Victor Zamora

Comprehensive Translational Assessment of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes for Evaluating Drug-Induced Arrhythmias

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) hold promise for assessment of drug-induced arrhythmias and are being considered for use under the comprehensive in vitro proarrhythmia assay (CiPA). We studied the effects of 26 drugs and 3 drug combinations on 2 commercially available iPSC-CM types using high-throughput voltage-sensitive dye and microelectrode-array assays being studied for the CiPA initiative and compared the results with clinical QT prolongation and torsade de pointes (TdP) risk. Concentration-dependent analysis comparing iPSC-CMs to clinical trial results demonstrated good correlation between drug-induced rate-corrected action potential duration and field potential duration (APDc and FPDc) prolongation and clinical trial QTc prolongation. Of 20 drugs studied that exhibit clinical QTc prolongation, 17 caused APDc prolongation (16 in Cor.4U and 13 in iCell cardiomyocytes) and 16 caused FPDc prolongation (16 in Cor.4U and 10 in iCell cardiomyocytes). Of 14 drugs that cause TdP, arrhythmias occurred with 10 drugs. Lack of arrhythmic beating in iPSC-CMs for the four remaining drugs could be due to differences in relative levels of expression of individual ion channels. iPSC-CMs responded consistently to human ether-a-go-go potassium channel blocking drugs (APD prolongation and arrhythmias) and calcium channel blocking drugs (APD shortening and prevention of arrhythmias), with a more variable response to late sodium current blocking drugs. Current results confirm the potential of iPSC-CMs for proarrhythmia prediction under CiPA, where iPSC-CM results would serve as a check to ion channel and in silico modeling prediction of proarrhythmic risk. A multi-site validation study is warranted.

The Use of Ratiometric Fluorescence Measurements of the Voltage Sensitive Dye Di-4-ANEPPS to Examine Action Potential Characteristics and Drug Effects on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and higher throughput platforms have emerged as potential tools to advance cardiac drug safety screening. This study evaluated the use of high bandwidth photometry applied to voltage-sensitive fluorescent dyes (VSDs) to assess drug-induced changes in action potential characteristics of spontaneously active hiPSC-CM. Human iPSC-CM from 2 commercial sources (Cor.4U and iCell Cardiomyocytes) were stained with the VSD di-4-ANEPPS and placed in a specialized photometry system that simultaneously monitors 2 wavebands of emitted fluorescence, allowing ratiometric measurement of membrane voltage. Signals were acquired at 10 kHz and analyzed using custom software. Action potential duration (APD) values were normally distributed in cardiomyocytes (CMC) from both sources though the mean and variance differed significantly (APD90: 229 ± 15 ms vs 427 ± 49 ms [mean ± SD, P < 0.01]; average spontaneous cycle length: 0.99 ± 0.02 s vs 1.47 ± 0.35 s [mean ± SD, P < 0.01], Cor.4U vs iCell CMC, respectively). The 10–90% rise time of the AP (Trise) was ∼6 ms and was normally distributed when expressed as 1/Trise2 in both cell preparations. Both cell types showed a rate dependence analogous to that of adult human cardiac cells. Furthermore, nifedipine, ranolazine, and E4031 had similar effects on cardiomyocyte electrophysiology in both cell types. However, ranolazine and E4031 induced early after depolarization-like events and high intrinsic firing rates at lower concentrations in iCell CMC. These data show that VSDs provide a minimally invasive, quantitative, and accurate method to assess hiPSC-CM electrophysiology and detect subtle drug-induced effects for drug safety screening while highlighting a need to standardize experimental protocols across preparations.

Application of optical action potentials in human induced pluripotent stem cells-derived cardiomyocytes to predict drug-induced cardiac arrhythmias

INTRODUCTION Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging as new and human-relevant source in vitro model for cardiac safety assessment that allow us to investigate a set of 20 reference drugs for predicting cardiac arrhythmogenic liability using optical action potential (oAP) assay. METHODS Here, we describe our examination of the oAP measurement using a voltage sensitive dye (Di-4-ANEPPS) to predict adverse compound effects using hiPS-CMs and 20 cardioactive reference compounds. Fluorescence signals were digitized at 10kHz and the records subsequently analyzed off-line. Cells were exposed to 30min incubation to vehicle or compound (n=5/dose, 4 doses/compound) that were blinded to the investigating laboratory. Action potential parameters were measured, including rise time (Trise) of the optical action potential duration (oAPD). RESULTS Significant effects on oAPD were sensitively detected with 11 QT-prolonging drugs, while oAPD shortening was observed with ICa-antagonists, IKr-activator or ATP-sensitive K+ channel (KATP)-opener. Additionally, the assay detected varied effects induced by 6 different sodium channel blockers. The detection threshold for these drug effects was at or below the published values of free effective therapeutic plasma levels or effective concentrations by other studies. DISCUSSION The results of this blinded study indicate that OAP is a sensitive method to accurately detect drug-induced effects (i.e., duration/QT-prolongation, shortening, beat rate, and incidence of early after depolarizations) in hiPS-CMs; therefore, this technique will potentially be useful in predicting drug-induced arrhythmogenic liabilities in early de-risking within the drug discovery phase.

Tailoring Mathematical Models to Stem-Cell Derived Cardiomyocyte Lines Can Improve Predictions of Drug-Induced Changes to Their Electrophysiology

Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) have applications in disease modeling, cell therapy, drug screening and personalized medicine. Computational models can be used to interpret experimental findings in iPSC-CMs, provide mechanistic insights, and translate these findings to adult cardiomyocyte (CM) electrophysiology. However, different cell lines display different expression of ion channels, pumps and receptors, and show differences in electrophysiology. In this exploratory study, we use a mathematical model based on iPSC-CMs from Cellular Dynamic International (CDI, iCell), and compare its predictions to novel experimental recordings made with the Axiogenesis Cor.4U line. We show that tailoring this model to the specific cell line, even using limited data and a relatively simple approach, leads to improved predictions of baseline behavior and response to drugs. This demonstrates the need and the feasibility to tailor models to individual cell lines, although a more refined approach will be needed to characterize individual currents, address differences in ion current kinetics, and further improve these results.

Electrophysiological characterization of iCell2 hiPSC derived cardiomyocytes: the new generation of CDI hiPSC-CMs

Abstract Most of the efforts in cardiac safety pharmacology are orientated towards the improvement of early stage drug screening. In this regard, the creation of in vitro human ventricular electrophysiology has been accelerated by the commercial availability hiPSC-CMs. Many studies have validated iCell cardiomycytes (Cellular Dynamics Inc.) as a robust model to carry out predictive cardiotoxicity, but the requirement of at least 10 days in vitro (DIV), increases the handling and associated risks. The electrophysiological characterization of 2nd generation of this cell type (iCell2 cardiomyocytes) with accelerated maturation processes that allows their use after 4-7 DIV is presented. Cellular electrophysiology was examined using CellOPTIQTM (Clyde Biosicences Ltd.). The cells are cultured in 96-well glass bottom plates. The electrical activity is recorded on cell monolayers loaded with di-4ANEPPS (transient incubation in serum-free media). Baseline electrical recordings are registered after 7 DIV using the CellOPTIQTM system (10kHz, 15s). A dosedependent APD shortening was evident in iCell2 following treatment with the L-type Ca2+ channel blocker nifedipine. A dose-dependent prolongation of APD is shown upon E4031 hERG blockade, with EADs evident following treatment with the highest concentration of E4031 (0.1μM). In summary, the electrophysiological characteristics of iCell2 cell line are easily recorded using the CellOPTIQTM system and are suitable for use in cardiotoxicology screening. Methodology iCell Cardiomyocytes were purchased from Cellular Dynamics International (USA) and were seeded in a fibronectin coated 96 well plate (50,000 cells/well) and cultured for 7 days. Following maturation, the cells were transferred to serum free media and transiently exposed to voltage sensitive dye (Di-4-ANEPPS). Measurements were obtained before and after drug treatment by exciting the cells with a 470nm LED. The emitted fluorescence was recorded at 10KHz from regions of iCell2 cardiomyocytes for periods up to 15s on the CellOPTIQTM electrophysiology platform (Clyde Biosciences Ltd). The records were subsequently analysed off-line using proprietary software (Clyde Biosciences Ltd). A Dunnett’s test was completed to test for statistical significance of all data (*p<0.05; **p<0.01; ***p<0.001).