The transition from pluripotency to differentiation is a key event in the life of all complex multicellular organisms. In the development of the mouse, the pluripotent epiblast undergoes gastrulation and gives rise to three multipotent germ layers, which will in turn form the tissues of the adult body. The events leading up to gastrulation have been extensively studied in vivo in developing embryos, and modelled in vitro making use of embryonic stem (ES) cells. Bone morphogenic protein (BMP) signalling plays a key role in these processes. BMP can in fact maintain ES cells in a self-renewing state by inhibiting their differentiation into neural ectoderm, whilst at the same time being required for the specification of mesoderm in the developing embryo (Winnier et al. 1995, Ying et al. 2003a). A key intracellular target of BMP is the transcriptional regulator Id1, which can recapitulate the effects of BMP in the preservation of ES cell pluripotency and in the inhibition of neural specification from pluripotent cells (Ying et al. 2003a). This thesis will focus on understanding the roles of this molecule in the early decisions affecting the transition from pluripotency to differentiation. In particular, I aim to study the expression pattern of Id1 in cultures of pluripotent cells, and to clarify which extracellular and intracellular molecules regulate the expression of the factor; I aim to understand how forced Id1 expression inhibits the differentiation of pluripotent cells, and whether Id1 may play a similar role in the regulation of the asynchronous exit from pluripotency observed in differentiating wild-type cells; finally, I aim to characterise the expression pattern of Id1 in the early stages of post-implantation development at the single-cell resolution, and to understand how the expression of the molecule correlates with the previously characterised expression patterns of key signalling molecules and transcription factors. The generation of a reporter ES cell line expressing the yellow fluorescent protein Venus fused to the C-terminus of Id1 allowed me to assess the expression of the factor in culture on a single-cell basis, making use of immunofluorescence and flow cytometry. I observed that expression of Id1 is reliant on active BMP signalling and low Activin/Nodal signalling, and I characterised the combinatory effects of the two pathways on Id1 expression. Furthermore, I demonstrated that high Nanog expression is incompatible with high Id1 expression in ES cell cultured in the presence of LIF and serum, which raises the possibility that Nanog may be affecting the expression of Id1 in vivo, both in pre-implantation and in post-implantation embryos. I generated ES cell lines overexpressing Id1 and observed that the factor inhibits differentiation of pluripotent cells into neural ectoderm by delaying their exit from a post-implantation epiblast-like pluripotent state, and ultimately favouring mesodermal specification. This suggests that Id1 is acting at a specific stage of differentiation and that the differentiation process itself is following a similar developmental pathway to what is observed in the peri-gastrulation stage embryo. I performed single-cell transcriptional analysis on differentiating wild-type ES cells and observed that Id1 is not expressed at an appropriate point in time to affect the asynchronous the exit from pluripotency observed in neural adherent monolayer differentiation, which suggests that other factors must be responsible for this phenomenon. Finally, I addressed the expression pattern of Id1 protein in the embryonic tissue of gastrulating mouse embryos by imaging chimaeric embryos generated using the Id1- Venus reporter ES cells. I observed that Id1 is expressed in the proximal regions of streak stage embryos; in the epiblast and migrating mesendoderm of bud stage embryos; in cardiac, lateral and allantoic mesoderm and in foregut endoderm in headfold stage embryos. These expression patterns fit with the reported expression of BMP molecules at these stages of development, and suggest that Id1 expression is primarily dependent on BMP expression in early post-implantation embryos. However, I also observed Id1 expression in a ring of cells surrounding the node in headfold stage embryos, a previously uncharacterised expression pattern not directly attributable to BMP expression. This raises the intriguing question of what is regulating Id1 expression and what roles Id1 may be playing in this key embryonic structure.

Abstract not available

Bibliography: leaves 113-126. / v, 126, [113] leaves, [10] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Studies the biological significance of the mouse Listone variant H2A.Z. Describes the isolation and characterisation of H2A.Z genomic clones from different mouse genomic libraries; H2A.Z gene targeting in mouse E14 embryonic stem cells; and an attempt to generatae ES cell lines and mice which lack the functional H2A.Z protein to investigate H2A.Z function in vitro and in vivo. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1995?

No description available.

Embryonic stem cells are pluripotent cells isolated from morula stage embryos or the inner cell mass of blastocyst stage embryos. They are capable of differentiating into tissues of all three primary germ layers. In recent years pluripotent cell lines have been created from somatic cell types using various methods, the primary method being viral transduction of exogenous Oct4, Sox2, Klf4, and c-Myc or Oct4, Sox2, Nanog, and Lin28 transgene constructs. The resulting cell lines are termed induced pluripotency stem cells, and are similar to embryonic stem cells in many ways. However, these cell lines are not acceptable for clinical applications due to the use of both modified viral vectors and insertion of exogenous transgenes in their production. Recently the small molecule RepSox, a TGF-ß pathway inhibitor, was used to replace Sox2 during cellular reprogramming of murine embryonic fibroblasts. We evaluated the effects of RepSox on expression of pathways related to pluripotency in murine embryonic fibroblast, murine embryonic stem, and bovine embryonic fibroblast cells. Each cell type was treated with RepSox for 72 hours and subjected to standard qPCR for gene expression analysis. PCR arrays specific to stem cell pathways were used to initially evaluate the effects of RepSox on candidate genes. A subset of genes was then selected for further analysis based on these initial results. We report that RepSox inhibition of the TGF-ß pathway in murine embryonic fibroblasts results in significant upregulation of components of the Wnt, Notch, and Hedgehog signaling pathways, all of which have been linked to stem cell maintenance. In addition, we observed significant upregulation of genes associated with embryonic, mesenchymal, stem cell, and neural cell lineages, indicating that RepSox may be useful in direct reprogramming of murine cells to other somatic cell types. RepSox treatment of murine embryonic stem cells did not result in consistent upregulation of Wnt, Notch, or Hedgehog pathway components, but did result in upregulation of Sox2 and Klf4 expression. Lastly, RepSox treatment of bovine embryonic fibroblasts did not result in the same effects as seen in murine fibroblasts, indicating a need for further analysis to determine the effects of RepSox on bovine cells.

SirT1 is a class III histone deacetylase that has beneficial roles in various diseases related to aging such as cancer, diabetes and neurodegenerative disease. Resveratrol is a natural compound that mimics most of the beneficial effects attributed to SirT1. Resveratrol has toxicity towards cancer cells and has been reported to be a direct activator of SirT1. Interestingly, SirT1 over-expression has also been reported to be toxic. We set out to determine if resveratrol toxicity is mediated through activation of SirT1. We have assessed resveratrol toxicity in embryonic stem cells and mouse embryonic fibroblast (MEFs) across different SirT1 genotypes. Our data indicates that SirT1 is not implicated in resveratrol toxicity in either normal or transformed MEFs. Thus, resveratrol toxicity does not appear to be mediated by SirT1.

Embryonic stem cells (ESC) possess immune privileged properties and have the capacity to modulate immune activation. ESCs can persist across allogeneic immunological barriers, prevent lymphocyte proliferation in mixed lymphocyte reaction (MLR) assays and can promote graft acceptance. However, clinical application of live ESC to treat immunological disorders is not feasible as live ESC can form teratoma in-vivo. In order to harness these properties of ESCs without adverse risk to patients, we hypothesized that ESC derived extracts may retain immune modulatory properties of whole cells and therefore could be used to abrogate allo-immune responses. We found addition of ESC-extracts from human lines H1 and H9, significantly prevented T cell proliferation in allogeneic MLRs. These results were confirmed using murine J1 ESC line. In-vitro studies showed human ESC EXT were able to modulate maturation of human monocyte derived dendritic cells (DC) by suppressing up-regulation of important co-stimulatory and maturation markers CD80, HLA-DR and CD83. In addition, DCs educated in the presence of human ESC extracts significantly lost their ability to stimulate purified allogeneic T cells compared to control extract treated DCs. We also determined that ESC extracts have an independent effect on T cells. ESC extracts prevented T cell proliferation in response to anti CD3/CD28 stimulation. In MLRs, ESC derived factors significantly down-regulated IL-2 and IFN-γ expression, while up-regulating TGF-β and Foxp3 expression. Furthermore, lymphocytes and purified T cells activated with anti-CD3/CD28, ConA and PMA proliferated poorly in the presence of ESC derived factors, while proliferation in response to ionomycin was not affected. Western blot analysis indicated that ESC derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. Therefore, ESC extracts have potent immune suppressive properties and may have clinical applications in ameliorating transplant rejection and autoimmune conditions.

Mouse embryonic stem cells (mESCs) are pluripotent cells capable of differentiating into all three germ layers present in the adult mouse. In this thesis, I have investigated the transcriptional changes that mESCs undergo as they are induced to differentiate towards the mesoderm lineage by 2i/LIF withdrawal and dimethyl sulfoxide (DMSO) treatment. 5 days of differentiation causes significant drops in expression of Sox2 and Oct4 primary transcript, while expression of Nanog and Kit significantly drops after only 1 day. It was determined that DMSO has no effect on the short-term changes in Nanog and Kit expression induced by 2i/LIF withdrawal. An expanded look at pluripotency-associated genes shows significant up-regulation of Oct4 and down-regulation of Klf4 and Stat3 following only 6 hours of 2i/LIF withdrawal. This data indicates that while some aspects of the transcriptional networks underlying pluripotency respond quickly to mesodermal differentiation cues, others remain unchanged for up to 5 days.

Mouse embryonic stem cells (mESCs) are pluripotent cells capable of differentiating into all three germ layers present in the adult mouse. In this thesis, I have investigated the transcriptional changes that mESCs undergo as they are induced to differentiate towards the mesoderm lineage by 2i/LIF withdrawal and dimethyl sulfoxide (DMSO) treatment. 5 days of differentiation causes significant drops in expression of Sox2 and Oct4 primary transcript, while expression of Nanog and Kit significantly drops after only 1 day. It was determined that DMSO has no effect on the short-term changes in Nanog and Kit expression induced by 2i/LIF withdrawal. An expanded look at pluripotency-associated genes shows significant up-regulation of Oct4 and down-regulation of Klf4 and Stat3 following only 6 hours of 2i/LIF withdrawal. This data indicates that while some aspects of the transcriptional networks underlying pluripotency respond quickly to mesodermal differentiation cues, others remain unchanged for up to 5 days.

SirT1 is a class III histone deacetylase that has beneficial roles in various diseases related to aging such as cancer, diabetes and neurodegenerative disease. Resveratrol is a natural compound that mimics most of the beneficial effects attributed to SirT1. Resveratrol has toxicity towards cancer cells and has been reported to be a direct activator of SirT1. Interestingly, SirT1 over-expression has also been reported to be toxic. We set out to determine if resveratrol toxicity is mediated through activation of SirT1. We have assessed resveratrol toxicity in embryonic stem cells and mouse embryonic fibroblast (MEFs) across different SirT1 genotypes. Our data indicates that SirT1 is not implicated in resveratrol toxicity in either normal or transformed MEFs. Thus, resveratrol toxicity does not appear to be mediated by SirT1.