Stem Cell & Regenerative Medicine Consortium, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong; Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong; School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong; Dr. Li Dak-Sum Research Centre, The University of Hong Kong, Hong Kong.
Stem Cell & Regenerative Medicine Consortium, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong; Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong; School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
Toxicol Lett. 2018 Sep 15;294:61-72. doi: 10.1016/j.toxlet.2018.05.006. Epub 2018 May 21.
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are emerging tools for applications such as drug discovery and screening for pro-arrhythmogenicity and cardiotoxicity as leading causes for drug attrition. Understanding the electrophysiology (EP) of hPSC-CMs is essential but conventional manual patch-clamping is highly laborious and low-throughput. Here we adapted hPSC-CMs derived from two human embryonic stem cell (hESC) lines, HES2 and H7, for a 16-channel automated planar-recording approach for single-cell EP characterization. Automated current- and voltage-clamping, with an overall success rate of 55.0 ± 11.3%, indicated that 90% of hPSC-CMs displayed ventricular-like action potential (AP) and the ventricular cardiomyocytes (VCMs) derived from the two hESC lines expressed similar levels of I, I, I and I and similarly lacked I and I. These well-characterized hPSC-VCMs could also be readily adapted for automated assays of pro-arrhythmic drug screening. As an example, we showed that flecainide (FLE) induced I blockade, leftward steady-state inactivation shift, slowed recovery from inactivation in our hPSC-VCMs. Since single-cell EP assay is insufficient to predict drug-induced reentrant arrhythmias, hPSC-VCMs were further reassembled into 2D human ventricular cardiac monolayers (hvCMLs) for multi-cellular electrophysiological assessments. Indeed, FLE significantly slowed the conduction velocity while causing AP prolongation. Our RNA-seq data suggested that cell-cell interaction enhanced the maturity of hPSC-VCMs. Taken collectively, a combinatorial approach using single-cell EP and hvCMLs is needed to comprehensively assess drug-induced arrhythmogenicity.
人多能干细胞衍生的心肌细胞（hPSC-CMs）正在成为药物发现和筛查致心律失常性和心脏毒性的新兴工具，这些毒性是导致药物淘汰的主要原因。了解 hPSC-CMs 的电生理学（EP）至关重要，但传统的手动膜片钳技术非常繁琐且通量低。在这里，我们对来自两个人类胚胎干细胞（hESC）系 HES2 和 H7 的 hPSC-CMs 进行了改编，用于 16 通道自动平面记录方法，以进行单细胞 EP 特征分析。自动电流和电压钳位，整体成功率为 55.0±11.3%，表明 90%的 hPSC-CMs 显示出心室样动作电位（AP），并且来自两个 hESC 系的心室心肌细胞（VCMs）表达相似水平的 I、I、I 和 I，并且同样缺乏 I 和 I。这些经过良好表征的 hPSC-VCMs 也可以很容易地适应自动化药物致心律失常性筛选测定。例如，我们表明氟卡尼（FLE）诱导 I 阻断、稳态失活左移、从失活中恢复减慢，在我们的 hPSC-VCMs 中。由于单细胞 EP 测定不足以预测药物诱导的折返性心律失常，因此 hPSC-VCMs 进一步重新组装成 2D 人类心室心脏单层（hvCMLs）进行多细胞电生理评估。事实上，FLE 显著减慢了传导速度，同时导致 AP 延长。我们的 RNA-seq 数据表明细胞间相互作用增强了 hPSC-VCMs 的成熟度。总之，需要使用单细胞 EP 和 hvCMLs 的组合方法来全面评估药物诱导的致心律失常性。
