Sala Luca, Ward-van Oostwaard Dorien, Tertoolen Leon G J, Mummery Christine L, Bellin Milena
Department of Anatomy and Embryology, Leiden University Medical Center.
Department of Anatomy and Embryology, Leiden University Medical Center; Department of Applied Stem Cell Technologies, University of Twente.
J Vis Exp. 2017 May 12(123):55587. doi: 10.3791/55587.
Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.
现在可以从人类胚胎干细胞和人类诱导多能干细胞（hPSC）高效地分化出心肌细胞。hPSC衍生的心肌细胞（hPSC-CM）在模拟人类心血管疾病，尤其是心律失常综合征方面的价值越来越受到认可。它们还作为预测药物反应的体外系统显示出相关性，这使其在药物筛选与发现、安全药理学以及最终可能在个性化医疗中具有潜在用途。通过从患者或易感个体中获取hPSC作为hiPSC来分化出hPSC-CM，这将有助于实现上述应用。然而，对于所有应用而言，精确测量和分析hPSC-CM的电特性对于识别由于心脏离子通道突变和/或靶向离子通道并可能导致心源性猝死的药物所引起的变化至关重要。与手动膜片钳相比，多电极阵列（MEA）设备具有允许进行中到高通量记录的优势。本方案描述了如何将hPSC-CM的二维细胞培养物解离为小聚集体和单细胞，并将它们接种到MEA上以记录其作为场电位的自发电活动。本文还介绍了分析记录数据以提取特定参数（如QT和RR间期）的方法。在携带导致心律失常的突变的hPSC-CM中以及添加特定药物后，预计这些参数会发生变化，从而能够检测出那些具有心脏毒性风险的情况。
