Abstract Thu046: Enhancing iPSC-Derived Cardiomyocyte Maturation to Express Disease Phenotypes

Background: A major challenge in iPSC-derived cardiomyocyte (iPSC-CM) research is the prolonged time required for maturation. Current in vitro maturation strategies are complex, time-intensive, and often require co-culture with other cell types, underscoring the need for a rapid and efficient method to enhance iPSC-CM maturity. Methods: We generated iPSC-CMs using the GiWi monolayer differentiation method for 14 days, followed by subculturing with TrypLE 10X and purification in a lactose-based medium for 4 days. Cells were then expanded in CHIR 99021-containing medium before transitioning to a maturation medium for 3 weeks. Functional maturation was assessed at weeks 1, 2, and 3 using the IonOptix MultiCell HTS system to measure contractility. Additionally, α-Actinin staining was performed to evaluate sarcomere length, organization, and cell morphology, while qPCR quantified the expression of maturation markers. Results: Our findings demonstrate that supplementing thyroid hormone, dexamethasone, insulin-like growth factor 1 (TDI), and secreted frizzled-related protein 2 (sFRP2) significantly enhances iPSC-CM maturation. Compared to TDI alone, 3 weeks of TDI + sFRP2 treatment resulted in improved contractility and relaxation stability, increased sarcomere length and organization, a higher proportion of rod-shaped cells, and upregulated maturation marker expression. Furthermore, in MYBPC3 (R502W) mutant iPSC-CMs, TDI + sFRP2 treatment induced a hypercontractile phenotype with impaired relaxation, recapitulating hypertrophic cardiomyopathy. Conclusions: This study presents a novel, efficient, and reproducible method for accelerating iPSC-CM maturation by combining sFRP2 with TDI factors. Achieving functional and structural maturity within three weeks in a monolayer system has significant implications for disease modeling, drug discovery, and regenerative medicine.