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.

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 dissertation, "Calcium Signaling in the Cardiac Differentiation of Mouse Embryonic Stem Cells" by Wenjie, Wei, 魏闻捷, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract:  Intracellular Ca2+ mobilization via secondary messengers modulates multiple cell functions. Cyclic Adenosine 5'-Diphosphate-Ribose (cADPR) is one of the most well recognized endogenous Ca2+ mobilizing messengers. In mammalian, cADPR is mainly formed by CD38, a multi-functional enzyme, from nicotinamide adenine dinucleotide (NAD). It has previously been shown that the cADPR/CD38/Ca2+pathway mediates many cardiac functions, such as regulating the excitation-contraction coupling in cardiac myocytes and modulating the Ca2+ homeostasis during the ischemia injury of the heart. Thus it is reasonable to propose that the cADPR/CD38/Ca2+ pathway plays a role in cardiogenesis. The pluripotent mouse embryonic stem (mES) cells which can be induced to differentiate into all cell types provide an ideal model for studying cardiogenesis. The first part of this dissertation is to determine the role of CD38/cADPR/Ca2+pathwayin the cardiomyogenesis of mES cells. The data showed that CD38 expression was markedly up-regulated during the in vitro embryoid body (EB) differentiation of mouse ES cells, which indicated a regulatory role of CD38 in the differentiation process. Lentivirus mediated shRNA provides a convenient method to knockdown the expression of CD38 in mES cells. Surprisingly, beating clusters appeared earlier and more in CD38 knockdown EBs than that in control EBs. Likewise, the expressions of several cardiac markers were up regulated in CD38 knockdown EBs. In addition, more cardiomyocytes (CMs) existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs than those in control EBs. On the other hand, over-expression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, we showed that CMs derived from the CD38 knock down mES cells possessed the functional properties characteristic of CMs derived fromnormal ES cells. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2cascade during CM differentiation of mES cells, and transiently inhibition of Erk1/2 blocked the enhancive effects of CD38 knockdown on the differentiation of CM from mES cells. Taken together, our data indicate that the CD38/cADPR/Ca2+ signaling pathway suppresses the cardiac differentiation of mES cells. One of the main goals of the researches on cardiac differentiation of ES cells is to enhance the production of CMs from ES cells, thereby providing sufficient amount of functional intact CMs for the treatment of severe heart disease. Nitric oxide (NO) has been found to be a powerful cardiogenesis inducer of mES cells, in that it can significantly increase the yield of ES-derived CM. The second objective of this dissertation is to explore the mechanism underlying the NO facilitated cardiomyogenesis of mES cells. We found that the NO did induce intracellular Ca2+ increases in mES cells, and this Ca2+ increase was due to internal Ca2+ release from ER through theIP3 pathway. Therefore, the expression of IP3 receptors (IP3Rs) in mES cells were knocked down by lentivirus-mediated shRNAs. Interestingly, only type 3 IP3R (IP3R3) knockdown significantly inhibited the NO induced Ca2+ release in mES cells. Moreover, NO facilitated cardiogensis of mES cells was abolished in IP3R3 knockdown EBs. In summary, our results indicate that the IP3R3-Ca2+ pathway is required for NO facilitated cardiomyogenesis of mES cells. DOI: 10.5353/th_b4961786 Subjects:

This dissertation, "The Fate of Undifferentiated Murine Embryonic Stem Cells in a Mouse Model With Acute Myocardial Infarction" by Chun-wai, Wong, 黃俊瑋, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled The Fate of Undifferentiated Murine Embryonic Stem Cells in a Mouse Model with Acute Myocardial Infarction Submitted by Wong Chun Wai for the degree of Master of Philosophy at The University of Hong Kong in August 2005 Myocardial infarction (MI) due to coronary artery diseases causes irreversible loss of heart muscle and is the leading cause of heart failure in developed countries. For patients with end-stage heart failure, current pharmacological and interventional treatments are only palliative and the option of heart transplantation is limited by the availability of donor organs. Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, can propagate indefinitely in culture to serve as an unlimited cell source and maintain their pluripotency to differentiate into all cell types, including cardiomyocyte. However, the signals that stimulate cardiac differentiation of injected ES cells in vivo under such pathophysiological environments remain unclear. To address this question, we investigated the in vitro and in vivo cardiac fate of ES cells under normal and pathophysiological conditions. Our experiments indicated that ES cells have distinct fates in normal and infarcted hearts. Transplantation of undifferentiated ES cells into normal hearts of syngeneic and allogeneic mice did not induce teratoma formation but resulted in limited engraftment of ES cells. In contrast, transplantation of undifferentiated ES cells after acute MI resulted in successful engraftment. Cardiomyocyte differentiation could be observed at the peri-infarct area but not at the infarct area as early as ten days after the transplantation since cardiomyocyte-like ES cells could only be found at the peri-infarct area while most of ES cells found at the infarct area still retained the morphology of undifferentiated ES cells. Endothelial cell-like and smooth muscle cell-like ES cells found in the fibrotic tissue of an infarcted heart twelve weeks after transplantation suggests that the enhancement of neovascularization could also be a long-term consequence after successful engraftment of ES cells at the peri-infarct and infarct areas. In vitro co-culture studies demonstrated that cardiomyocyte differentiation of ES cells in the presence of primary neonatal cardiomyocytes was not adversely affected by short-term hypoxia. Furthermore, the short-term hypoxia appears to be a stimulant that triggers the anti-apoptotic effects of ES cells on cardiomyocytes since the percentage of apoptotic cardiomyocytes was significantly reduced by half when co-culture with ES cells compared with the culture of neonatal cardiomyocytes alone. Exposure to short-term hypoxia also resulted in the up-regulation of connexin 43 and Flk-1 in ES cells which may involve in preventing the apoptosis of neonatal cardiomyocytes. These results were also discussed in relation to the importance of microenvironments in cardiac differentiation, and the potential implications on the applications of ES cells for cardiac regeneration after MI. DOI: 10.5353/th_b3192763 Subjects: Embryonic stem cells Myocardial infarction - Pathophysiology Mice as laboratory animals

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

In their classic paper in 1929, Drury and Szent-Gyorgyi described a number of the important cardiovascular actions of adenosine. Another thirty years were to pass before the possible physiological role of adenosine in coronary vasodilation was studied by Berne and others. Since then, there has been a tremendous increase in research into the actions of adenosine. Workers from many disciplines have employed a wide variety of techniques, since adenosine is a product of and a substrate for a number of metabolic pathways, is transported into cells, and acts at discrete receptor sites to modulate the activity of adenylate cyclase and to produce important actions on many cells and tissues including platelets, adipo cytes, heart, blood vessels, and other smooth muscles. International symposia on the actions of adenosine were held in 1978, 1981, and 1982, and the proceedings of these symposia have been published (Baer and Drummond, 1979; Daly et at., 1983; Berne et at., 1983). Since it is not the primary purpose of the present volume to review our current understanding of the nu merous actions of adenosine, these volumes should be consulted for such details. Rather, the present volume has been planned to provide both graduate students and investigators in pharmacology and related disciplines with a summary of some of the methods now available for the study of the actions of adenosine and, in particular, to highlight their possible uses and limitations.