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.

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Univ., Diss.--Göteborg, 2005. / Literaturverz. S. [289] - 313.

Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 107-130).

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Biomedical Engineering." Includes bibliographical references (p. 53-55).

Embryonic stem (ES) cells provide a potentially unlimited cell source for cellular therapies; however, reliable methods must be developed to provide clinically-relevant numbers of homogeneous therapeutic cell populations. Dynamic cultures may encourage ES cell differentiation and amenable to large-scale cell production. Our goal was to optimize dynamic culture parameters (bioreactor type, speed, cell seeding density, conditioned medium, and hypoxia) to maximize the generation of hematopoietic stem and progenitor cells (HSPCs) from ES cells and also to investigate the ability of dynamic culture-derived HSPCs to generate terminally differentiated hematopoietic cells. Our results indicate that varying cell seeding density and speed in two different bioreactors significantly affects embryoid body formation and ES cell differentiation efficiency into progenitor cells. In general, increased cell seeding density generated higher percentages of HSPCs in both bioreactors. In addition, rotary (Synthecon) bioreactors produced more sca-1⁺ progenitors, and spinner flasks generated more c-kit⁺ progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that unique gene expression profiles were observed in the two bioreactors with the expression of specific hematopoietic genes more up regulated in the Synthecon cultures compared to spinner flasks. Combining bioreactor cultures with directed differentiation strategies via conditioned medium and hypoxic culture may further encourage hematopoietic differentiation. Dynamically cultured ES cell-derived hematopoietic stem and progenitor cells were further differentiated into a phenotype typical of dendritic cells which had the ability to process antigen. Additionally, microarray analysis of isolated ES cell-derived HSPCs demonstrated differences in the gene expression from native HSCs isolated from the fetal liver or bone marrow of mice. Insight gained from this work should be continued by comparing the differentiation efficiency of HSPCs derived in dynamic and traditional static culture methods into functional, terminally differentiated hematopoietic cells to generate clinically-relevant numbers of transplantable, therapeutic cells. / text

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy