Glioblastoma (GBM) is a very aggressive and invasive tumour that relapses within nine months of diagnosis and remains incurable despite advances in multimodal therapy including surgical resection, chemotherapy and radiation. Poor patient outcome has been correlated to specific markers of brain tumour initiating cells (BTIC) and intratumoural heterogeneity (ITH), which have also been associated with treatment resistance and tumour recurrence. ITH can be explained at the cellular level by the existence of multiple populations of cancer cells, including some which have acquired stemness properties like self-renewal, proliferation, and multilineage differentiation, also known as cancer stem cells (CSCs). In brain tumours, CSCs or BTICs, have been shown to be resistant to both chemotherapy and radiation treatment, allowing them to escape therapy and consequently generate for tumour recurrence. As a result, therapies that focus on targeting the BTIC compartment within the bulk GBM tumour would provide better treatment and prognosis for patients. To profile GBM BTICs we conducted two transcriptomic screens. The first compared GBM BTICs to neural stem cells (NSCs), their healthy counterparts, and for the second we developed a pipeline utilizing a dynamic BTIC patient-derived xenograft (PDX) model of human GBM recurrence allowing for the profiling of GBM BTICs at engraftment, after chemoradiotherapy delivery in a phase we have termed "minimal residual disease" (MRD), and at tumour recurrence. In this study, Dickkopf-1 (DKK1) was identified as a potential therapeutic target for GBM from each transcriptomic screen and was studied using short hairpin knockdowns, blockade with monoclonal antibodies, and subsequent functional stem cell assays. / Thesis / Master of Science (MSc) / Glioblastoma (GBM) is a very aggressive tumour that relapses within nine months of diagnosis and remains incurable despite chemotherapy, radiation, and surgery. Relapse is believed to be caused by the presence of a wide variety of cell types, including cancer stem cells (CSCs), which have been shown to be resistant to both chemotherapy and radiation in GBM. As a result, therapies that focus on targeting the CSCs within the bulk GBM tumour would provide better treatment for patients. In this study, we analyzed this cell population by conducting two screens. The first compared the level at which genes are expressed in GBM CSCs in comparison to how they are expressed in their healthy counterparts, neural stem cells, whereas the second compared the primary patient GBM tumour to its relapsed form in a mouse model of the disease. In this study, the protein Dickkopf-1 (DKK1) was identified and validated as a potential therapeutic target of GBM using well established molecular and stem cell functional assays.

Hematopoiesis is the highly regenerative process of producing billions of blood cells each day, including white blood cells, red blood cells, and platelets. Given the relatively short life span of these mature cells, hematopoiesis is dependent on stem and progenitor cells to generate renewed progeny, which represents a tightly regulated process. This includes cell intrinsic and external factors, and where dysregulation can lead to anemia and cancer. As such, the hematopoietic hierarchy has been intensely studied for nearly a century and represents a gold standard model of cell fate and developmental biology, in research and clinical applications. Cellular models, such as in vitro culture and human-mouse xenografts in vivo, have been developed to explain complex phenomena pertaining to hematopoiesis and also interrogate processes which are too invasive to study in humans. Hematopoietic generation is required beyond sustaining homeostasis, and progenitors can be damaged through cytotoxic injuries such as radiation and standard chemotherapy, and also undergo leukemic transformation. There are two main treatment modalities for leukemia patients (a) receiving a stem cell transplant, and (b) drug or radiation-based therapy. In the former, shortages of donors and stem cells has remained an unmet clinical need for decades. In the latter, selective targeting of genetic mutations has become a successful standard-of-care in leukemias such as chronic myelogenous leukemia and acute promyelocytic leukemia. However, in the most common adult hematologic malignancy, chronic lymphocytic leukemia (CLL), similar targeting therapies have not been developed. Altogether, shortages of stem cells from healthy donors, chemotherapy-induced immune dysfunction, and a lack of targeted therapies, all reinforce the immediate need for innovative cellular models to address these clinical problems. To generate additional sources of human hematopoietic progenitors for laboratory study, human PSCs have been used. Unlike hematopoietic progenitor cells collected from healthy and leukemic donors, human pluripotent stem cells (PSC) can be easily propagated and expanded in vitro. PSCs can generate hematopoietic progenitor cells, but they remain poorly understood and have not been robustly applied to solve the aforementioned deficiencies related to patient treatment. Importantly, the biological regulation of both hematopoiesis and PSCs has been experimentally confirmed to significantly deviate between humans and other animals, such as mice, further reinforcing the importance of human-specific cell models of hematopoiesis. Therefore, I hypothesized that human stem cell models provide a focused approach to interrogate the regulation of hematopoiesis from the apex of the hierarchy, which can be used to understand the promotion of healthy hematopoiesis and understand malignant transformation. Collectively, the data presented within this thesis offer a deeper conceptualization of human stem cell models and the deconvolution of several complex components of hematopoietic regulation. This work has revealed novel, clinically relevant, and actionable targets to ultimately enable the promotion of healthy hematopoiesis on multiple fronts. / Thesis / Doctor of Philosophy (PhD) / This thesis presents research on novel molecular and genetic regulatory pathways of self-renewal and differentiation in models of healthy and malignant human hematopoiesis. The origin of healthy hematopoietic regulation stems from a large body of work spanning decades and encompasses many efforts by others to derive hematopoietic stem cells from human pluripotent cells. The development of a genetic model for the malignant regulation of CLL was truly serendipitous, was propelled through robust and intriguing results that begged for further exploration, and filled a clinical gap in identifying actionable targets in CLL. Lastly, these two projects, along with my supportive roles in other published works throughout my graduate studies, instructed me to develop a human-mouse transplant model to uncover the biology of regenerating healthy hematopoiesis during injury.