Differentiation, Maintenance, and Contraction Profiling of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

The development of patient-derived cardiac disease models has advanced rapidly due to the progress of human-induced pluripotent stem cell (hiPSC) technologies. Many protocols detail individual parts of the entire workflow, from handling hiPSCs and differentiating them into cardiomyocytes to live contraction imaging via widefield/phase-contrast and fluorescence microscopy. Here, we propose a streamlined protocol that guides users through hiPSC culture, differentiation, expansion, and functional imaging of hiPSC cardiomyocytes. First, hiPSC maintenance and handling procedures are outlined. Differentiation occurs over a two-week period, followed by selective expansion to increase the yield of hiPSC cardiomyocytes. Comprehensive characterization and quantification enable detailed contraction profiling of these cells. Designed to be low-cost, this protocol is suited for applications in drug discovery, screening, and clinical testing of patient-specific phenotypes. The addition of cardiomyocyte expansion and automated analysis distinguishes our protocol from current approaches. Key features • Protocol for the maintenance, differentiation, and expansion of hiPSC cardiomyocytes. • Detailed guidance for characterization and functional imaging of hiPSC cardiomyocytes. • Streamlined workflow integrating state-of-the-art protocols streamlined into a cost-effective approach. • A complete timeline from hiPSC culture to contraction imaging achievable in as little as three weeks.