Leung, Sze Lee Cecilia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 144-153). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / Abbreviations --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.1.1 --- Adult stem cells --- p.2 / Chapter 1.1.2 --- Embryonic stem cells --- p.2 / Chapter 1.1.3 --- Pros and cons of embryonic and adult stem cells --- p.5 / Chapter 1.1.4 --- Human embryonic stem cells (hESCs) --- p.6 / Chapter 1.1.5 --- Mouse embryonic stem cells (mESCs) --- p.7 / Chapter 1.1.6 --- Characteristics of ESC-derived cardiomyocytes --- p.7 / Chapter 1.2 --- Cardiovascular Diseases (CVD) --- p.9 / Chapter 1.2.1 --- Causes and statistics of CVD --- p.9 / Chapter 1.2.2 --- Current treatment for CVD --- p.10 / Chapter 1.2.3 --- Current hurdles of putting hESC-CMs into clinical use --- p.11 / Chapter 1.3 --- Myosin light chain2v --- p.13 / Chapter 1.4 --- Genetic-engineering of hESCs & their cardiac derivatives by lentiviral-mediate gene transfer --- p.14 / Chapter 1.5 --- Cytokines secretion during myocardial infarction --- p.15 / Chapter 1.6 --- Aims of the Project --- p.19 / Chapter 1.7 --- Significance of the Project --- p.19 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Subcloning --- p.20 / Chapter 2.1.1 --- Amplification of MLC-2v --- p.20 / Chapter 2.1.2 --- Purification of DNA product --- p.21 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.21 / Chapter 2.1.4 --- Ligation of MLC-2v promoter with DuetO 11 vector --- p.22 / Chapter 2.1.5 --- Transformation of ligation product into competent cells --- p.22 / Chapter 2.1.6 --- PCR confirmation of successful ligation --- p.23 / Chapter 2.1.7 --- Small-scale preparation of bacterial plasmid DNA --- p.23 / Chapter 2.1.8 --- Restriction enzyme digestions to reconfirm positive clones --- p.24 / Chapter 2.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.25 / Chapter 2.1.10 --- Large-scale preparation of target recombinant expression vector --- p.25 / Chapter 2.2 --- Mouse Embryonic Fibroblast (MEF) Culture --- p.26 / Chapter 2.2.1 --- Derivation of MEF --- p.26 / Chapter 2.2.2 --- Mouse embryonic fibroblast cells culture --- p.27 / Chapter 2.2.3 --- Irradiation of mouse embryonic fibroblast --- p.28 / Chapter 2.3 --- HESC culture --- p.29 / Chapter 2.3.1 --- Thawing and Plating hESCs --- p.29 / Chapter 2.3.2 --- Splitting hESCs --- p.30 / Chapter 2.3.3 --- "Culture maintainence, selection and colony removal" --- p.31 / Chapter a) --- Distinguish differentiated and undifferentiated cells and colonies / Chapter b) --- "Remove differentiated cells by ""Picking to Remove""" / Chapter c) --- "Remove undifferentiated cells by ""Picking to Keep""" / Chapter 2.3.4 --- Freezing hESCs --- p.31 / Chapter 2.3.5 --- Differentiation of hESCs --- p.32 / Chapter 2.3.6 --- "HESC culture on feeder free system, mTeSR TM1" --- p.34 / Chapter a) --- Preparation of mTeSRTMl / Chapter b) --- Preparation of BD MatrigelTM hESC-qualified Matrix aliquots / Chapter c) --- Coating plates with BD MatrigelTM hESC-qualified Matrix / Chapter d) --- Human Embryonic stem cells culture in mTeSRTMl / Chapter 2.4 --- ES Cell Characterization (Chemicon Cat# SCR001) --- p.36 / Chapter 2.4.1 --- Alkaline Phosphatase Staining --- p.36 / Chapter 2.4.2 --- Immunofluorescence staining --- p.37 / Chapter 2.5 --- MESC culture --- p.38 / Chapter 2.5.1 --- Thawing and Plating mESCs --- p.38 / Chapter 2.5.2 --- Splitting mESCs --- p.38 / Chapter 2.5.3 --- Differentiation of mESCs --- p.39 / Chapter 2.5.4 --- To study the effects of cytokines on mESC differentiation --- p.40 / Chapter 2.6 --- Lentivirus (LV) Packaging --- p.41 / Chapter 2.6.1 --- Transfection of lentiviral vectors into HEK293FT cells --- p.41 / Chapter 2.6.2 --- LV titering --- p.42 / Chapter 2.7 --- MultipleTransduction --- p.43 / Chapter 2.8 --- Selection of transduced cells by hygromycin --- p.43 / Chapter 2.8.1 --- Determination of hygromycin selection dosage --- p.43 / Chapter 2.8.2 --- Selection of stable clones --- p.44 / Chapter 2.9 --- Isolation of green fluorescent cardiomyocytes derived from differentiated hESCs --- p.45 / Chapter 2.9.1 --- Collagenase digestion of embryoid bodies into single cells --- p.45 / Chapter 2.9.2 --- FACS --- p.46 / Chapter 2.10 --- Gene expression study / Chapter 2.10.1 --- Primer design --- p.46 / Chapter 2.10.2 --- RNA extraction --- p.46 / Chapter 2.10.3 --- DNase Treatment --- p.47 / Chapter 2.10.4 --- Synthesis of Double-stranded cDNA from Total RNA --- p.47 / Chapter 2.10.5 --- Quantitative real-time PCR --- p.48 / Chapter 2.10.6 --- Quantification of mRNA expression --- p.49 / Chapter 2.11 --- Protein Expression study --- p.49 / Chapter 2.11.1 --- Crude protein extraction --- p.49 / Chapter 2.11.2 --- Quantitation of protein samples --- p.50 / Chapter 2.11.3 --- SDS-PAGE --- p.50 / Chapter 2.11.4 --- Western Blot --- p.51 / Chapter 2.11.5 --- Western blot luminal detection --- p.52 / Chapter 2.11.6 --- Quantification of protein expression --- p.52 / Chapter CHAPTER 3 --- PURIFICATION OF CARDIOMYOCYTES DERIVED FROM DIFFERENTIATED HESCs / Chapter 3.1 --- Subcloning --- p.57 / Chapter 3.1.1 --- Linearization of DuetO11 and excision of UBC promoter --- p.58 / Chapter 3.1.2 --- PCR cloning of MLC-2V --- p.59 / Chapter 3.1.3 --- Ligation of MLC-2v promoter to linearized DuetO11 --- p.60 / Chapter 3.1.3.1 --- Colony PCR to screen for positive clones --- p.61 / Chapter 3.1.3.2 --- Restriction digestion to confirm the success of ligation --- p.61 / Chapter 3.2 --- Lentivirus (LV) packaging --- p.62 / Chapter 3.2.1 --- Transfection --- p.63 / Chapter 3.2.2 --- LV titering --- p.64 / Chapter 3.3 --- HESC culture --- p.66 / Chapter 3.4 --- Multi-transduction of hESCs with LVs --- p.67 / Chapter 3.5 --- Differentiation after transduction --- p.69 / Chapter 3.6 --- Antibiotic selection --- p.71 / Chapter 3.6.1 --- Characterization of hESCs on feeder free system --- p.72 / Chapter 3.6.1.1 --- Alkaline Phosphatase (AP) staining --- p.72 / Chapter 3.6.1.2 --- Immunostaining with pluripotency marker --- p.73 / Chapter 3.6.2 --- Determination of hygromycin dosage by MTT assay --- p.74 / Chapter 3.6.3 --- HESCs after selection in feeder free system --- p.75 / Chapter 3.7 --- Differentiation of hESCs after selection --- p.76 / Chapter 3.8 --- FACS --- p.77 / Chapter 3.9 --- QPCR of cells after FACS --- p.80 / Chapter 3.9.1 --- Gene expression of Nkx2.5 --- p.81 / Chapter 3.9.2 --- Gene expression of c-Tnl --- p.82 / Chapter 3.9.3 --- Gene expression of c-TnT --- p.83 / Chapter 3.9.3 --- Gene expression of MLC-2v --- p.84 / Chapter CHAPTER 4 --- THE STUDY OF CYTOKINES' EFFECT ON MESC DIFFERENTIATION / Chapter 4.1 --- mESC culture --- p.85 / Chapter 4.2 --- The effect of cytokines on the differentiation of mESCs --- p.86 / Chapter 4.2.1 --- Beating curves of mESCs treated with different concentrations of cytokines at differentiation day 2 to 6 before attachment --- p.88 / Chapter 4.2.2 --- qPCR to determine the cytokines' effect on the differentiation of mESCs --- p.94 / Chapter 4.2.2.1 --- The effect of IL-1α on the expression of cardiac specific genes --- p.95 / Chapter 4.2.2.2 --- The effect of IL-1β on the expression of cardiac specific genes --- p.98 / Chapter 4.2.2.3 --- The effect of IL-6 on the expression of cardiac specific genes --- p.101 / Chapter 4.2.2.4 --- The effect of IL-10 on the expression of cardiac specific genes --- p.104 / Chapter 4.2.2.5 --- The effect of IL-18 on the expression of cardiac specific genes --- p.107 / Chapter 4.2.2.6 --- The effect of TNF-α on the expression of cardiac specific genes --- p.110 / Chapter 4.2.3 --- Western blot analysis of the cytokines' effect on the differentiation of mESCs --- p.113 / Chapter 4.2.3.1 --- The effect of IL-lα on the abundance of cardiac specific proteins --- p.114 / Chapter 4.2.3.2 --- The effect of IL-1β on the abundance of cardiac specific proteins --- p.116 / Chapter 4.2.3.3 --- The effect of IL-6 on the abundance of cardiac specific proteins --- p.118 / Chapter 4.2.3.4 --- The effect of IL-10 on the abundance of cardiac specific proteins --- p.120 / Chapter 4.2.3.5 --- The effect of IL-18 on the abundance of cardiac specific proteins --- p.122 / Chapter 4.2.3.6 --- The effect of TNF-α on the abundance of cardiac specific proteins --- p.124 / Chapter CHAPTER 5 --- DISCUSSION / Chapter 5.1 --- Purification of cardiomyocytes derived from differentiated hESCs --- p.127 / Chapter 5.2 --- Study on the effect of cytokines on mESC differentiation --- p.135 / Chapter 5.3 --- Conclusion --- p.142 / REFERENCES --- p.144

Thesis (doctoral)--Humboldt-Universiẗat, Berlin, 2005. / Includes bibliographical references (p. 231-260) and index.

Source: Masters Abstracts International, Volume: 47-02, page: 0981. Adviser: Luisa Franzini. Includes bibliographical references.

The loss of regenerative capacity of bone, from fetal to adult to aged animals, has been attributed not only to a decline in the function of cells involved in bone formation but also to alterations in the bone microenvironment that occur through development and aging, including extracellular matrix (ECM) composition and growth/trophic factor content. In the development of novel treatments for bone repair, one potential therapeutic goal is the restoration of a more regenerative microenvironment, as found during embryonic development. One approach to creating such a microenvironment is through the use of stem cells. In addition to serving as a differentiated cell source, pluripotent stem cells, such as embryonic stem cells (ESCs), may possess the unique potential to modulate tissue environments via local production of ECM and growth factors. ESC-produced factors may be harnessed and delivered to promote functional tissue regeneration. Such an approach to generate a naturally derived, acelluar therapy has been employed successfully to deliver osteoinductive factors found within adult bone, in the form of demineralized bone matrix (DBM), but the development of treatments derived instead from developing, more regenerative tissues or cells remains attractive. Furthermore, the derivation of regenerative materials from an ESC source also presents the added benefit of eliminating donor to donor variability of adult, cadaveric tissue derived materials, such as DBM. Thus, the objective of this project was to examine the osteoinductive potential harbored within the embryonic microenvironment, in vitro and in vivo. The osteogenic differentiation of mouse ESCs as embryoid bodies (EBs) was evaluated in response to phosphate treatment, in vitro, including osteoinductive growth factor production. The osteoinductivity of EB-derived material (EBM) was then compared to that of adult tissue-derived DBM, in vivo. Phosphate treatment enhanced osteogenic differentiation of EBs. EBM derived from phosphate treated EBs retained bioactive, osteoinductive factors and induced new bone formation, demonstrating that the microenvironment within osteogenic EBs can be harnessed in an acellular material to yield in vivo osteoinductivity. This work not only provides new insights into the dynamic microenvironments of differentiating stem cells but also establishes an approach for the development of an ESC-derived, tissue specific therapy.

Embryonic stem cells (ESCs) are being considered as a cell source for cardiac regeneration because of their potency and availability. We studied the electrophysiological implications using co-cultures of ESCs and neonatal rat ventricular myocytes (NRVM) grown on a multi-electrode array (MEA). To mimic expected engraftment rates 5% mouse ESCs were co-cultured with NRVMs. Comparing cultures without and with 5% ESCs at 4 days, the mean bipolar field potential duration (FPD) of NRVMs increased from 26.3 ± 2.2 ms (n=10) to 44.3 ± 6.2 ms (n=9; p < 0.05), the interspike interval (ISI) increased from 358.3 ± 62.8 ms (n=10) to 947.8 ± 214.6 ms (n=7; p < 0.01), and conduction velocity (CV) decreased from 14.2 ± 1.3 cm/s (n=8) to 4.6 ± 1.2 cm/s (n=5; p < 0.01). To evaluate whether ESC were having direct or paracrine effects on NRVMs, media conditioned by 3x106 ESCs for 24 hr was diluted 1:1 with fresh media and then introduced to NRVM cultures on the day of plating. Conditioned media was changed daily and altered mean FPD, ISI, and CV to 46.1 ± 7.8 ms, ISI to 682.0 ± 128.5 ms, and 4.2 ± 0.4 cm/s (n=8; p < 0.01 for each measure), respectively at 4 days. However, changes were not seen in media that was incubated for 24hrs and diluted 1:1 with fresh media and introduced to NRVM cultures in a similar fashion (n=7; p > 0.05). Slowed CV is associated with increased arrhythmic risk and reports demonstrate an inverse relationship between CV and nonphosphorylated Cx43(NP-Cx43). Western blots for total Cx43 expression revealed a decrease in ratio of P-Cx43/NP-Cx43 in the 5% mouse ESCs and ESC conditioned media cultures as compared to controls (n=8; p < 0.01 for each). There was not significant increase in the total Cx43 expression (n=6; p > 0.05). Culturing ESCs with NRVMs resulted in a decreased ISI, prolonged FPD, and slowed CV of the co-cultures as compared to controls leading to pro-arrhythmic conditions. Similar effects on NRVMs were observed when applying media conditioned by ESCs, suggesting that the electrophysiological changes were mediated by soluble factors. The increase in NP-Cx43 leads to gap junction uncoupling being a potential mechanism for these arrhythmogenic substrates. Further research into preventing NP-Cx43 in cultures is currently underway.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI) / Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine neurotransmitter that is synthesized from the amino acid L-tryptophan and is reported to localize in mitochondria of embryonic stem cells. Even before its role as a neurotransmitter in mature brain was discovered, 5-HT has been shown to play an important role in regulating brain development. However, there is a lack of knowledge about the downstream target genes regulated by serotonin in embryonic stem (ES) cells. Towards this end, our study helps in understanding transcriptional regulatory mechanisms of 5-HT responsive genes in ES cells. By combining the gene expression data with motif prediction algorithms, literature validation and comparison with public domain data, gene targets specific to endogenous or exogenous 5-HT in ES cells were identified. By performing one-way ANOVA, and volcano plots using GeneSpring software, we identified 44 5-HT induced and 29 5-HT suppressed genes, likely to be transcriptionally regulated by 4 & 2 TFs respectively. Motif enrichment analysis on these target genes using MotifScanner revealed that the transcription factor TFAP2A plays a key role in regulating the expression of 5-HT responsive genes. Furthermore, by comparing our dataset with published expression profiles of ES cells, we observed a number of 5-HT responsive target genes showing enrichment in ES cells. Genes such as Nanog, Slc38a5, Hoxb1 and Eif2s1 from this analysis have been observed to be components of ‘stemness’ phenotypes reported in literature. Functional annotation of the 5-HT responsive genes identified gene ontologies such as regulation of translation in response to stress and energy derivation by oxidation, suggesting a regulatory role for 5-HT in mitochondrial functions of ES cells. Additionally, enrichment of other biological process terms such as development of various parts of nervous system, cell adhesion, and apoptosis suggests that 5-HT target genes may play an important role in ES cell differentiation. Our study implemented a new combinatorial approach for identifying gene regulatory mechanisms involved in 5-HT responsive genes and proposed potential mediatory role for serotonin in ES cell differentiation and growth. Thus, this study provides potential 5-HT target genes in ES cells for biological validation.

Thesis (DPhil (Philosophy))--University of Stellenbosch, 2007. / Should surplus embryos which are destined to be discarded be protected at all cost, to the extent that they cannot contribute to medical knowledge - knowledge which could benefit society at large? Are embryos people or merely items of property? Different moral theories address these questions in different ways. Deontologists argue that the end never justifies the means and that the right not to be killed is more fundamental than the obligation to save. Utilitarians, on the other hand, argue that certain criteria should be met before moral significance can be contributed to an entity. The question of the moral status of the embryo is, as my discussion will show, one of the most widely discussed issues in the history of bioethics. Extensive literature exists on the topic. This study holds that an Ethics of Responsibility (ER) should by applied when answering the questions posed above as it encourages one to accept responsibility for the choices or decisions made and to defend them accordingly. I have endeavoured to answer the question of the personhood and rights of the embryo within the framework of the Ethics of Responsibility. Although these concepts overlap in many ways they remain central to the debate surrounding the sanctioning or prevention of the use of human embryonic stem cells in research. After identifying the micro-issues surrounding the human embryonic stem cell debate and explaining why both the deontologist and utilitarians fail to provide any adequate answers in this respect, I turn my attention to macro-issues such as safety concerns surrounding the usages and storage of stem cells. Commercialization, power issues, accessibility and the allocation of limited resources are also examined. Living in a society such as South Africa one cannot be blind to the inequalities of our health system. On a macro level I cannot but conclude that stem cell research does not seem to be a viable exercise within the South African context. South Africa faces a health care crisis far greater than the benefits stem cell research currently has to offer. However, the need still exists for a policy to guide future lawmakers who might need to address stem cell research and to guide decisions and actions. This brings me to my final chapter, namely proposing a morally justified policy for South Africa. I propose a policy which respects and values the autonomy of the progenitors’ choices (provided they have not been coerced) and which focuses on the beneficence of the greater society. Furthermore, it is paramount that the goal of any stem cell research should be for therapeutic use ONLY. Before commencing with the extraction of the stem cells, scientists should be obligated first to present convincing evidence that they have tried alternative ways to reach the same result. Once this has been proven, a regulatory body could issue the scientist/team with a license to undertake the specific research with a specific therapy as goal in order to prevent abuse. If they are found guilty of any unethical conduct their licenses should be revoked and an investigation launched.

Embryonic stem cells (ESCs) are a unique cell population that can differentiate into all three embryonic germ layers (endoderm, mesoderm, and ectoderm), rendering them an invaluable cell source for studying the molecular mechanisms of embryogenesis. Signaling molecules that direct tissue patterning during embryonic development are secreted by ESC aggregates, known as embryoid bodies (EBs). As many of these signaling proteins interact with the extracellular matrix (ECM), manipulation of the ESC extracellular environment provides a means to direct differentiation. ECM components, such as glycosaminoglycans (GAGs), play crucial roles in cell signaling and regulation of morphogen gradients during early development through binding and concentration of secreted growth factors. Thus, engineered biomaterials fabricated from highly sulfated GAGs, such as heparin, provide matrices for manipulation and efficient capture of ESC morphogens via reversible electrostatic and affinity interactions. Ultimately, biomaterials designed to efficiently capture and retain morphogenic factors offer an attractive platform to enhance the differentiation of ESCs toward defined cell types. The overall objective of this work was to examine the ability of microparticles synthesized from both synthetic and naturally-derived materials to enhance the local presentation of morphogens to direct ESC differentiation. The overall hypothesis was that microparticles that mimic the ECM can modulate ESC differentiation through sequestration of endogenous morphogens present within the EB microenvironment.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy