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.

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

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, "Combinatorial Expression of Critical Ca⁺ Handling Proteins in Human Embryonic Stem Cell-derived Cardiomyocytes" by Li, Li, 李力, 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: The self-renewable human embryonic stem cells (hESCs) represent a potential unlimited ex vivo source of human cardiomyocytes that could be used for applications in disease modeling, cardiotoxicity screening, drug discovery, and cell-based therapies. The rise and decay of 〖Ca〗 DEGREES(2+) during the excitation-contraction process is known as the 〖Ca〗 DEGREES(2+) transient and a precise modulation of such transient plays an important part of the cardiomyocytes contractility. An array of 〖Ca〗 DEGREES(2+)-handling proteins is responsible for the regulation of the 〖Ca〗 DEGREES(2+) transient. Many of these proteins, are, however, sub-optimally expressed or even absent in hESC-CMs, comparing to normal adult CMs. Previous efforts made on the individual overexpression of 〖Ca〗 DEGREES(2+)-handling proteins like Calsequestrin (CSQ), Phospholamban (PLB) and Sarcoplasmic Reticulum Calcium ATPase 2a (SERCA2a) showed positive maturation of 〖Ca〗 DEGREES(2+) handling, but the effect of combinatorial overexpression of multiple 〖Ca〗 DEGREES(2+)-handling proteins simultaneously in hESC-CMs have never been examined. In this work, I have taken a lentiviral-based transgenes delivery approach to try to simultaneously overexpress 3 critical 〖Ca〗 DEGREES(2+) handling proteins (CSQ, PLB, SERCA2a) that are insufficiently expressed in hESC-CMs and subsequently examined the functional consequences following successful combinatorial overexpression. My results indicate that overexpressing 〖Ca〗 DEGREES(2+) handling proteins can definitely cause to consequential phenotypic changes regarding the 〖Ca〗 DEGREES(2+) handling of hESC-CMs but a carefully titration for an optimal combination of different proteins will be needed in order to fulfill an overall driven maturation of the 〖Ca〗 DEGREES(2+) handling of hESC-CMs. Subjects: Embryonic stem cells

Defined as, “The science about the development of an embryo from the fertilization of the ovum to the fetus stage,” embryology has been a mainstay at universities throughout the world for many years. Throughout the last century, embryology became overshadowed by experimental-based genetics and cell biology, transforming the field into developmental biology, which replaced embryology in Biology departments in many universities. Major contributions in this young century in the fields of molecular biology, biochemistry and genomics were integrated with both embryology and developmental biology to provide an understanding of the molecular portrait of a “development cell.” That new integrated approach is known as stem-cell biology; it is an understanding of the embryology and development together at the molecular level using engineering, imaging and cell culture principles, and it is at the heart of this seminal book. Stem Cells and Regenerative Medicine: From Molecular Embryology to Tissue Engineering is completely devoted to the basic developmental, cellular and molecular biological aspects of stem cells as well as their clinical applications in tissue engineering and regenerative medicine. It focuses on the basic biology of embryonic and cancer cells plus their key involvement in self-renewal, muscle repair, epigenetic processes, and therapeutic applications. In addition, it covers other key relevant topics such as nuclear reprogramming induced pluripotency and stem cell culture techniques using novel biomaterials. A thorough introduction to stem-cell biology, this reference is aimed at graduate students, post-docs, and professors as well as executives and scientists in biotech and pharmaceutical companies.

To date, the need for effective treatments to tackle ischaemic diseases such as CHD and PAD remains unmet. As such, there has been a great deal of interest in developing cell therapies in order to address these important pathologies. The main goals of a cell therapy strategy for ischaemic disease remain prompt restoration of blood supply to the affected areas in order to salvage tissue and/or regeneration of tissues previously lost to ischaemia. Derived from the Inner Cell Mass (ICM) of an embryo at the blastocyst stage, hESC have been proposed as a potential source of functional, transplantable cells for a variety of cell therapy applications. Although successful differentiation of multiple cell types from hESC has been demonstrated, the molecular processes governing the cell commitment process remain poorly understood, and differentiation efficiency often fails to provide the number of cells required to see clinical benefit in the patient. As such, a more thorough transcriptional characterisation of cardiovascular cell types derived from hESC was the goal of this study. MicroRNAs (miRNA; miR) are small (~22nt), non-coding RNAs which negatively regulate mRNA. MiR-1 and miR-133 were previously shown to play a role in regulating cardiac differentiation with miR-1 potentiating cardiac differentiation and miR-133 having an inhibitory effect. Optimisation of lentiviral vectors showed generation of single pre-miR overexpression lentiviruses for miR-1 and miR-133 in a construct using the SFFV promoter to be possible. Furthermore, it was realised SA461 hESC were unsuitable for cardiac differentiation, however, using a modified version of the LaFlamme protocol in a monolayer system resulted in beating cells with a cardiomyocyte phenotype in H1 hESC. Despite successful overexpression of miR-1 and miR-133, there was very little effect on cardiac differentiation over no virus control. Previously published methods for the generation of vascular endothelial cells (EC) have reported varying efficiency and target cell population purity (~3 - 30%). This laboratory recently reported the successful generation of functional EC-like cells from hESC in a feeder-free manner. HESC-EC were analysed by LC Sciences miRNA microarray at early time points day 0, day 2, day 4 and day 10 after initiation of differentiation with time-matched pluripotent controls. An induction of miR-99b, -181a and -181b over time was observed, and validated in H1 hESC. In addition, miR-99b, -181a and -181b were also found to be expressed in other mesodermal cell types including adult human saphenous vein endothelial cells (HSVEC). No statistically significant expression of these miRNAs could be found in representative cell types of ectoderm and endoderm germ layers, therefore it was hypothesised that these miRNAs were largely mesoderm specific. Despite initial data showing a significant difference in expression between HSVEC from control patients and patients undergoing coronary artery bypass grafting (CABG), classical pathophysiological stimuli to cause endothelial cell stress did not change the expression of miR-99b, -181a and -181b in vitro. In order to understand more about gene expression in early lineage commitment, hESC-EC were analysed by Illumina microarray at early timepoints day 0, day 2, day 4 and day 10 after initiation of differentiation with time-matched pluripotent controls. In parallel, primary human saphenous vein endothelial cells (HSVEC) were analysed. Illumina technology permitted whole-genome profiling in a high throughput chip format. Due to overall expression levels being lower intensity than expected, no cut-off of fold-change was applied to the dataset. Analysis of the dataset showed a large number of significantly differentially expressed probes at each time point: Day 2 of endothelial differentiation compared to Day 0 pluripotent control showed 1040 significant differentially expressed probe changes, Day 4 of endothelial differentiation compared to Day 0 pluripotent control showed 2400 significant differentially expressed probe changes and Day 10 of endothelial differentiation compared to Day 0 pluripotent control showed 2157 significant differentially expressed probe changes (all False Discovery Rate