Fig. 2.

Cardiomyocyte differentiation capacity depends on telomerase activity of the stem cells. A Schematic describing the proposed in vitro model to generate hiPSC-cardiomyocytes with long and short telomeres. Schematic also depicts the CRISPRi TERT hiPSC maintenance and differentiation protocol used in the study. CRISPRi TERT hiPSCs treated with doxycycline possess shorter telomeres and should result in hiPSC-cardiomyocytes with shorter telomere lengths as opposed to the untreated CRISPRi TERT hiPSCs. B Telomere length measured via TEL-qFISH in hiPSC-cardiomyocytes arising from CRISPRi TERT hiPSCs with long (− Doxycycline) and short telomeres (passage 2 and passage 5 + Doxycycline, red bars) (n ≥ 45 nuclei per group). The dotted grey line indicates the nuclei region used for qFISH analysis. C 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 and 5) (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 doxycycline treated hiPSCs are depicted in red and the ones arising from control hiPSCs (− Doxycycline) are depicted in dark grey. Representative plots with mean ± SEM are depicted. D Quantification of hiPSC-cardiomyocyte differentiation efficiency amongst the CRISPRi hiPSCs with and without doxycycline treatment is represented in terms of fold change. All data are mean fold change relative to control ± SEM (n = 3 independent differentiation experiments); hiPSC-CM = human induced pluripotent stem cell derived cardiomyocyte; *p < 0.05; **p < 0.01; ***p < 0.001; One-way ANOVA, Tukey multiple-comparisons test