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.

SFWT Zebrafish were exposed to various concentrations of EtOH at 24 hours post-fertilization for a period of two hours. When fish reached maturity they were placed in individual tanks in a larger open field. A preliminary strain comparison was also conducted using control (EtOH untreated) SFWT and AB fish. The behaviour of fish was recorded for 24 hours during a normal light:dark cycle. Motor patterns and general activity were quantified and analyzed and several behaviors were found to change significantly throughout the daytime and nighttime period. Also, fish exposed to the highest concentration of alcohol were found to exhibit significantly reduced amount of thrashing towards other subjects as compared to fish in the control group confirming previous results that demonstrated reduction of shoaling after early embryonic alcohol exposure.

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.

SFWT Zebrafish were exposed to various concentrations of EtOH at 24 hours post-fertilization for a period of two hours. When fish reached maturity they were placed in individual tanks in a larger open field. A preliminary strain comparison was also conducted using control (EtOH untreated) SFWT and AB fish. The behaviour of fish was recorded for 24 hours during a normal light:dark cycle. Motor patterns and general activity were quantified and analyzed and several behaviors were found to change significantly throughout the daytime and nighttime period. Also, fish exposed to the highest concentration of alcohol were found to exhibit significantly reduced amount of thrashing towards other subjects as compared to fish in the control group confirming previous results that demonstrated reduction of shoaling after early embryonic alcohol exposure.

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.

No description available.

Androgen receptor (AR) is a member of the steroid hormone receptor family of molecules, and expansion of a CAG repeat encoding polyglutamine (poly-Q) in AR gene are associated with a progressive neuromuscular disease known as spinal bulbar muscular atrophy (SBMA) or Kennedy disease. The hallmark of SBMA diseases is formation of juxtanuclear AR inclusions that have been termed ¡¥AR aggregates¡¦.Previous studies showed that transgenic mice overexpressing wild-type AR exclusively in the skeletal muscle fibers display similar abnormalities to those observed in models of SBMA disease. To elucidate the mechanisms underlying toxicity conferred by wild-type protein aggregation within normal cells, a mouse embryonic stem cell (ESC) model with non-genetic modified settings in AR overexpression was used to display the common features of polyglutamine disease in this experiment. It was found that wild-type AR proteins are highly expressed and form nuclear aggregate inclusions in response to androgen treatment in ES cells, the formation of AR aggregates inhibit the differentiation of embryonic bodys and enhanced caspase-3 activity in androgens -induced apoptosis. In addition, it was also investigated that relation between chaperones¡BAR and the endoplasmic reticulum (ER) stress-induced pathways in ES cells in this study, and it was found that chaperones could colocalize with AR aggregates, these findings may help us to better understand the roles of the chaperones on AR aggregates.