published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

abstract / Medicine / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--George Mason University, 2007. / Title from PDF t.p. (viewed Jan. 17, 2008). Thesis director: Richard E. Rubenstein. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Conflict Analysis and Resolution. Vita: p. 228. Includes bibliographical references (p. 222-227). Also available in print.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2010. / Härtill 4 uppsatser.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

The prognosis for patients diagnosed with glioma has changed little over the past two decades. Many therapies that appeared promising in preclinical studies have been unsuccessful in the clinic. In an attempt to address this problem I developed a method for the efficient derivation of glioma primary cultures from fresh human brain tumours. These cultures are enriched for putative cancer stem-like cells that are thought to be responsible for glioma initiation, therapy resistance and recurrence. This mechanism of tumour development is a departure from the traditional multistep model of cancer. It is hoped that preclinical models incorporating glioma stem-like cells will more effectively recapitulate the biology of human disease and so better predict the likely clinical efficacy of inhibitor compounds tested in vitro and in the preclinical setting. In contrast to the majority of the existing literature, I identified two distinct tumourderived glioma stem-like cell phenotypes in my primary cultures that I have called ‘branched’ and ‘flat.’ The branched cells had similarities to the radial glia-like cells previously described in glioma stem-like cultures. In contrast, the flat cells had mesenchymal-like features. I discuss the implications of these observations for understanding glioma cell biology. I describe the development of high content phenotypic assays that incorporate these putative glioma stem-like cells. I screened inhibitor compounds of the PI3 kinase pathway, which is important in glioma cell behaviour, and identified that PIK75, a drug that targets the p110α catalytic subunit of PI3 kinase, inhibited growth of all the primary cells tested. I examined PIK75 activity in some detail. In vivo models of glioma are used to validate the findings of in vitro compound screening, so I describe my attempt to develop a novel genetically engineered mouse model designed to initiate glioma formation from the glioma stem-like cell. Surprisingly, these mice actually developed malignant peripheral nerve sheath tumours and that gave me a novel insight into the pathogenesis of this rare disease. This also informed future work on my long-term goal of generating a genetic model of glioma that recapitulates human disease.

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Embryonic stem cells are defined by two key characteristics; apparently symmetrical self-renewing cell division and the ability to differentiate into cells of all three germ layers. Self-renewal depends on several extrinsic and intrinsic cues including a gene regulatory network centered around Oct4, Sox2 and Nanog that has been hypothesized to be reinforced by positive reciprocal interactions. Studies measuring Nanog expression by fluorescent reporters and immunoflourescence have shown that some undifferentiated Oct4 positive cells do not express Nanog (Chambers et al., 2007). However, the mechanisms responsible for generating this heterogeneity in Nanog expression are unknown. Here I show that Oct4 heterozygote ES cells lack Nanog-negative cells. Consistent with a model in which ES cell differentiation proceeds effectively through Nanog-negative cells, these Oct4 heterozygotes are retarded in their differentiation kinetics. Importantly, restoring Oct4 levels towards wild type reestablished both heterogenous Nanog expression and rapid differentiation. Analysis of ES cells carrying a mutation in the Oct4 binding site in the proximal Nanog promoter showed that Oct4 acts as a positive activator on the endogenous Nanog. Finally, comparison of gene expression in Nanog expressing and Nanog non-expressing ES cells has identified candidate genes that may be responsible for the switch in Nanog expression.

Mesenchymal stem cells are multipotent cells that have the ability to differentiate down multiple lineages as well as secrete trophic and anti-inflammatory factors. These qualities make MSCs a promising cell source for cell-based therapies to treat a variety of injuries and pathologies. Biomaterials are often used to control and direct stem cell behavior by engineering a desired environment around the cells. Recent research has focused on using the naturally derived sulfated glycosaminoglycan (GAG), heparin as a biomaterial due to its negative charge and ability to sequester and bind positively charged growth factors. Engineering a heparin coating that can mimic the native heparan sulfate proteoglycan structure found at cell surfaces can be used as a novel platform to present GAGs to cells to direct cell behavior. The overall goal of this dissertation was to develop GAG-based coatings on MSC spheroids in order to study the role of heparin and its derivatives on MSC culture applications. To investigate the role of heparin in coating form on MSC behavior, the ability of the coating to sequester positively charged growth factors was characterized. Given the role of sulfation in the negative charge density of heparin and growth factor interactions, a desulfated heparin coating was develop and used to examine how presentation of coatings with native and no sulfation levels could potentiate response to growth factors in the surrounding environment. Additionally, heparin and growth factor binding in coating presentation was explored to develop a novel platform to assemble MSC-based microtissues. Together these studies provided valuable insight into a novel approach to direct cell behavior by engineering a coating that harnesses heparin interactions with the surrounding environment.