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.

Schwann cells are the glial cells of the peripheral nervous system. When transplanted into the injured central or peripheral nervous systems they promote repair. Traditionally Schwann cells have been isolated from the sciatic nerve, creating nerve-SC. An alternative Schwann cell source is from the differentiation of skin-derived precursors (SKPs), stem cells found in the skin, to Schwann cells (SKP-SC). SKP-SC have shown enhanced regenerative ability compared to nerve-SC. This study compares nerve-SC with SKP-SC at the functional and gene expression level to determine their degree of similarity and find their sources of variance. The functional ability of both Schwann cell types appeared similar. Their gene expression, as assessed by microarray, was similar but not identical. Genes that differed between nerve-SC and SKP-SC may represent differences important to regeneration. The similarity of SKP-SC to nerve-SC supports the use of SKP-SC for repair, and reasons for enhanced regeneration by SKP-SC are suggested.

In embryonic stem cells (ESCs) neural differentiation is elicited in the absence of extrinsic signaling in minimal conditions. This ‘default mechanism’ in ESCs produces neural stem cells termed primitive neural stem cells, which can subsequently yield FGF2-dependent definitive neural stem cells (dNSCs). We hypothesized that dNSCs have properties similar to neural stem/progenitor cells derived from the adult brain (aNPCs). The neural differentiation profile of the cell-types was characterized in vitro and in vivo following transplantation into the Shiverer mouse. The dNSCs produced a differentiation profile similar to that of aNPCs and both cell-types produced oligodendrocytes. This is the first demonstration of the in vivo differentiation of neural stem cells, derived from ESCs through the default mechanism, into the oligodendrocyte lineage. We conclude that dNSCs are a similar cell population to aNPCs. The default mechanism is a promising approach to generate neural stem cells and their progeny from pluripotent cell populations.

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.

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In this thesis, I describe studies identifying and characterizing two putative stem cell populations of the neural and pancreatic lineages. The mechanisms governing the emergence of the earliest mammalian neural cells during development and the ontogeny of neural stem cells remain incompletely characterized. A default mechanism has been suggested to underlie neural fate acquisition, however an instructive process has also been proposed. I utilized mouse ES cells to explore the fundamental issue of how an uncommitted, pluripotent mammalian cell will self-organize in the absence of extrinsic signals, and what cellular fate will result. Individual ES cells were found to rapidly transition directly into neural cells by a default mechanism, a process shown to be independent of suggested instructive factors. Further, I provide evidence that the default neural identity is that of a primitive neural stem cell, the earliest identified stem cell of the neural lineage. The exiguous conditions used to reveal the default state were found to present primitive neural stem cells with a survival challenge, which could be mitigated by survival factors or genetic interference with apoptosis. I also report the clonal identification of multipotent precursor cells, PMPs, from the adult mouse and human pancreas. These cells proliferate in vitro to form clonal colonies and display both pancreatic and neural cell multipotentiality. Importantly, the newly generated beta cells demonstrate glucose-dependent calcium responsiveness and regulated insulin release. PMP colonies do not express markers of embryonic stem cells, nor genes suggestive of mesodermal or neural crest origins. Moreover, genetic lineage-labeling experiments excluded the neural crest, and established the embryonic pancreatic lineage, as the developmental source of PMPs. The PMP cell was further found to express insulin in vivo, and insulin+ stem cells were shown to contribute to multiple pancreatic and neural cell populations in vivo. These findings demonstrate that the adult mammalian pancreas contains a population of insulin+ multipotent stem cells, capable of contributing to the pancreatic and neural lineages. In the final section of this thesis, I consider the relationships between neural and pancreatic tissues, as well as discussing the relevance of these two novel stem cell populations.

Cryopresevation is the process of cooling biological materials to low sub-zero temperatures for storage purposes. Numerous medical and technical applications, such as hematopoeitic stem cell transplantation and sperm banking, sometimes require the use of cryopreserved cells. Cryopreservation, however, can induce cell injury and reduce the yields of viable functional cells. Ice recrystallization is a mechanism of cryopreservation injury, but is rarely addressd in strategies to optimize cell cryopreservation. The results from this thesis demonstrate an association between the potency of carbohydrate-mediated ice recrystallization inhibition used in the cryopreservation of umbilical cord blood and recovery of viable non-apoptotic cells and hematopoietic progenitor function. Furthermore, increased numbers of apoptotic cells in hematopoeitic stem cell grafts were associated with reduced hematopoietic function and delayed hematopoietic recovery in patients undergoing blood stem cell transplantation. These findings provide a basis for pursuing further studies assessing ice recrystallization inhibition as a strategy for improving cell cryopreservation.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

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.

The conventional treatments for craniofacial bone defects currently are unsatisfactory due to several drawbacks. Replacement of lost bone by autografts typically causes donor site morbidity while allografts, xenografts, and demineralized bone matrix all have a chance of disease transmission. Current synthetic implants placed within the defect site generally lack osseointegration and biodegradability. There are several methods of generating a hybrid extracellular matrix (ECM) and synthetic material construct. These include coating the synthetic material scaffold with collagen and calcium phosphate, incorporating acellular biological tissue within the scaffold material, and using cells to generate an ECM coating on the synthetic material scaffold. The research performed for this thesis developed and characterized mesenchymal stem cell (MSC)-generated extracellular matrix poly(ε-caprolactone) constructs (PCL/ECM) for the replacement of bone tissue. The osteogenic potential of the PCL/ECM constructs was explored by culturing i) MSCs and ii) whole marrow cells combined with MSCs onto the construct with or without the osteogenic differentiation supplement, dexamethasone. It was established that the osteogenic differentiation of MSCs seeded onto ECM-containing constructs was maintained even in the absence of dexamethasone and that the co-culture of MSCs and whole bone marrow cells without dexamethasone and ECM enhances the proliferation of a cell population (or populations) present in the whole bone marrow. The osteogenicity of the constructs encouraged the characterization of the protein and mineral composition of the ECM coating on the PCL/ECM constructs. Characterization revealed that at short culture durations the MSCs used to generate the ECM deposited cellular adhesion proteins that are a prerequisite protein network for further bone formation. At the later culture durations, it was determined that the ECM was composed of collagen 1, hydroxyapatite, matrix remodeling proteins, and regulatory proteins. The prior studies on the PCL/ECM constructs persuaded exploration of the effect of various devitalization and demineralization processes on the retention of the ECM components within and the osteogenicity of the PCL/ECM constructs. Analysis demonstrated that the freeze-thaw technique is a milder method of devitalization of cell-generated ECM constructs as compared to other methods, but it reduced the osteogenicity of the constructs. In addition, it was elucidated that void spaces in the surface of the constructs are important for allowing access of MSCs into the interior of the constructs.