Functional Maturation of Human iPSC-Derived Cardiomyocytes by Prolonged 3D Culture in Engineered Cardiac Tissue Constructs

The advent of human induced pluripotent stem cell (hiPSC) technology has revolutionized the way we study inherited diseases, as it allows us to study the effect of mutations in a human context. This is particularly true in the heart were species-specific differences has dramatic effects on heart rate, and expression of protein isoforms involved in excitation-contraction coupling. A major drawback of using hiPSCs is, however that cardiomyocytes derived from hiPSC (hiPSC-CM) are relatively immature, limiting their utility as a tool to study adult onset cardiac diseases. We postulated that culturing hiPSC-CM in a 3D environment in engineered cardiac tissue constructs (ECT) would promote hiPSC-CM maturation. ECTs were generated from lactate-selected day 30 hiPSCs that were mixed with isogenic hiPSC derived cardiac fibroblasts in a 10:1 ratio. Cells were mixed with fibrinogen and thrombin and seeded into molds under vacuum in FlexCell dishes to form ECT. ECTs were harvested 14-21 or 42-52 days later and subjected to functional testing, transcriptional analysis and top-down mass-spectrometry of sarcomeric proteins to assess maturation. Functionally, we found that prolonged culture of hiPSCs in ECT resulted in increased calcium transient amplitude (0.856 vs. 0.462; P < 0.01) and an acceleration of calcium kinetics (calcium release time 67.7 ms vs. 109.3 ms; P < 0.01 and calcium decay time 148.3 ms vs. 188.0 ms; P < 0.05). This acceleration of calcium kinetics with time in culture was also true for twitch force kinetics (time to peak twitch force 178.6 ms vs. 208.0 ms; P < 0.05). Furthermore, beta adrenergic stimulation had a much greater effect on twitch kinetics in older ECTs (reduction in contraction time of 26.5% vs. 7.1%; P < 0.01). Transcriptional analysis revealed an increase in expression level of the beta 1 adrenergic receptor in older ECTs, while top-down mass spectrometry showed increased expression and phosphorylation of cardiac troponin I (cTnI) and mono-phosphorylated cTnI, as well as decreased phosphorylation of alpha-tropomyocin, all markers of myocardial maturation. Taken together, these data supports our hypothesis that prolonged culture of hiPSC-CM in ECT promotes maturation of the calcium handling system and the contractile apparatus.