal and 83-year old donors, respectively (Trokovic et al., in press-a,b), exhibited normal pluripotent stem cell properties, including the expression of the stem cell markers NANOG, TRA-1-60, OCT4, and SSEA3, as shown by immunocytochemistry (Supplementary Fig. 1A). All iPSC lines had activated the transcription of the endogenous pluripotency genes OCT4, SOX2, NANOG, and TDGF1 (Supplementary Fig. 1B). Both iPSC lines were negative for Sendai virus vectors as shown by immunocytochemistry and qPCR (Supplementary Fig. 1A and B). Multi-lineage differentiation capacity into all three-germ line lineages was demonstrated by teratoma formation (Supplementary Fig. 1C). Karyotype of iPSC line HEL24.3 (p14) was 46,XY and HEL47.2 (p14) 46,X, abn (Y). The karyotype of the respective donor fibroblasts of HEL47.2 was also found to be 46,X, abn (Y). Cellular aging in vitro was determined by cell proliferation and telomere length. Telomere length, which has been associated with human aging (Marion et al., 2009), was significantly shortened by prolonged culture of all fibroblasts (Fig. 2E). In accordance with telomere length, proliferation rate of the fibroblasts, examined by live-cell imaging, was reduced in all long-term cultured samples (Fig. 2F and Supplementary Fig. 2B). With increasing time in culture, the population doubling times of fibroblasts from and 1-year old donors increased from 18 to 20 and 18 to 33h, respectively. Similarly, fibroblasts from the 83-year old donor showed a marked elongation of doubling time, from 21 to 33h. We then assessed the proliferation rate of all donor fibroblast lines, at passages 6–8, and observed a significant positive correlation between increasing age and doubling time (Fig. 2G). To uncover the possible mechanisms underlying the differences in reprogramming efficiency, expression of selected genes controlling cell cycle and apoptosis were investigated. The genes were selected based on previous reports of their role in iPSC reprogramming (Hong et al., 2009; Li et al., 2013). In mammalian cells, permanent growth arrest is achieved through one of the two signaling pathways p53/p21 and p16/Rb (Choudhury et al., 2007; Wright and Shay, 1992). Notably, p21 was dramatically upregulated in long term-cultured fibroblasts, while there was no change in p16, p53 or PUMA expression (Fig. 3A). Besides the upregulation of p21, long term-cultured fibroblasts displayed significant shortening of telomeres (Fig. 2E). This observation was in line with the role of p21 in mediating senescence in response to telomere dysfunction (Brown et al., 1997). As p21 inhibition has been demonstrated to enhance reprogramming efficiency predominantly by the cell division rate-dependent mechanisms (Hanna et al., 2009), it is likely that the reduction of cell proliferation and reprogramming efficiency coinciding with the increase in p21 expression are mechanistically related in reducing reprogramming efficiency. Our data show that both donor age and passage number are strongly correlated with increasing p21 expression (Fig. 3B and C), indicating cellular senescence-induced upregulation of p21.