Fig. 6.

HiPSC-cardiomyocytes derived from hiPSCs with restored telomerase activity and telomere length exhibit improved function. A Schematic describing the reversal of the doxycycline treatment at the hiPSC stage to restore telomerase activity and telomeres and further differentiating them into hiPSC-cardiomyocytes; B HiPSC-cardiomyocyte differentiation efficiency measured by cTnT expression via flow cytometry; at day 12 of the cardiomyocyte differentiation protocol; from CRISPRi hiPSCs (with and without doxycycline treatment until passage 2) plus the hiPSCs after removal of doxycycline treatment for one passage to reverse the telomeres (n ≥ 5000 events per sample). Isotype controls (grey dotted line, filled) and unstained cells (light grey dotted line, filled) were used as controls. CRISPRi TERT hiPSC-cardiomyocytes arising from reverse doxycycline hiPSC group are depicted in green whereas doxycycline treated hiPSCs are depicted in red and the ones arising from control hiPSCs (− Doxycycline) are depicted in dark grey. C Quantification of hiPSC-cardiomyocyte differentiation efficiency amongst the CRISPRi hiPSCs with, without plus reversal of doxycycline treatment is represented in terms of fold change. All data are mean fold change relative to control ± SEM (n = 3 independent differentiation experiments); D Assessment of contractility (beats per minute, bpm) on day 7 of the RTCA analysis between hiPSC-cardiomyocytes with long (black), short (red) and reversed (green) telomeres (n = 12–16 wells from one CardioECR Xcelligence experiment). One-way ANOVA, Kruskal–Wallis test with Dunn’s multiple comparison test. E The strong apoptotic effects of Doxorubicin (1 µM) treatment as measured by caspase activity in CRISPRi TERT hiPSC-cardiomyocytes with shorter telomeres (+ Doxycycline) is diminished in the CRISPRi TERT hiPSC-cardiomyocytes with reversed telomere lengths (n = 3 samples per group, from one differentiation round). Level of caspase activity represented as fold change to the basal level of CRISPRi TERT hiPSC-cardiomyocytes with respective long (black bar), short (red bar) or reversed (green bar) telomeres. All data are mean fold change relative to control ± SEM; hiPSC-CM = human induced pluripotent stem cell derived cardiomyocyte; *p < 0.05; **p < 0.01; ***p < 0.001; unpaired 2-tailed t-test was performed to calculate significance between 2 groups, One-way ANOVA, Tukey multiple-comparisons test