Serum-assisted metabolic priming enables robust structural maturation of hiPSC-cardiomyocytes in 2D and 3D systems

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are crucial for modeling cardiovascular disease and drug development, yet their immature phenotype remains a significant challenge. Cardiomyocyte maturation is an energy-demanding process involving a metabolic shift from glycolysis to fatty acid oxidation. Fetal Bovine Serum (FBS), a common cell culture supplement, contains a complex array of growth factors, hormones, and nutrients, including various fatty acids. While FBS supports initial differentiation and survival, its role in comprehensive hiPSC-CM maturation remains underexplored. We hypothesized that the diverse fatty acid profile within FBS, acting individually or synergistically, could accelerate the structural, functional, and metabolic maturation of hiPSC-CMs. In this study, hiPSC-CMs were cultured for 9 days in three media supplemented with FBS. Results demonstrated significant morphological changes, including a shift to a rod-like shape, increased cell size, enhanced cell fusion, and decreased circularity. The expression of mature genes was elevated, and the distribution of mitochondria was rearranged. FBS-treated hiPSC-CMs exhibited highly organized sarcomere structures, intercalated disk-like formations, and increased sarcomere length. Functional improvements were evident in increased beating intensity and beating units. Notably, these maturation effects were observed in both 2D monolayers and 3D spheroid cultures, with improved cell viability in 3D spheroids. These findings suggest FBS, particularly its fatty acid components, promotes hiPSC-CM maturation, warranting further mechanistic investigation into the precise roles of specific FBS-derived fatty acids.