Se on the stabilization of p53 by telomeric repeats (Milyavsky et al., 2001). Nevertheless, activation of p53 was not increased in WS-MSCtert despite the greater basal level (Figure S4I). Yet another senescence marker p16, as anticipated, was decreased in WS-MSCtert. When WS MSCs were exposed to H2O2, 53BP1 was activated at low oxidative stress (50 mM), whereas gH2AX was induced at high oxidative anxiety (250 mM) accompanied by activation of ATM (p-ATM) (Figure S4E). The expression of hTERT in WS MSCs appears to rescue senescence by way of reduction from the p16 level (but not of p53/p21) along with the DNA damage marker gH2AX. These data support the critical part of telomerase in cell proliferation plus the cell’s replicativepotential, at the same time as in stopping DNA harm and premature senescence in WRN-deficient cells. We recommend that, with no protection on the telomere by telomerase, WS cells rapidly enter senescence by way of the p53 pathway. To verify this postulation, we derived steady p53 knockdown cells by RNAi (p53i) in WS fibroblasts. When these p53i WS cells have been reprogrammed to iPSCs, they showed tiny distinction from unmodified iPSCs; nevertheless genomic instability was present (Table S2). Genomic instability on account of p53 depletion in iPSCs has been previously reported (Kawamura et al., 2009; Marion et al., 2009a). Upon differentiation to MSCs (WS-MSCp53i), p53 protein remains low, evidence of persistent expression of p53 shRNA (Figure S4F). As a consequence in MSCs, p53i enhanced their proliferative potential and rescued the premature senescence phenotype devoid of the will need for high telomerase activity and extended telomere length (Figures 4BD). As anticipated, WS-MSCp53i expressed less p21 and phosphorylated p53 (Figure S4G). Next, we examined the telomere status in these genetically modified cells. Longer telomere length was identified in WS-MSCtert, but not in WS-MSCp53i, suggesting a rescue of your accelerated telomere attrition by telomerase (Figure 4E). CO-FISH evaluation revealed a reduction of defective synthesis for the lagging strand telomeres in WS-MSCtert, but not in WS-MSCp53i (Figures 4F and 4G). Collectively, these data help the crucial function of telomerase in stopping premature senescence in MSCs by restoring telomere function. p53 seems to become a downstream effector due to the fact a equivalent effect was accomplished as a consequence of depleting p53 and bypassing the senescence pathway.Stem Cell Reports j Vol. two j 53446 j April eight, 2014 j 014 The AuthorsStem Cell ReportsTelomerase AOH1160 Protocol protects against Lineage-Specific AgingFigure 3. Recurrence of Premature Senescence and Telomere Dysfunction in WS MSCs (A) Reduced cell proliferation and replication possible in WS MSCs with continuous culture for 76 days. (B) Quantitative evaluation for percentage of senescent cells in MSCs right after 44 days of culture (p11). A substantial difference is discovered amongst normal and WS MSCs (p 0.05).Values represent mean of technical replicates SD (n = 3). (C) Representative pictures for standard and WS MSCs by SA-b-galactosidase staining. (legend continued on subsequent page)538 Stem Cell Reports j Vol. 2 j 53446 j April eight, 2014 j 014 The AuthorsStem Cell ReportsTelomerase Protects against Lineage-Specific AgingTelomerase Activity in NPCs and Its Function in Guarding DNA Harm Since telomerase has a critical function in protection of telomere erosion in MSCs, we speculate that the neural lineage telomerase is differentially regulated and protects neural lineage cells from accelerated senescence. To test.