Functional characterisation of cardiomyocytes derived from mouse and human embryonic stem cells
thesisposted on 06.02.2017, 05:52 by Lagerqvist, Ebba Louise
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.