Optical Recording of Action Potentials in Human Induced Pluripotent Stem Cell-Derived Cardiac Single Cells and Monolayers Generated from Long QT Syndrome Type 1 Patients

Abstract

Induced pluripotent stem cells (iPSCs) from type 1 long QT (LQT1) patients can differentiate into cardiomyocytes (CMs) including ventricular cells to recapitulate the disease phenotype. Although optical recordings using membrane potential dyes to monitor action potentials (APs) were reported, no study has investigated the disease phenotypes of cardiac channelopathy in association with the cardiac subtype at the single-cell level. We induced iPSC-CMs from three control and three LQT1 patients. Single-cell analysis using a fast-responding dye confirmed that ventricular cells were the dominant subtype (control-iPSC-CMs: 98%, 88%, 91%; LQT1-iPSC-CMs: 95%, 79%, 92%). In addition, LQT1-iPSC-ventricular cells displayed an increased frequency of early afterdepolarizations (pvalue=0.031). Cardiomyocyte monolayers constituted mostly of ventricular cells derived from LQT1-iPSCs showed prolonged AP duration (APD) (pvalue=0.000096). High-throughput assays using cardiomyocyte monolayers in 96-well plates demonstrated that IKr inhibitors prolonged APDs in both control- and LQT1-iPSC-CM monolayers. We confirmed that the optical recordings of APs in single cells and monolayers derived from control- and LQT1-iPSC-CMs can be used to assess arrhythmogenicity, supporting the feasibility of membrane potential dye-based high-throughput screening to study ventricular arrhythmias caused by genetic channelopathy or cardiotoxic drugs.

An intrinsic, label-free signal for identifying stem cell-derived cardiomyocyte subtype

“…The subtype of each hiPSC-CMs was first determined by its AP profile through the clustering analysis, an established alternative method to patch clamp for subtype classification. 8,[17][18][19] The AP of a ventricularlike cell shows a more prominent plateau phase than that of an atriallike and pacemaker-like cell ( Figure 2A1-A3). Moreover, ventricularlike cells have an average AP duration (APD50, AP duration at 50% repolarization) near 324 ± 20 ms, which is longer than that of atriallike cells (166 ± 4 ms) and pacemaker-like cells (171 ± 8 ms).…”

“…The subtype of each hiPSC‐CMs was first determined by its AP profile through the clustering analysis, an established alternative method to patch clamp for subtype classification . The AP of a ventricular‐like cell shows a more prominent plateau phase than that of an atrial‐like and pacemaker‐like cell (Figure A1‐A3).…”

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor

“…Although current protocols for cryopreserving hiPSC-CMs have already been established by using CryStor CS10 (BioLife Solutions Inc.) [61], our preliminary results on cryopreservation and post-thaw efficacy were very low. To adequately address this question, more studies that directly compare the physiological properties of freshly derived and cryopreserved hiPSC-CMs by using other freezing solutions, such as STEM-CELLBANKER from Nippon Zenyaku Kogyo, Japan [56], and methods, such as addition of activin A and bone morphogenetic protein-4 [63], should be further explored.…”

hiPSCs Derived Cardiac Cells for Drug and Toxicity Screening and Disease Modeling: What Micro- Electrode-Array Analyses Can Tell Us

“…Several studies have proven feasibility of voltage sensor probes for drug screening experiments in iPSC-CMs [34,35,36]. Recently, Takaki and colleagues [37] applied voltage sensitive dyes for the identification of distinct cardiac subtypes in an iPSC-CM population. Further, the authors were able to detect differences in the AP pattern in iPSC-CMs obtained from patients suffering from the long QT syndrome, compared to control cells.…”