br Experimental Procedures br Acknowledgments br Introductio

Experimental Procedures
Acknowledgments
Introduction Patients with Werner syndrome (WS) exhibit premature aging and early onset of a cancer during adulthood (third to fourth decade) (Chen and Oshima, 2002; Muftuoglu et al., 2008). Autosomal recessive mutations of the RecQ helicase WRN are commonly found in the majority of WS patients, although non-WRN mutations have been documented (Chen et al., 2003). WRN is a critical protein for DNA replication, repair, recombination, and telomere maintenance (Crabbe et al., 2004; Opresko et al., 2004). An important molecular event seen in WS pathology is dysfunction of telomeres. It results in accelerated telomere attrition and failure to fully synthesize the lagging strand sister telomeres (Brosh et al., 2001; Crabbe et al., 2004). Critically short telomeres have been known to elicit a DNA damage response and trigger cellular senescence (Abdallah et al., 2009; d’Adda di Fagagna et al., 2003; Takai et al., 2003). Another observed phenotype in WS Exendin-4 manufacturer is genome instability, perhaps due to deficient DNA repair and uncapping of chromosome ends. WRN is known to interact with a number of proteins involved in DNA repair, recombination, and telomere protection (Crabbe et al., 2007; Laud et al., 2005; Li and Comai, 2001). These biological functions and molecular mechanisms of WRN serve as the basis for our understanding of the clinical pathology of accelerated aging (Rossi et al., 2010). A fascinating clinical feature of WS is the predominant aging that fibroblasts and mesenchymal tissues exhibit (Chen and Oshima, 2002; Goto et al., 2013). Although the spectrum of the aging disorder in many aspects resembles natural aging (such as graying hair, cataract, osteoporosis, and atherosclerosis), neurodegeneration, such as that which occurs in Alzheimer and Parkinson diseases, is seemingly not associated with WS (Goto et al., 2013; Mori et al., 2003). These observations suggest WS is not simply an accelerated aging phenomenon, such as that observed in normally aged people; however, the reason for this selectively accelerated aging remains unclear.
Results
Discussion Our data demonstrate premature senescence caused by WRN loss can be reversed by nuclear reprogramming, possibly as a consequence of reactivation of telomerase machinery that corrects the telomere defect. Reprogramming of normally aged fibroblasts or diseases of laminopathies and Hutchinson-Gilford Progeria syndrome have been reported (Lapasset et al., 2011; Liu et al., 2011; Zhang et al., 2011). Our observations highlight telomere function in WS cells, because abnormal telomere homeostasis is a critical molecular event in WS pathology (Chang et al., 2004; Crabbe et al., 2004; Ishikawa et al., 2011; Laud et al., 2005; Multani and Chang, 2007). Telomere length in pluripotent WS cells appears to be normal. With differentiation premature senescence recurs, and aberrant telomere synthesis is found in derived MSCs, but not in NPCs, indicative of a lineage-specific aging phenomenon. This observation is consistent with the clinical phenotype of WS, where mesenchymal tissues are severely affected but mild or no symptoms are associated with neural lineages (Goto et al., 2013). The inability to perform systematic studies of different cell types or tissues during embryonic development and in adulthood validates iPSC technology as a valuable tool to study its pathogenesis. By comparing the different stem/progenitor cells, we identified a dramatic difference in telomerase activity. In line with other studies, telomerase activity is high in embryonic cells, and its activity declines with differentiation (Armstrong et al., 2000; Yang et al., 2008). MSCs and fibroblasts express low telomerase activity, which explains the vulnerability of these cells to replication-induced senescence and telomere dysfunction. The present study supports the critical role for telomerase in preventing specific lineages of cells from accelerated aging, and it may affect stem cell renewal and their capacity for regeneration (Blasco, 2007). Our observation is consistent with the Wrn knockout mouse model, which does not recapitulate the pathogenesis of the disease unless it is expressed on a background of Terc−/− (Chang et al., 2004; Lombard et al., 2000). How telomerase rescues telomere dysfunction is not clear; however, telomerase was reported to extend telomeres and rescue premature aging and reverse tissue degeneration in aged Terc−/− mice with shortened telomeres (Jaskelioff et al., 2011; Samper et al., 2001). An unexpected result from our study is the moderate upregulation of p53 and hence p21 in hTERT-expressing WS cells (Figure S4E). Similar moderate increase of p53 upon hTERT overexpression was also observed in wild-type MSCs (Figure S4H). Shortened telomeres are postulated to activate p53, leading primary cells to senesce (d’Adda di Fagagna et al., 2003). Intriguingly, our data show that hTERT expression in WS MSCs, albeit enhancing their proliferation potential and slowing telomere erosion, increased p53 and p21 levels. Consistently, inhibition of telomerase in WS NPCs decreased p53/p21 (Figure S5F). One possible explanation for this phenomenon is that longer telomere repeats stabilize p53 and thus slow their turnover (Milyavsky et al., 2001). However, the detailed mechanism remains to be elucidated.