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1 / Connor MacCrimmon

Pregnancy succeeds because the fetal allograft survives in the presence of a fully functional maternal immune system. The placenta, especially its trophoblast, provides the initial barrier between the maternal and fetal environment and, due to their location, trophoblast cells could be expected to be immune-privileged. Yet in the ectopic sites tested thus far, trophoblast stem cell transplants have failed to show noticeable immune privilege and appear to lack physiological support. However in this study, portal vein injected green fluorescent protein-labeled trophoblast stem cells were able to survive for several months in the livers of allogeneic female (14/14), but not male (0/4), mice. Gonadectomy experiments revealed that this gender-dependent survival does not require the presence of ovarian hormones (4/4) but the absence of testicular factors (5/5). In contrast, similarly labeled allogeneic embryonic stem cells were reliably rejected (11/11); these same embryonic stem cells survived when mixed with unlabeled trophoblast stem cells (13/13). The protective effect offered by the trophoblast stem cells did not require any immunological similarity with the co-injected embryonic stem cells. Neither the trophoblast stem cells nor the co-injected embryonic stem cells gave rise to tumors during the study period. Thus, this study demonstrates that, provided a suitable location and hormonal context, ectopic trophoblast stem cells may exhibit and confer immune privilege. These findings suggest applications in cell and gene therapy as well as provide a new model for studying trophoblast physiology and immunology.

The interplay between extrinsic and intrinsic processes as they influence a cell’s behaviour is a perennial question in both cellular and developmental biology. In this thesis these two issues are examined in the context of adult stem cells, a somatic stem cell present in the adult murine brain and a germline stem cell present in the adult Drosophila melanogaster ovary. I find that both of these distinct cell types exhibit patterns of non-random chromatid segregation in which the stem cells retain chromosomes carrying the older DNA strands. This unusual behaviour seems to exclusively occur in the context of differentiation, when one cell remains a stem cell and the other goes on to differentiate. Following these studies, the effects of extrinsic processes are tested in adult murine stem cells. It is determined that such cells can only produce neural progeny regardless of their association with foreign environments. These results argue against the phenomenon of stem cell plasticity which is proposed in several other systems and seem to support a primarily intrinsic-centered view of stem cell behaviour. However, the role of adhesion mediating proteins is next studied in such cells to determine their requirement for specific environments. The results of these experiments suggest that adult murine neural stem cells require association with support cells expressing E-Cadherin. Because the loss of such association results in a loss of stem cell number, these data show that intrinsic processes are insufficient to account for all stem cell behaviour. Indeed, based on these data and the results of other studies, it is hypothesized that the extrinsic association of stem cells in these diverse systems determines their polarity and subsequent intrinsic processes that enable these to divide asymmetrically. The implications of this theory are discussed with a view to general biological issues, the proximate mechanisms underlying these phenomena and the ultimate reasons these occur.

The interplay between extrinsic and intrinsic processes as they influence a cell’s behaviour is a perennial question in both cellular and developmental biology. In this thesis these two issues are examined in the context of adult stem cells, a somatic stem cell present in the adult murine brain and a germline stem cell present in the adult Drosophila melanogaster ovary. I find that both of these distinct cell types exhibit patterns of non-random chromatid segregation in which the stem cells retain chromosomes carrying the older DNA strands. This unusual behaviour seems to exclusively occur in the context of differentiation, when one cell remains a stem cell and the other goes on to differentiate. Following these studies, the effects of extrinsic processes are tested in adult murine stem cells. It is determined that such cells can only produce neural progeny regardless of their association with foreign environments. These results argue against the phenomenon of stem cell plasticity which is proposed in several other systems and seem to support a primarily intrinsic-centered view of stem cell behaviour. However, the role of adhesion mediating proteins is next studied in such cells to determine their requirement for specific environments. The results of these experiments suggest that adult murine neural stem cells require association with support cells expressing E-Cadherin. Because the loss of such association results in a loss of stem cell number, these data show that intrinsic processes are insufficient to account for all stem cell behaviour. Indeed, based on these data and the results of other studies, it is hypothesized that the extrinsic association of stem cells in these diverse systems determines their polarity and subsequent intrinsic processes that enable these to divide asymmetrically. The implications of this theory are discussed with a view to general biological issues, the proximate mechanisms underlying these phenomena and the ultimate reasons these occur.

Pregnancy succeeds because the fetal allograft survives in the presence of a fully functional maternal immune system. The placenta, especially its trophoblast, provides the initial barrier between the maternal and fetal environment and, due to their location, trophoblast cells could be expected to be immune-privileged. Yet in the ectopic sites tested thus far, trophoblast stem cell transplants have failed to show noticeable immune privilege and appear to lack physiological support. However in this study, portal vein injected green fluorescent protein-labeled trophoblast stem cells were able to survive for several months in the livers of allogeneic female (14/14), but not male (0/4), mice. Gonadectomy experiments revealed that this gender-dependent survival does not require the presence of ovarian hormones (4/4) but the absence of testicular factors (5/5). In contrast, similarly labeled allogeneic embryonic stem cells were reliably rejected (11/11); these same embryonic stem cells survived when mixed with unlabeled trophoblast stem cells (13/13). The protective effect offered by the trophoblast stem cells did not require any immunological similarity with the co-injected embryonic stem cells. Neither the trophoblast stem cells nor the co-injected embryonic stem cells gave rise to tumors during the study period. Thus, this study demonstrates that, provided a suitable location and hormonal context, ectopic trophoblast stem cells may exhibit and confer immune privilege. These findings suggest applications in cell and gene therapy as well as provide a new model for studying trophoblast physiology and immunology.

This thesis explores the unequal partitioning of damaged proteins during mitosis and its implications for cell fate. Initially described in unicellular organisms, it was unclear if this method was used in vivo in multicellular organisms and had functional consequences in mammalian cells. To determine if this asymmetry was conserved in multicellular organisms, I studied three stem/progenitor populations in Drosophila: the larval neuroblast, adult female germline stem cell, and adult intestinal stem cell. Each cell type was found to asymmetrically segregate damaged proteins identified by the 2,4-hydroxynonenal (HNE) modification, which are associated with oxidative stress and age. Both the larval neuroblast and female germline stem cell were found to retain damaged proteins during division, whereas the intestinal stem cell segregated damaged proteins to differentiating progeny. I suggest that functional lifespan, and not cell type, determines the cell that receives the majority of damaged proteins during division. In each cell type, damaged proteins were associated with DE-Cadherin, a common component of the stem cell niche and removal from the niche was associated with reduced damaged protein polarization. Interestingly, when larval neuroblasts were mechanically dissociated from their niche and placed in culture, the internal polarization of damaged proteins was found to increase with progression through the cell-cycle. Therefore, I suggest that both the niche and intrinsic factors play a role in the asymmetric division of damaged proteins. To determine if an asymmetric division of damaged proteins influenced cell fate, I used a mammalian cell line with inducible expression of misfolded Huntingtin, which shares similar properties to damaged proteins. This study also revealed that the conformation of damaged proteins impacts cell fate: cells with diffuse Huntingtin displayed greater proliferation and reduced resistance to stress. Tracking cells containing an aggregate with live-imaging revealed that the cell that inherits the aggregate has a longer cell-cycle and an enhanced capacity to differentiate. Therefore, the asymmetric inheritance of damaged proteins impacts cell fate. In the final chapter of this thesis, I discuss the implications of an asymmetric division of damaged proteins on cell fate and how this information can be applied to cancer treatments.

This thesis explores the unequal partitioning of damaged proteins during mitosis and its implications for cell fate. Initially described in unicellular organisms, it was unclear if this method was used in vivo in multicellular organisms and had functional consequences in mammalian cells. To determine if this asymmetry was conserved in multicellular organisms, I studied three stem/progenitor populations in Drosophila: the larval neuroblast, adult female germline stem cell, and adult intestinal stem cell. Each cell type was found to asymmetrically segregate damaged proteins identified by the 2,4-hydroxynonenal (HNE) modification, which are associated with oxidative stress and age. Both the larval neuroblast and female germline stem cell were found to retain damaged proteins during division, whereas the intestinal stem cell segregated damaged proteins to differentiating progeny. I suggest that functional lifespan, and not cell type, determines the cell that receives the majority of damaged proteins during division. In each cell type, damaged proteins were associated with DE-Cadherin, a common component of the stem cell niche and removal from the niche was associated with reduced damaged protein polarization. Interestingly, when larval neuroblasts were mechanically dissociated from their niche and placed in culture, the internal polarization of damaged proteins was found to increase with progression through the cell-cycle. Therefore, I suggest that both the niche and intrinsic factors play a role in the asymmetric division of damaged proteins. To determine if an asymmetric division of damaged proteins influenced cell fate, I used a mammalian cell line with inducible expression of misfolded Huntingtin, which shares similar properties to damaged proteins. This study also revealed that the conformation of damaged proteins impacts cell fate: cells with diffuse Huntingtin displayed greater proliferation and reduced resistance to stress. Tracking cells containing an aggregate with live-imaging revealed that the cell that inherits the aggregate has a longer cell-cycle and an enhanced capacity to differentiate. Therefore, the asymmetric inheritance of damaged proteins impacts cell fate. In the final chapter of this thesis, I discuss the implications of an asymmetric division of damaged proteins on cell fate and how this information can be applied to cancer treatments.

Despite the importance of inter-cellular (between cell) communication networks in regulating homeostasis in multicellular organisms, very little is known about their topology, dynamics, or functional significance. Inter-cellular communication networks are particularly relevant in stem cell biology, as stem cell fate decisions (self-renewal, proliferation, lineage specification) are tightly regulated based on physiological demand. Using human blood stem cell cultures as an experimental paradigm, we present an integrated experimental and computational approach to interrogate a hierarchically organized tissue network. We have developed a novel mathematical model of blood stem cell development incorporating cell-level kinetic parameters as functions of secreted molecule-mediated inter-cellular networks. By relation to quantitative cellular assays, our model is capable of predictively simulating many disparate features of both normal and malignant hematopoiesis, relating internal parameters and microenvironmental variables to measurable cell fate outcomes. Through integrated in silico and experimental analyses we show blood stem and progenitor cell fate is regulated by cell-cell feedback, and can be controlled non-cell autonomously by dynamically perturbing inter-cellular signalling. Furthermore, we have compiled genome-scale molecular profiles (transcriptome and secretome), publicly available databases, and literature mining to reconstruct soluble factor-mediated inter-cellular signalling networks regulating cell fate decisions. We find that dynamic interactions between positive and negative regulators, in the context of tuneable cell culture parameters, tip the balance between stem cell supportive vs. non-supportive conditions. The cell-cytokine interactions can be summarized as an antagonistic positive-negative feedback circuit wherein stem cell self-renewal is regulated by a balance of megakaryocyte-derived stimulatory factors vs. monocyte-derived inhibitory factors. To understand how the experimentally identified positive and negative regulatory signals are integrated at the intra-cellular level, we define a literature-derived blood stem cell self-renewal network wherein these extracellular signals converge for coherent processing into cell fate decisions. In summary, this work demonstrates the utility of integrating experimental and computational methods to explore complex cellular systems, and represents the first attempt to comprehensively elucidate non-autonomous signals balancing stem cell homeostasis and regeneration.