The potential for drug-induced cardiac arrhythmias is a vital component of the toxicological and safety pharmacological profile of new chemical entities (NCEs). Traditional methods for electrophysiological assessment of NCE arrhythmogenic propensity employ human ion channels heterologously expressed in non-cardiac cell lines and/or animal cardiac models. Neither system completely reflects the human condition; heterologous expression systems do not typically express ancillary channel subunits and may miss the functional impact of NCEs affecting multiple channels, while animal models do not express human proteins and may miss species-specific effects. Human induced pluripotent stem (hIPS) cell-derived cardiomyocytes provide a human-based model in which ion channels are expressed in their native environment and thus surmount many of the vagaries associated with traditional models. The suitability of hIPs cell-derived cardiomyocytes and microelectrode array (MEA) recording technology as a cardiac arrhthymogenicity assessment platform was tested by measuring changes, or lack thereof, in MEA-recorded field potential waveforms from spontaneously electrically active syncitia otf hIPS-derived cardiomyocytes before and during cardioactive compound exposure. Beta-adrenergic activation by 100nM Isoproteranol produced an approximate 2-fold increase in beating frequency. Sodium channel block by 10 – 30 mM tetrodotoxin decreased spontaneous beating frequency. Calcium channel block by 10 – 30nM nifedipine shortened the spontaneous field potential duration. hERG channel block by 3 to 100nM E-4031 or KCNQ channel block by 3 – 10mM chromanol 293B prolonged the spontaneous field potential duration. These results demonstrate that a hIPS-cell derived cardiomyocyte / MEA platform is suitable for assessing drug-induced changes in electrical activity of a relevant human-based cardiac model.

Identifying off-target cardiac ion channel blocking effects of new chemical entities (NCEs) is typically accomplished through voltage clamp analysis of non-cardiac cell lines (CHO, HEK293) transiently or stably expressing the ion channel under investigation. While such cell lines are amenable to the preferred higher throughput automated voltage clamp methodologies, they may not fully recapitulate the native environment of the ion channel. Primary cardiomyocyte cultures provide a native environment, but have met with limited success on automated voltage clamp platforms. Human induced pluripotent stem (hIPs) cell-derived cardiomyocytes can be produced in industrialized quantities, possess attributes amenable to automated voltage clamp methodologies, and may enable higher throughput screening for NCE effects on human cardiac ion channels in a native environment. The feasibility of automated voltage clamp analysis of hIPs cell-derived cardiomyocytes and their suitability for use in determining the effects of NCE on cardiac ion channels was assessed by comparing biophysical characterizations of cells at days 28 to 35 post-differentiation generated with the automated PatchXpress 7000A and the gold-standard conventional voltage clamp. The automated patch clamp success rate approached that observed with commonly used non-native cell lines (> 50%) and both methodologies produced similar biophysical profiles for all tested ion channels, including the highly regulated native Ca2+ channel. These results demonstrate that hIPs cell-derived cardiomyocytes represent a native human system suitable for automated examination of NCE effects on cardiac ion channels.

A high content imaging strategy was implemented to compare the toxicological properties of a set of compounds across two distinct cell systems, the rat H9C2 cell line and human induced pluripotent stem (hiPS) cell-derived cardiomyocytes. The use of a multiplexed approach includes parameters suitable for detecting DNA damage, nuclear morphology, mitochondrial membrane potential, oxidative stress, effects on lysosomal integrity, glutathione redox status, and apoptotic events. A test panel of compounds with known in vivo cardiotoxicological attributes including doxorubicin and many well characterized kinase inhibitors were used as reference set to assist in interpretation of the high content imaging assay results. The toxicity profile investigations revealed that cardiotoxicants are more readily identified using hiPS cell-derived cardiomyocytes compared to H9C2 cells. In particular doxirubicin and other overt cardiotoxicants induced a dose responsive change in cellular ATP, caspase 3/7 activity, and mitochondrial membrane potential in hiPS derived cardiomyocytes. This study supports the continued use of high content imaging and stem-cell derived models as a part of a safety evaluation strategy implemented during the early phase of drug discovery. As the availability of additional differentiated hIPs cell types are developed, high content imaging techniques can then be rapidly deployed to provide more complete insight into the early safety profile of a molecule.

The potential of utilizing human primary cardiomyoctes in early safety programs for compound assessment has been historically limited by cell availability and technical challenges. However, recently, human induced pluripotent stem (hIPS) cell-derived cardiomyocytes have been established in suitable scale sufficient for the conduct of characterization experiments. During bulk production of hIPS cell-derived cardiomyocytes, particular emphasis was placed upon defining appropriate quality controls for consistent manufacturing including cardiomyocyte purity and post-cryopreservation viability. From a functional utility purpose, initial sets of validation experiments assessed the hiPS cell derived cardiomyocytes for their suitability to profiling molecules for their safety pharmacological and toxicological properties. In doing so, a set of specific cytotoxic endpoints were identified. This presentation will highlight the method development for cell handling and provide select examples of the high-content screening (HCS) assays and feature measurements that have been adapted for such studies. Illustrated features include, but are not limited to, cell viability, cell purity, nuclear morphology, DNA damage, mitochondrial membrane potential, oxidative stress, reactive oxygen species, lysosomal integrity, and apoptotic events.