Abstract Body (Do not enter title and authors here): Introduction: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold great promise for cell therapy and drug discovery. However, their inherent immaturity, characterized by spontaneous beating, limits their utility. Interestingly, previous data showed that proliferating hPSC-CMs exhibited a slower beat rate, but their genetic and maturation status was not reported. Here, we show that prolonged proliferation of hPSC-derived ventricular cardiomyocytes (hPSC-VCMs) reduces beat rate and pacemaker gene expression along with the increased expression of genes associated with cardiomyocyte maturation.
Hypothesis: Prolonged proliferation of early-stage hPSC-VCMs leads to a reduction in beat rate and pacemaker gene expression and improves maturation.
Methods: Four hPSC lines were differentiated to early-stage hPSC-VCMs. On day 11 of differentiation, early-stage hPSC-VCMs were harvested, replated, and proliferated weekly for 5 weeks alongside time-matched, non-proliferated control hPSC-VCMs. Protein expression, gene expression, and electrophysiological activity were evaluated using flow cytometry, RNA sequencing, and microelectrode array analysis, respectively. Data are shown as mean ± SEM.
Results: After 5 weeks, the cardiomyocyte marker cTnT increased from 88 ± 4% (day 11, n = 15) to 94 ± 3% (n = 15) in proliferated hPCS-VCMs, which was higher than the non-proliferated control (60 ± 7%, n = 15), and resulted in an ~80-fold cell expansion. Proliferated hPSC-VCMs exhibited a stepwise decrease in beat rate over five passages (P1 = 28 ± 1 BPM, P2 = 16 ± 1 BPM, P3 = 11 ± 1 BPM, P4 = 8 ± 3 BPM, P5 = 10 ± 1 BPM; n = 4), a trend not observed in control hPSC-VCMs (P1 = 23 ± 2 BPM, P2 = 29 ± 3 BPM, P3 = 33 ± 6 BPM, P4 = 20 ± 4 BPM, P5 = 37 ± 1 BPM; n = 4). Passages 2, 3, and 5 had significantly lower beat rates compared to their time-matched controls (p < 0.05). RNA sequencing revealed a corresponding stepwise downregulation of pacemaker genes (HCN1, HCN4, CACNA1H) and upregulation of maturation-related genes (MYL2, KCNH2, CACNA1C, SCN5A, KCNJ2, JPH2) in proliferated cells. Notably, expression of cardiac ion channel genes KCNH2, KCNQ1, and CACNA1C in proliferated hPSC-VCMs were comparable to those found in the adult human ventricle.
Conclusion: Prolonged proliferation of hPSC-VCMs effectively reduces intrinsic pacemaking activity and promotes a mature phenotype which will improve their utility in basic and translational cardiovascular research.