Live-Cell Monitoring and Transcriptomic Profiling Identify Distinct Temporal Patterns of Cell-Cycle Activity and Maturation in Human iPSC-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show decreasing cell-cycle activity during prolonged culture. However, it remains unclear whether changes in cell-cycle activity occur before, after, or concurrently with transcriptional changes in cell-cycle and maturation-associated programs. Here, we combined a TNNT2-FUCCI reporter, Ki67 immunostaining, and bulk RNA sequencing to define how cell-cycle activity changes during prolonged human iPSC-cardiomyocyte culture. FUCCI and Ki67 analyses showed similar levels of cell-cycle activity at days 20 and 30 of cardiomyocyte differentiation but declined more clearly by day 40. In contrast, transcriptomic profiling revealed that canonical cell-cycle and DNA-replication programs were already reduced by day 30. The subsequent day-30-to-day-40 interval was characterized by increased expression of sarcomeric, contractile, and calcium-handling gene programs rather than a further broad loss of mitotic pathways. These findings support a staged model of human iPSC-cardiomyocyte maturation-associated transcriptional remodeling in which a reduction in cell-cycle-associated transcription is detectable before the subsequent decline in cardiomyocyte cell-cycle activity, thereby identifying distinct temporal patterns of cell-cycle and maturation-associated changes during prolonged hiPSC-CM culture.