Multi-omic analysis reveals maturation programs in human pluripotent stem cell-derived cardiomyocytes during long-term culture

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold tremendous promise for disease modeling, drug discovery, and cardiac regenerative therapies. However, the immature phenotype of hPSC-CMs remains a major barrier limiting their translational utility. Here, we performed integrated multi-omic profiling to define the molecular mechanisms underlying hPSC-CM maturation during extended culture. hPSC-CMs were cultured for 113 days and analyzed using metabolomics, proteomics, and transcriptomics across progressive stages of maturation. Extended culture induced widespread multi-omic remodeling, including significant changes in 142 metabolites, 550 proteins, and 2,892 transcripts. Metabolomic analyses revealed early increases in phospholipid biosynthesis and mitochondrial fatty acid β-oxidation from Day 30 to Day 60, suggesting metabolic priming precedes later maturation events. In contrast, proteomic remodeling occurred predominantly during later stages of maturation and was characterized by enhanced calcium handling and cell cycle exit. Transcriptomic analyses demonstrated progressive increases in ion channel expression, t-tubule organization, fatty acid metabolism, creatine shuttle pathways, and cell cycle arrest programs. Integrative pathway analyses identified coordinated suppression of TGFβ, MAPK, Wnt, and Hedgehog signaling together with activation of integrin-related, respiratory electron transport, muscle contraction, and Slit-Robo signaling pathways during maturation. Moreover, integrated transcription factor network analysis prioritized a GATA4-centered regulatory program involving SOX7, SOX18, TBX2, and ZFPM2 (FOG2) as candidate drivers of cardiomyocyte maturation. Together, these findings reveal coordinated temporal regulatory programs underlying human cardiomyocyte maturation and establish an integrated multi-omic framework for identifying mechanisms to accelerate hPSC-CM maturation.