Drug discovery and development requires preclinical models to eliminate flawed compounds from development pipelines. Unfortunately, current models have limitations, occasionally resulting in toxic or ineffective compounds progressing to the clinic at great cost and patient risk. Utilising stem cell technology, it is now possible to generate sophisticated models with human biology and physiological context, potentially overcoming the limitations of more established preclinical models. In this thesis I have investigated the use of embryonic stem cell derived cardiac cells for use as models in pharmacology, physiology and toxicology studies. Mouse embryonic stem cells were differentiated to generate cardiomyocytes in multicellular aggregates containing not only myocytes, but also pacemaker cells, fibroblasts and endothelium. These aggregates were used for pharmacology studies, where the signaling resulting from [beta]-adrenoceptor and adenosine receptor stimulation was explored. Using the same differentiation method, in combination with a pan-cardiac reporter for cell enrichment, the function of individual cardiac cells was measured using calcium imaging. Following extensive method development, multiple phenotypes were identified in the enriched population based on spontaneous calcium oscillations. These distinct phenotypes were characterised based on calcium oscillation kinetics, pharmacology and immunocytochemistry. Using human stem cell derived cardiomyocyte aggregates I studied the effects of doxorubicin (a known cardio-toxin) and Trastuzumab, a humanised antibody with disputed cardio-toxicity. Following extensive method development, toxicity was observed for both doxorubicin and Trastuzumab. Furthermore, mechanistic studies implicate multiple cell types mediating Trastuzumab toxicity via a complicated signaling pathway. Based on my results, as models of pharmacology stem cell derived cardiomyocytes provide access to a physiologically heterogeneous model that may be useful for the screening of compounds for non-specific cardiac activity. Unfortunately, the complexity of multicellular aggregates limits their use in characterizing less established, or complicated receptor signaling pathways. Results from calcium imaging studies indicate that at a single cell level, there is considerable heterogeneity of stem cell derived cardiac cells. Focusing on cells with spontaneous calcium oscillations, presumably pacemaker cells, it may be possible to gain greater insight into the mechanisms required to maintain spontaneous cardiac activity, and identify drugs that disrupt it. The results of the Trastuzumab toxicity study provide evidence of a novel mechanism of Trastuzumab cardio-toxicity. More importantly, these results support the use of stem cell derived models for toxicology screening, particularly of humanised antibodies whose toxicity may be missed in classical models. The work presented in this thesis identified novel pacemaker phenotypes previously unreported, and a novel mechanism for Trastuzumab toxicity. Furthermore, this thesis highlights the strengths and weaknesses of stem cell derived models for use in pharmacology, physiology and toxicology assays.

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia that causes the irregular and uncoordinated contractions of the atrial chambers. Current first-line pharmacological treatments are limited in efficacy with side effects including ventricular proarrhythmia. Thus, it is imperative to find novel treatments for better management of the disease. However, current preclinical assays such as heterologous expression and animal models do not recapitulate the entirety of human cardiac physiology. As such, the ability to generate hiPSC-derived atrial-like CMs (hiPSC-aCMs) and ventricular-like CMs (hiPSC-vCMs) can provide a more robust physiological system to assess drug effects for AF treatment in vitro. The objective of this thesis is to develop a preclinical assay system using optical mapping technique and human induced pluripotent stem cells (hiPSCs). Here, I characterized the function of hiPSC-aCMs and demonstrated the sensitivity and specificity of the assay system in capturing the effects of atrial-selective compounds.

This volume provides methodologies for ES and iPS cell technology on the study of cardiovascular diseases. Chapters guide readers through protocols on cardiomyocyte generation from pluripotent stem cells, physiological measurements, bioinformatic analysis, gene editing technology, and cell transplantation studies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Pluripotent Stem-Cell Derived Cardiomyocytes aims to help researchers set up experiments using pluripotent stem cell-derived cardiac cells.

Embryonic stem cell-derived cardiomyocytes (ESC-CMs) have applications in understanding cardiac disease pathophysiology, pharmacology and toxicology. However, a comprehensive characterisation of their basic physiological and pharmacological properties is critical in determining their suitability as models of cardiac activity.Initially, video microscopy and motion analysis software were used to investigate the responses of mouse ESC-derived beating bodies (BBs) to isoprenaline (Iso) and the cardio-active peptides angiotensin II (Ang II) and endothelin-1 (ET-1). Whilst all of these agonists mediated changes in contraction amplitude, indicating the presence of functional ß-adrenoceptor, ETA, AT1 and AT2 receptors, the BBs could be divided on the basis of their contraction frequency responses to the peptide agonists, Ang II and ET-1. This indicated functional heterogeneity amongst the pacemaker cells within the differentiated CM population.An Nkx2.5-eGFP ESC reporter cell line was used to facilitate the isolation of pacemaker cells of the cardiac lineage through live single cell high acquisition rate calcium imaging. Multiple kinetically distinct, previously unreported intracellular Ca2+ ([Ca2+]i) waveforms were observed, most of which were markedly sensitive to reactive oxygen species generation during confocal imaging. By modifying the imaging medium to contain an anti-oxidant cocktail, the activities of six distinct [Ca2+]i waveforms were preserved. On the basis of their kinetics and immunocytochemical profiles, the single cells exhibiting these distinct [Ca2+]i waveforms could be crudely localised to specific regions of the secondary cardiac conduction system. Through investigation of [Ca2+]i handling mechanisms, as well as responsiveness to various cardio-active agonists, this study has demonstrated that automaticity in different spontaneously active Nkx2.5-eGFP+ pacemaker-like populations is governed by varying mechanisms and each population exhibits distinct agonist response profiles.Through collaboration with David Elliott at the Monash Immunology and Stem Cell Laboratories, the pharmacological modulation and [Ca2+]i handling properties of NKX2.5-GFP+ human ESC-BBs was investigated. Only a maximum of 60% of BBs responded to Iso, carbachol, Ang II and ET-1. Investigation of second messenger signalling activation indicated that this was due to ineffective receptor-second messenger coupling during early differentiation stages. Furthermore, confocal calcium imaging on sorted, spontaneously active NKX2.5-GFP+ hESC-cardiac cells indicated the presence of a single, homogeneous pacemaker-like population within these BBs. Unlike the mESC-derived cardiac system, the human BBs were differentiated using a defined exogenous growth factor induced approach which may have biased the differentiation of a particular cardiac conduction system cell type. The signalling cues required for the differentiation of these distinct cardiac subpopulations is under continued investigation.Due to the technical challenges of their investigation from in vivo sources, little is known regarding the function of secondary cardiac conduction system cells, particularly with respect to the mechanisms by which arrhythmias manifest themselves. The ability to isolate and characterise distinct populations of the cardiac conduction system is, therefore, highly clinically relevant. The results from this thesis provide strong support for the potential use of ESCs in conduction system disease modelling, as well as drug discovery and screening platforms.

July 02-04, 2018 Berlin, Germany Key topics : Toxicology, Clinical & Medical Toxicology, Food and Nutritional Toxicology, Environmental Toxicology, Industrial & Occupational Toxicology, Systems Toxicology, Immunotoxicology, Chemical Carcinogenesis, Methods for Toxicity Testing, Risk Assessment, Toxicity Testing Markets, Emerging Toxicology Concepts, Molecular and Biochemical Toxicology, Reproductive and Developmental Toxicology, Genetic Toxicology, Drug Toxicology, Product Development Toxicology, Pharmacology, Developmental Pharmacology, Applied Pharmacology,