Functional analysis of human induced pluripotent stem cell-derived cardiac tissue during endothelial cell network changes.

Introduction: Tissue structure stability is essential for effective contractile function in cardiac tissue. Although endothelial cells (ECs) serve as critical sources for functional bioengineered cardiac tissue, the process of network changes in ECs affects the cardiac tissue structure. However, how the process precisely influences the synchrony of cardiomyocytes (CMs) contraction and the contraction regularity in the cardiac tissue remains unclear. Methods: Human induced pluripotent stem cell-derived CMs (hiPSC-CMs) were cultured in well-plates for 9-10 days until they displayed stable spontaneous beating. Human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs), or NHDFs alone, were then seeded and cultured with hiPSC-CMs for 3 days (NHDF + HUVEC + CM, NHDF + CM). Changes in the EC network and cardiac functionality were analyzed over the 3-day period following seeding. Results: The EC network expanded until day 2, after which it stabilized. The EC network was observed at the layer of hiPSC-CMs on day 3 in NHDF + HUVEC + CM cultures. Motion capture analysis revealed that hiPSC-CMs in NHDF + HUVEC + CM exhibited more synchronous contractions on day 2 than those in NHDF + CM, with no notable differences on days 1 and 3. The irregularity of spontaneous beat rates, measured using cardiac calcium imaging, was significantly higher in NHDF + HUVEC + CM than in NHDF + CM on day 1. However, the irregularity of spontaneous beating in NHDF + HUVEC + CM was more stabilized on day 3 compared with day1. Other parameters, including contraction amplitude, spontaneous beat rate, and calcium uptake and release, did not significantly differ between NHDF + HUVEC + CM and NHDF + CM over time. Conclusions: Alterations in the EC network may induce irregular spontaneous contractions in hiPSC-CMs at the early phase of co-culture followed by more regular contractions over time, with improving synchrony in the contractions within the cardiac tissue transiently.