Figure 5. In vitro characterization of MEDUSA hESC-CMs.

(A) qRT-PCR of gene-edited ion channels in MEDUSA hESC-CMs compared to WT control. Data shown as mean ± SEM of 3 independent experiments. Statistical differences are reported vs. WT hESC-CMs by unpaired t-test (* p <0.05, ** p < 0.01 and *** p <0.001). Insert showing western blotting analysis for NCX1 in MEDUSA hESC-CMs, see also Supplementary Figs. 5E, F. (B) Onset of beating and beating rate during cardiac differentiation of MEDUSA hESC-CMs. Data shown as mean ± SEM of 2 independent batches of monolayer differentiation (12-wells/cell line per batch of differentiation). See Methods section for details on quantification. (C) Representative MEA analysis of MEDUSA hESC-CMs cultured for 2 weeks on MEA plates. Data shown as average ± SEM of total beats recorded for 5 min every hour. Spontaneous activity at day 35 is shown as continuous recording for 20 hours, panel below. Differences vs. WT hESC-CMs by two-way ANOVA with Sidak correction (*** p < 0.001). (D) Distribution of quiescent and spontaneously depolarizing cells in patch clamp preparation. (E) Quantification of patch clamp metrics under pacing condition (RMP: resting membrane potential; MDP: maximum diastolic potential). Data shown as violin plots of 16 WT and 11 MEDUSA hESC-CMs. Differences vs. WT hESC-CMs by unpaired t-test with Welch’s correction (**p<0.01, ***p<0.001). (F) AP traces of WT and MEDUSA hESC-CMs after electrical stimulation (RMP, MDP as in 5E; APD₉₀: action potential at 90% repolarization). (G) Representative AP traces of MEDUSA hESC-CMs stimulated at 0.5 – 3 Hz, during patch clamp experiments. (H) Calcium transient analysis of WT and MEDUSA hESC-CMs during pacing at 1 Hz. Data shown as mean ± SEM of 12 individual cells/cell line. Statistical differences are shown by multiple unpaired t-test (***p<0.001). Representative traces shown in Supplementary Fig. 5H.