Human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hESC-CM and hiPSC-CM, respectively) have held considerable scientific interest for their potential applications in the fields of drug screening, disease modeling, and tissue engineering. One of the most significant roadblocks currently hindering the use of hESC-CMs and hiPSC-CMs concerns their inability to achieve cellular phenotypic maturity. This roadblock is referred to as the "maturation block" problem: cardiomyocytes derived from stem cells are known to experience limitations in phenotypic expression, in which the cells demonstrate characteristics analogous to late fetal stage cells, rather than adult cells. If hESC-CM and hiPSC-CM cultures are to achieve their full potential in pharmacology and regenerative medicine applications, the maturation block problem must be resolved. This dissertation sought to improve upon the current understanding of the mechanisms involved in stem cell-derived cardiomyocyte maturation. Here, analysis of microelectrode array (MEA) recordings of hESC-CM and hiPSC-CM cultures revealed that the pacemaker region often moves (translocates) across the MEA. The variable length of the quiescent period between translocation events was found to obey a power law probability distribution. Such distributions are a characteristic of critical systems, or systems that demonstrate complex spatiotemporal dynamics and emergent properties. The power law exponent obtained for pacemaker translocation quiescent periods ([alpha] = -1.58) closely mirrors the power law exponent observed in several critical systems ([alpha] = -1.5), indicating that critical dynamics may play a crucial role in the development of a stable pacemaker region in the cardiomyocyte culture. The computational tools developed for cardiomyocyte power law analysis were expanded to investigate a variety of cardiomyocyte properties, including local activation time and conduction velocity, as well as spatial relationships between the pacemaker region and cardiomyocyte electrical properties. This led to the development of Cardio PyMEA, a free and open source, graphical user interface-based program that was written in Python for the analysis of MEA cardiomyocyte data. Cardio PyMEA was made available on Github for any interested individual to use for MEA-based cardiomyocyte analysis and could serve as an evolving platform for such analyses in the future

This volume covers all aspects of embryonic stem cell differentiation, including mouse embryonic stem cells, mouse embryonic germ cells, monkey and human embryonic stem cells, and gene discovery.* Early commitment steps and generation of chimeric mice* Differentiation to mesoderm derivatives* Gene discovery by manipulation of mouse embryonic stem cells

Calcium is crucial in governing contractile activities of myofilaments in cardiomyocytes, any defeats in calcium homeostasis of the cells would adversely affect heart pumping action. The characterization of calcium handling properties in human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMCs) is of significant interest and pertinent to the stem cell and cardiac regenerative field because of their potential patient-specific therapeutic use.

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.

This volume will cover a series of reviews on stem cells including adult and embryonic stem cells. Speakers were invited to present these talks during the Stem Cell Symposia in fall of 2010, in Samsun, Turkey. Unique aspect of this volume is that it brings a multidisciplinary aspect of stem cells extracted from a symposium.