Machine-learning-based quality control of contractility of cultured human-induced pluripotent stem-cell-derived cardiomyocytes.

The precise and early assessment of cardiotoxicity is fundamental to bring forward novel drug candidates to the pharmaceutical market and to avoid their withdrawal from the market. Recent preclinical studies have attempted to use human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) to predict clinical cardiotoxicity, but the heterogeneity and inconsistency in the functional qualities of the spontaneous contractility of hiPSC-CMs across cell culture wells and product lots still matter. To rapidly assess the functional qualities of hiPSC-CMs without histological labeling, we optically detected the contractility of confluently cultured hiPSC-CMs using bright-field microscopy. Using a method that consisted of data preprocessing, data augmentation, dimensionality reduction, and supervised learning, we succeeded in precisely discriminating between functionally normal and abnormal contractions of hiPSC-CMs.

International Multisite Study of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Proarrhythmic Potential Assessment.

To assess the utility of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an in vitro proarrhythmia model, we evaluated the concentration dependence and sources of variability of electrophysiologic responses to 28 drugs linked to low, intermediate, and high torsades de pointes (TdP) risk categories using two commercial cell lines and standardized protocols in a blinded multisite study using multielectrode array or voltage-sensing optical approaches. Logistical and ordinal linear regression models were constructed using drug responses as predictors and TdP risk categories as outcomes. Three of seven predictors (drug-induced arrhythmia-like events and prolongation of repolarization at either maximum tested or maximal clinical exposures) categorized drugs with reasonable accuracy (area under the curve values of receiver operator curves ∼0.8). hiPSC-CM line, test site, and platform had minimal influence on drug categorization. These results demonstrate the utility of hiPSC-CMs to detect drug-induced proarrhythmic effects as part of the evolving Comprehensive In Vitro Proarrhythmia Assay paradigm.

Characterization of Human Hippocampal Neural Stem/Progenitor Cells and Their Application to Physiologically Relevant Assays for Multiple Ionotropic Glutamate Receptors.

The hippocampus is an important brain region that is involved in neurological disorders such as Alzheimer disease, schizophrenia, and epilepsy. Ionotropic glutamate receptors-namely,N-methyl-D-aspartate (NMDA) receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs), and kainic acid (KA) receptors (KARs)-are well known to be involved in these diseases by mediating long-term potentiation, excitotoxicity, or both. To predict the therapeutic efficacy and neuronal toxicity of drug candidates acting on these receptors, physiologically relevant systems for assaying brain region-specific human neural cells are necessary. Here, we characterized the functional differentiation of human fetal hippocampus-derived neural stem/progenitor cells-namely, HIP-009 cells. Calcium rise assay demonstrated that, after a 4-week differentiation, the cells responded to NMDA (EC50= 7.5 ± 0.4 µM; n= 4), AMPA (EC50= 2.5 ± 0.1 µM; n= 3), or KA (EC50= 33.5 ± 1.1 µM; n= 3) in a concentration-dependent manner. An AMPA-evoked calcium rise was observed in the absence of the desensitization inhibitor cyclothiazide. In addition, the calcium rise induced by these agonists was inhibited by antagonists for each receptor-namely, MK-801 for NMDA stimulation (IC50= 0.6 ± 0.1 µM; n= 4) and NBQX for AMPA and KA stimulation (IC50= 0.7 ± 0.1 and 0.7 ± 0.03 µM, respectively; n= 3). The gene expression profile of differentiated HIP-009 cells was distinct from that of undifferentiated cells and closely resembled that of the human adult hippocampus. Our results show that HIP-009 cells are a unique tool for obtaining human hippocampal neural cells and are applicable to systems for assay of ionotropic glutamate receptors as a physiologically relevant in vitro model.

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.

Screening quality for Ca2+-activated potassium channel in IonWorks Quattro is greatly improved by using BAPTA-AM and ionomycin.

INTRODUCTION: IonWorks automated patch clamp systems are being widely used for ion channel drug discovery, but the perforated patch mode of these systems makes it difficult to obtain a steady intracellular Ca(2+) concentration ([Ca(2+)](i)). This difficulty prevents obtaining high-quality data regarding Ca(2+)-activated channels such as BK and SK channels. We examined the methods for stabilizing [Ca(2+)](i) in the IonWorks Quattro automated patch clamp system to evaluate BK channels. METHODS: Electrophysiological recordings were performed using the single-hole or population patch clamp mode of IonWorks Quattro. To increase [Ca(2+)](i), ionomycin was used. The variation in the BK current and the effect of BK channel modulators were examined in the presence and absence of an intracellular Ca(2+) chelator, BAPTA-AM (20µM). RESULTS: BK current activated by step pulses to +100mV in the presence of ionomycin exhibited large variation (ranging from 0.086 to 11nA). In individual cells, oscillation of the current amplitude was observed when five repetitive pulses were applied at 0.1Hz. Approximately 30% of cells exhibited current variation exceeding 20% when the variation was calculated using the first and third pulses. However, BAPTA-AM treatment before current measurement decreased the number of cells displaying large variation (>20%) to 5%. In the presence of BAPTA-AM, the BK channel modulators NS1619 and 12,14-dichlorodehydroabietic acid increased the BK current at concentrations of 10µM or more showing clear concentration dependency, whereas in its absence, the effect of both compounds was detected only at 30µM. DISCUSSION: The main finding of this study is that the [Ca(2+)](i) variation in the basal condition is very large and hinders the accurate evaluation of compounds in Ca(2+)-activated ion channels. The application of BAPTA-AM and ionomycin greatly improved the precision of BK channel screening, and this method should be applicable to other Ca(2+)-activated ion channels such as SK channels.

A novel method of selecting human embryonic stem cell-derived cardiomyocyte clusters for assessment of potential to influence QT interval.

Physiologically relevant assessment of delayed repolarization is necessary in drug development. In our preliminary experiments on the evaluation using a multielectrode recording system, we had found that the responsiveness of field potential duration (FPD), as QT-like intervals, to hERG channel blockers differed greatly from non-responders to excessive responders in human embryonic stem cell-derived cardiomyocyte clusters. Thus, we report a novel method of selecting clusters suitable for evaluating compounds for the assessment. Clusters were treated with cisapride, a hERG channel blocker, at 100nM, and selected with criteria of 5-20% of corrected FPD (FPDc) prolongation. Then, selected clusters were treated with reference compounds. FPDc was prolonged by blockade of the hERG channel (E-4031 and dl-sotalol) and KvLQT1 channel (chromanol 293B and HMR1556), and by activation of the sodium channel (veratridine) and calcium channel (Bay K8644). FPDc was shortened by calcium channel blockage (verapamil, nifedipine and diltiazem) and by K(ATP) channel activation (pinacidil). Class Ia antiarrhythmic drugs, quinidine and disopyramide, prolonged FPDc. Selected clusters are appropriate for assessing the effects of compounds on ion channels affecting QT intervals. This is the first report of the establishment of an assessment system of potential to influence QT interval, using pharmacologically selected clusters.