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

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

The limited ability of the human heart to regenerate has made myocardial infarction and heart failure debilitating conditions. Recently, an approach using pluri- or multi-potent stem cells to repair damaged heart tissue is being explored for its potential to regenerate tissue as a tailored, patient-specific treatment. However, the mechanisms of integration remain unclear, and many cardiac grafting procedures utilizing both embryonic and adult stem cells have been met with limited success. While current evidence suggests that grafts are likely viable in host myocardium, clinical studies have reported pro arrhythmic side-effects following transplantation, which arise from disrupted propagation patterns. These issues may be attributed to grafts lacking cardiac differentiation, or possessing conduction properties inconsistent with the host tissue. Consequently, understanding the role of the electrical environment throughout the engraftment process is necessary, but infeasible due to a lack of proper tools. Elucidating the electrical aspects of stem cell transplantation aims to ensure proper integration of the transplanted cells to prevent aberrant electrical pathways in the heart. In this work, a set of in vitro tools were developed to study the potential mechanisms underlying the risk of arrhythmia following stem cell transplantation. A planar microelectrode array was first used to investigate the possibility of conduction block if undifferentiated or non cardiomyocyte stem cells, such as mesenchymal stem cells, are used as grafts. Conduction in murine cardiomyocytes was purposely blocked by co-culture with non-conducting murine fibroblasts, and a novel mathematical transform known as a co occurrence matrix was developed to quantitatively analyze the uniformity of conduction. The observed sensitivity of cardiomyocyte conduction illustrated the risk of grafting non-cardiomyocyte cell types despite any potential of differentiating into muscle-like cells. Unlike non-conducting fibroblasts, stem cell grafts are expected to electrically conduct if proper cardiac differentiation takes place. However, possible differences in the conduction properties of these grafts may still lead to arrhythmia. To perform a controlled study of such conduction mismatch, an in vitro co-culture system coupled to microelectrode arrays was developed. Spatially separated cultures representing the host and the graft were allowed to gradually merge above the microelectrode array, allowing the measurement of conduction throughout the integration process. Modeled host and graft cell populations were evaluated by analyzing the co occurrence matrix and conduction velocity for the quality and speed of conduction over time. Co cultures between murine cardiomyocytes (host) and murine skeletal myoblasts (graft) exhibited significant differences in conduction despite synchronous electrical activity. In contrast, conduction was well matched when the same host cells were co cultured with murine embryonic stem cells (mESC). A model using murine cardiomyocytes (host) and differentiating human embryonic stem cells (graft) allowed the characterization of conduction properties relevant to current trans-species animal models, and demonstrate the co-culture device as a screening platform for candidate graft cells. The limited region of the graft that supported conduction exhibited differences in the co-occurrence matrix as well as conduction velocity when compared to the host region. In an effort to improve the effects of conduction mismatch, both host and graft cell populations were electrically paced over the length of time the cultures remained viable (4-5 days). Although a difference between conduction velocities between host and graft was still observed, the overall uniformity of conduction improved in paced co-cultures, implying increased cardiac differentiation. A preliminary study of genomic changes due to paced mESCs resulted in a significant upregulation of several important cardiac genes and a significant downregulation of many embryonic genes. Further efforts are currently underway to examine gene expression with paced hESCs to optimize integration in the host-graft model, and ultimately to understand how the electrical environment influences stem cell transplantation.

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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.

The Research Topic is organized in the framework of the project BIORECAR (grant number: 772168; http://www.biorecar.polito.it/index.html)