Abstract：
Cardiovascular diseases have emerged as one of the leading causes of human mortality, but the discovery of new drugs has been hindered by the absence of suitable in vitro platforms. In recent decades, continuously refined protocols for differentiating human induced pluripotent stem cells (hiPSCs) into hiPSC-derived cardiomyocytes (hiPSC-CMs) have significantly advanced disease modeling and drug screening; however, this has led to an increasing need to monitor the function of hiPSC-CMs. The precise regulation of action potentials (APs) and intracellular calcium (Ca2+) transients is critical for proper excitation-contraction coupling and cardiomyocyte function. These important parameters are usually adversely affected in cardiovascular diseases or under cardiotoxic conditions and can be measured using optical imaging-based techniques. However, this procedure is complex and technologically challenging. We have adapted the IonOptix system to simultaneously measure APs and Ca2+ transients in hiPSC-CMs loaded with the fluorescent dyes FluoVolt and Rhod 2, respectively. This system serves as a powerful high-throughput platform to facilitate the discovery of new compounds to treat cardiovascular diseases with the cellular phenotypes of abnormal APs and Ca2+ handling. Here, we present a comprehensive protocol for hiPSC-CM preparation, device setup, optical imaging, and data analysis. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Maintenance and seeding of hiPSC-CMs Basic Protocol 2: Simultaneous detection of action potentials and Ca2+ transients in hiPSC-CMs.
摘要：
心血管疾病已成为人类死亡的主要原因之一，但是由于缺乏合适的体外平台，新药的发现受到了阻碍。近几十年来,将人诱导多能干细胞(hiPSC)分化为hiPSC衍生的心肌细胞(hiPSC-CMs)的不断细化的方案已经显著提高了疾病建模和药物筛选的水平;然而,这导致越来越需要监测hiPSC-CM的功能。动作电位（AP）和细胞内钙（Ca2）瞬变的精确调节对于适当的兴奋-收缩耦合和心肌细胞功能至关重要。这些重要参数通常在心血管疾病中或在心脏毒性条件下受到不利影响，并且可以使用基于光学成像的技术进行测量。然而,此程序复杂且技术上具有挑战性。我们已经调整了IonOptix系统，以同时测量分别装有荧光染料FluxVolt和Rhod2的hiPSC-CM中的AP和Ca2瞬变。该系统可作为强大的高通量平台，以促进发现具有异常AP和Ca2处理的细胞表型的治疗心血管疾病的新化合物。这里,我们提出了一个全面的HiPSC-CM制备方案，设备设置,光学成像,和数据分析。©2024Wiley期刊有限责任公司。基本方案1:hiPSC-CM的维持和播种基本方案2:同时检测hiPSC-CM中的动作电位和Ca2+瞬变。
