[Method for MEA Data Analysis of Drug-treated Rat Primary Neurons and Human iPSC-derived Neurons to Evaluate the Risk of Drug-induced Seizures].

　Use of the microelectrode array (MEA) system to record spontaneous neuron activity from networks of cultured neurons has potential as a good risk evaluation method for drug-induced seizure events. Spontaneous electrical activity in neural networks consists of action potential spikes and organized patterns of action potential bursts. In both potentiated rodent primary neurons and human induced pluripotent stem cell (iPSC)-derived neurons, an epileptogenic response pattern manifests as a synchronized burst from spatially separated neurons. Here, we delineate how to perform MEA experiments using cultured neurons, and how to analyze the MEA data to detect drug-induced seizurogenic abnormalities. Usually, a drug's effects, as shown by MEA data, include changes in spike frequency, inter-spike intervals (ISI), burst frequency, burst duration, spikes in a burst, etc. Subsequently, seizurogenic events are evidenced by changes in synchronized burst phenotypes from spatially separated multiple channels in an MEA probe, such as a change in the cross correlation of the spike events from all channels in an MEA probe, or a change in histogram from the sum of ISI for all channels in a probe, etc. We attempted to depict an epileptogenic marker using a histogram of the sum of spikes for all channels in an MEA probe. Verification of these metrics for drug induced abnormalities is ongoing in various collaboration organizations, including the Consortium for Safety Assessment using Human iPS Cells (CSAHi), iPS Non-clinical Experiments for the Nervous System (iNCENS). Collaboration networks for the utilization of iPSC-derived cells during drug development are also summarized here.

A new paradigm for drug-induced torsadogenic risk assessment using human iPS cell-derived cardiomyocytes.

INTRODUCTION: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are anticipated to be a useful tool for conducting proarrhythmia risk assessments of drug candidates. However, a torsadogenic risk prediction paradigm using hiPSC-CMs has not yet been fully established. METHODS: Extracellular field potentials (FPs) were recorded from hiPSC-CMs using the multi-electrode array (MEA) system. The effects on FPs were evaluated with 60 drugs, including 57 with various clinical torsadogenic risks. Actual drug concentrations in medium were measured using the equilibrium dialysis method with a Rapid Equilibrium Dialysis device. Relative torsade de pointes (TdP) scores were determined for each drug according to the degree of FP duration prolongation and early afterdepolarization occurrence. The margins were calculated from the free concentration in medium and free effective therapeutic plasma concentration. Each drug's results were plotted on a two-dimensional map of relative TdP risk scores versus margins. RESULTS: Each drug was categorised as high, intermediate, or low risk based on its location within predefined areas of the two-dimensional map. We categorised 19 drugs as high risk; 18 as intermediate risk; and 17 as low risk. We examined the concordance between our categorisation of high and low risk drugs against the torsadogenic risk categorisation in CredibleMeds®. Our system demonstrated high concordance, as reflected in a sensitivity of 81%, specificity of 87%, and accuracy of 83%. DISCUSSION: These results indicate that our torsadogenic risk assessment is reliable and has a potential to replace the hERG assay for torsadogenic risk prediction, however, this system needs to be improved for the accurate of prediction of clinical TdP risk. Here, we propose a novel drug induced torsadogenic risk categorising system using hiPSC-CMs and the MEA system.

Electrophysiological Characteristics of Human iPSC-Derived Cardiomyocytes for the Assessment of Drug-Induced Proarrhythmic Potential.

The aims of this study were to (1) characterize basic electrophysiological elements of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that correspond to clinical properties such as QT-RR relationship, (2) determine the applicability of QT correction and analysis methods, and (3) determine if and how these in-vitro parameters could be used in risk assessment for adverse drug-induced effects such as Torsades de pointes (TdP). Field potential recordings were obtained from commercially available hiPSC-CMs using multi-electrode array (MEA) platform with and without ion channel antagonists in the recording solution. Under control conditions, MEA-measured interspike interval and field potential duration (FPD) ranged widely from 1049 to 1635 ms and from 334 to 527 ms, respectively and provided positive linear regression coefficients similar to native QT-RR plots obtained from human electrocardiogram (ECG) analyses in the ongoing cardiovascular-based Framingham Heart Study. Similar to minimizing the effect of heart rate on the QT interval, Fridericia's and Bazett's corrections reduced the influence of beat rate on hiPSC-CM FPD. In the presence of E-4031 and cisapride, inhibitors of the rapid delayed rectifier potassium current, hiPSC-CMs showed reverse use-dependent FPD prolongation. Categorical analysis, which is usually applied to clinical QT studies, was applicable to hiPSC-CMs for evaluating torsadogenic risks with FPD and/or corrected FPD. Together, this results of this study links hiPSC-CM electrophysiological endpoints to native ECG endpoints, demonstrates the appropriateness of clinical analytical practices as applied to hiPSC-CMs, and suggests that hiPSC-CMs are a reliable models for assessing the arrhythmogenic potential of drug candidates in human.

CSAHi study: Evaluation of multi-electrode array in combination with human iPS cell-derived cardiomyocytes to predict drug-induced QT prolongation and arrhythmia--effects of 7 reference compounds at 10 facilities.

INTRODUCTION: Drug-induced QT prolongation is a major safety issue during drug development because it may lead to lethal ventricular arrhythmias. In this study, we evaluated the utility of multi-electrode arrays (MEA) with human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to predict drug-induced QT prolongation and arrhythmia. METHODS: Ten facilities evaluated the effects of 7 reference drugs (E-4031, moxifloxacin, flecainide, terfenadine, chromanol 293B, verapamil, and aspirin) using a MED64 MEA system with commercially available hiPS-CMs. Field potential duration (FPD), beat rate, FPD corrected by Fridericia's formula (FPDc), concentration inducing FPDc prolongation by 10% (FPDc10), and incidence of arrhythmia-like waveform were evaluated. RESULTS: The inter-facility variability of absolute values before drug application was similar to the intra-facility variability for FPD, beat rate, and FPDc. The inter-facility variability of FPDc10 for 5 reference drugs ranged from 1.8- to 5.8-fold. At all 10 facilities, E-4031, moxifloxacin, and flecainide prolonged FPDc and induced arrhythmia-like waveforms at concentrations 1.8- to 6.1-fold higher than their FPDc10. Terfenadine prolonged FPDc and induced beating arrest at 8.0 times the FPDc10. The average FPDc10 values for E-4031, moxifloxacin, and terfenadine were comparable to reported plasma concentrations that caused QT prolongation or Torsade de Pointes in humans. Chromanol 293B, a IKs blocker, also prolonged FPDc but did not induce arrhythmia-like waveforms, even at 7.4 times the FPDc10. In contrast, verapamil shortened FPDc and aspirin did not affect FPDc or FP waveforms. DISCUSSION: MEA with hiPS-CMs can be a generalizable method for accurately predicting both QT prolongation and arrhythmogenic liability in humans.

Improvement of acquisition and analysis methods in multi-electrode array experiments with iPS cell-derived cardiomyocytes.

INTRODUCTION: Multi-electrode array (MEA) systems and human induced pluripotent stem (iPS) cell-derived cardiomyocytes are frequently used to characterize the electrophysiological effects of drug candidates for the prediction of QT prolongation and proarrhythmic potential. However, the optimal experimental conditions for obtaining reliable experimental data, such as high-pass filter (HPF) frequency and cell plating density, remain to be determined. METHODS: Extracellular field potentials (FPs) were recorded from iPS cell-derived cardiomyocyte sheets by using the MED64 and MEA2100 multi-electrode array systems. Effects of HPF frequency (0.1 or 1Hz) on FP duration (FPD) were assessed in the presence and absence of moxifloxacin, terfenadine, and aspirin. The influence of cell density on FP characteristics recorded through a 0.1-Hz HPF was examined. The relationship between FP and action potential (AP) was elucidated by simultaneous recording of FP and AP using a membrane potential dye. RESULTS: Many of the FP waveforms recorded through a 1-Hz HPF were markedly deformed and appeared differentiated compared with those recorded through a 0.1-Hz HPF. The concentration-response curves for FPD in the presence of terfenadine reached a steady state at concentrations of 0.1 and 0.3µM when a 0.1-Hz HPF was used. In contrast, FPD decreased at a concentration of 0.3µM with a characteristic bell-shaped concentration-response curve when a 1-Hz HPF was used. The amplitude of the first and second peaks in the FP waveform increased with increasing cell plating density. The second peak of the FP waveform roughly coincided with AP signal at 50% repolarization, and the negative deflection at the second peak of the FP waveform in the presence of E-4031 corresponded to early afterdepolarization and triggered activity. DISCUSSION: FP can be used to assess the QT prolongation and proarrhythmic potential of drug candidates; however, experimental conditions such as HPF frequency are important for obtaining reliable data.

Assessment of testing methods for drug-induced repolarization delay and arrhythmias in an iPS cell-derived cardiomyocyte sheet: multi-site validation study.

A prospective comparison study across 3 independent research laboratories of a pure IKr blocker E-4031 was conducted by using the same batch of human iPS cell-derived cardiomyocytes in order to verify the utility and reliability of our original standard protocol. Field potential waveforms were recorded with a multi-electrode array system to measure the inter-spike interval and field potential duration. The effects of E-4031 at concentrations of 1 to 100 nM were sequentially examined every 10 min. In each facility, E-4031 significantly prolonged the field potential duration corrected by Fridericia's formula and caused early afterdepolarizations occasionally resulting in triggered activities, whereas it tended to decrease the rate of spontaneous contraction. These results were qualitatively and quantitatively consistent with previous non-clinical in vitro and in vivo studies as well as clinical reports. There were inter-facility differences in some absolute values of the results, which were not observed when the values were normalized as percentage change. Information described in this paper may serve as a guide when predicting the drug-induced repolarization delay and arrhythmias with this new technology of stem cells.

Low-density plating is sufficient to induce cardiac hypertrophy and electrical remodeling in highly purified human iPS cell-derived cardiomyocytes.

INTRODUCTION: Cardiac hypertrophy is a leading cause of many cardiovascular diseases, including heart failure, but its pathological mechanism is not fully understood. This study used highly purified human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes to produce an in vitro hypertrophy model and characterize its gene expression and electrophysiological properties. METHODS: For 7 days we cultured hiPSC-derived cardiomyocytes plated at high (2800-4800 cells/mm(2)) or low (500-1200 cells/mm(2)) cell density and assessed their cell size with confocal and fluorescence microscopy, their electrophysiological and pharmacological responses with multi-electrode array systems, and their gene expression patterns by using DNA microarray technology and quantitative PCR. We used quantitative PCR and Western blotting to compare the expression of potassium-channel genes between the hiPSC-derived cardiomyocytes and human fetal and adult hearts. RESULTS: The hiPSC-derived cardiomyocytes showed spontaneous beating and similar pattern of α-actinin molecules regardless of plating density. However, cells plated at low density had the following characteristics compared with those at high density: 1) significant enlargement in size; 2) significant increase or decrease in expression of the cardiac hypertrophy-characteristic genes NPPA, ATP2A2, ANKRD1 and MYL2 in accordance with the progression of hypertrophy; 3) significant reduction in responses to the inhibitors of cardiac slow delayed-rectifier K(+) current (IKs), chromanol 293B and HMR1556, in a cell-density-dependent manner; and 4) significant reduction in the expression of the KCNQ1 and KCNJ2 genes coding the K(+) ion channels conducting each IKs and cardiac inward rectifier outward K(+) current (IK1). DISCUSSION: The enlargement, hypertrophy-characteristic and potassium ion channels gene expression of hiPSC-derived cardiomyocytes suggest that low-density plating was sufficient to induce cardiac hypertrophy. This model may be useful in elucidating mechanisms underlying the onset and progress of cardiac hypertrophy, because these cells can be cultured for several weeks.