Embryonic stem cells (ESCs) represent a promising source of cells for cell replacement therapy in the context of muscle diseases; however, before ESC-based cell therapy can be translated to the clinic, we must learn to modulate cell-fate decisions in order to maximize the yield of myocytes from this systems. In order to gain a better understanding of the myogenic cell fate, we sought to define the molecular mechanisms underlying the specification and differentiation of ESCs into cardiac and skeletal muscle. More specifically, the central hypothesis of the thesis is that myogenic signalling cascades modulate cell fate via regulation of transcription factors. Retinoic acid (RA) is known to promote skeletal myogenesis, however the molecular basis for this remains unknown. We showed that RA expands the premyogenic progenitor population in mouse stem cells by directly activating pro-myogenic transcription factors such as Pax3 and Meox1. RA also acts indirectly by activating the pro-myogenic Wnt signalling cascade while simultaneously inhibiting the anti-myogenic influence of BMP4. This ultimately resulted in a significant enhancement of skeletal myogenesis. Furthermore, we showed that this effect was conserved in human embryonic stem cells, with implications for directed differentiation and cell therapy. The regulation of cardiomyogenesis by the Wnt pathway was also investigated. We identified a novel interaction between the cardiomyogenic transcription factor Nkx2.5 and the myosin phosphatase (MP) enzyme complex. Interaction with MP resulted in exclusion of Nkx2.5 from the nucleus and inhibition of its transcriptional activity. Finally, we showed that this interaction was modulated by phosphorylation of the Mypt1 subunit of MP by ROCK, downstream of Wnt3a. Treatment of differentiating mouse ESCs with Wnt3a resulted in exclusion of Nkx2.5 from the nucleus and a subsequent failure to undergo terminal differentiation into cardiomyocytes. This likely represents part of the molecular basis for Wnt-mediated inhibition of terminal differentiation of cardiomyocytes. Taken together, our results provide novel insight into the relationship between myogenic signalling cascades and downstream transcription factors and into how they function together to orchestrate the myogenic cell fate in stem cells.

A complex network of transcription factors, which are regulated by signalling molecules, is responsible in coordinating the formation of differentiated skeletal and cardiac myocytes from undifferentiated stem cells. The present study aims to understand and compare the transcriptional regulation of skeletal and/or cardiac muscle development in the absence of Sox7 or in the presence of a collagen-based matrix in P19 embyonal carcinoma (EC) and mouse embryonic stem (ES) cells. First, knock-down of Sox7 , by shRNA, in muscle inducing conditions (+DMSO) and in the absence of RA (-RA), decreased muscle progenitor transcription factor and myogenic regulatory factor (MRF) levels, suggesting that Sox7 is necessary for myogenesis. However, knock-down of Sox7 in the presence of RA (+RA) and DMSO increased expression of muscle progenitor markers and MRFs, suggesting that Sox7 is inhibitory for myogenesis +RA. Furthermore, Sox7 overexpression enhanced myogenesis -RA, but inhibited myogenesis and enhanced neurogenesis +RA. These results suggest an important interplay between RA signalling and Sox7 function during P19 differentiation. Second, Q-PCR analysis showed that compared to the mouse ES cells differentiated on the regular TC plates, differentiation on the collagen matrices had a higher expression of skeletal and cardiac precursors, MRFs and terminal differentiation markers. Collagen alone enhanced myotube formation. The enhanced collagen matrix, containing the oligosaccharide sialyl LewisX (sLeX), specifically enhanced cardiomyogenesis. These studies have added to our understanding of the transcriptional regulation of premyogenic mesoderm factors and the role of Sox7 in this process. In addition these studies provide a vision for possible use of biomaterials in directed differentiation of stem cells for the purpose of cell therapy.

Insulin-like growth factor (IGF-1) is a potent pro-survival cytokine that is not robustly expressed by human cardiac stem cells (CSCs). Here, we explore the mechanism underlying IGF-1 enhanced cardiac repair by CSCs. Human CSCs underwent lentiviral- mediated somatic gene transfer of IGF-1 to boost cytokine secretion without adversely blunting the overall cytokine signature of CSCs. In vitro studies demonstrated that IGF-1 provided paracrine and autocrine support that reduced apoptosis by CSCs and cardiomyoctes. In vivo experiments demonstrated that IGF-1 increased CSC-mediated cardiac repair by enhancing salvage of reversibly damaged myocardium and transplanted cell survival.

Despite advances in treatment, heart failure remains one of the top killers in Canada. This recognition motivates a new research focus to harness the fundamental repair properties of the human heart, with human cardiac stem cells (CSCs) emerging as a promising cell candidate to regenerate damaged myocardium. The rationale of this approach is simple with ex vivo amplification of CSCs from clinical grade biopsies, followed by delivery to areas of injury, where they engraft and regenerate the heart. Currently, outcomes are limited by modest engraftment and poor long-term survival of the injected CSCs due to on-going cell loss during transplantation. As such, we explored the effect of cell encapsulation to increase CSC engraftment and survival after myocardial injection. Transcript and protein profiling of human atrial appendage sourced CSCs revealed strong expression the pro-survival integrin dimers αVβ3 and α5β1- thus rationalizing the integration of fibronectin and fibrinogen into a supportive intra-capsular matrix. Encapsulation maintained CSC viability and expression of pro-survival transcripts when compared to standard suspended CSCs. Media conditioned by encapsulated CSCs demonstrated superior production of pro-angiogenic/ cardioprotective cytokines, angiogenesis and recruitment of circulating angiogenic cells. Intra-myocardial injection of encapsulated CSCs after experimental myocardial infarction favorably affected long-term retention of CSCs, reduced scar burden and improved overall cardiac function. Taken together, cell encapsulation of CSCs prevents detachment induced cell death while boosting the mechanical retention of CSCs to enhance repair of damaged myocardium.

Several studies have proposed the fallopian tube epithelium as a site of origin of ovarian cancer. The discovery of precursor lesions in the fallopian tube in patients at risk for ovarian cancer supports a probable origin for high-grade serous ovarian carcinoma in this tissue. While the fallopian tube epithelium consists of three distinct cell types, the paired box protein 2 (PAX2) positive cells and potentially the CD44 positive stem-like cells are most relevant to ovarian cancer. Loss of PAX2 expression in the fallopian tube cells is considered to be an early event in epithelial transformation, but the specific role of PAX2 in this transition is unknown. The aim of this study was to define the role of PAX2 in oviductal epithelial cells (OVE) cells and in mouse ovarian surface epithelial cells (MOSE), and to understand its contribution to the formation of serous precursor lesions in the fallopian tubes. Herein, we studied the OVE response to transforming growth factor β (TGFβ, a cytokine found in follicular fluid) and provide evidence of its potential involvement in the regulation of stem cell-like behaviors that may contribute to formation of cancer-initiating cells. Treatment of primary cultures of OVE cells with TGFβ at concentrations found in ovulatory follicular fluid induced an epithelial-mesenchymal transition (EMT) with expected changes in proliferation, cell morphology and expression of SNAIL, Vimentin and E-cadherin. EMT was also associated with decreased expression of PAX2 and an increase in the fraction of cells expressing CD44. Pax2 knockdown in OVE cells and overexpression in ovarian epithelial cells confirmed that PAX2 inhibits CD44 expression and regulates the degree of epithelial differentiation of OVE cells. These results suggest that the loss of PAX2 seen in serous tubal intraepithelial carcinomas (STIC) leads to a shift to a more mesenchymal phenotype associated with stem-like features. Pax2 overexpression in MOSE cells also induced the formation of vascular channels both in vitro and in vivo, which indicate a possible contribution of PAX2 to ovarian cancer progression by increasing the vascular channels to supply nutrients to the tumor cells. Furthermore, since loss of PAX2 in STIC was found associated with P53 and BRCA1 mutations, OVE cells with mutations of the tumor suppressor genes Trp53 and Brca1 were studied. We found that loss of Trp53 with or without loss of Brca1 increased cell proliferation and colony formation in vitro. In addition, loss of Trp53 induced OVE cells to undergo EMT and induced the expression of stem cell–associated genes. We therefore suggest a potential contribution of stem cells in initiating the precursor lesions in the fallopian tubes in combination with tumor suppressor gene mutation.

Cultures of peripheral blood mononuclear cells (MNCs) give rise to at least two different variants of endothelial progenitor cells (EPCs), early and late outgrowth EPCs. We investigated whether microRNAs in early and late EPCs could serve as markers of internal processes that can be exploited to distinguish cell identity and functional capacity. We hypothesized that as MNCs give rise to early and late EPCs, there is a gradual change in total microRNA profile, reflecting a total change in processes within the predominant cell population. Using a candidate microRNA array, early and late EPCs showed vastly different microRNA expression profiles. MiR-146a expression increased progressively as early EPCs emerged around 5-7 days (p<0.05). Through targeting TRAF6 and IRAK1, miR-146a conferred inflammatory tolerance in early EPCs, likely contributing to their purported ability to suppress inflammation. MiR-146a knock down (KD) in endotoxin-stimulated early EPCs reduced anti-inflammatory cytokine IL-1RA (p<0.001), and increased expression of pro-inflammatory cytokines IL-1 (p<0.001) and IL-8 (p<0.01). Interestingly, the microRNA expression profile of late EPCs was highly congruent to mature endothelial cells, with 100-fold greater miR-126 expression than monocytes and early EPCs (p<0.01). MiR-126KD in late EPCs abolished matrigel-network formation (p<0.05); while overexpression (OE) in early EPC augmented network formation (p<0.05) and chemotactic migration (p<0.001). We also found that the melanoma cell adhesion molecule or MCAM (CD146) identified late EPC precursors. Only MCAM+MNCs from adult blood (<5% of total MNCs) yielded late EPC-like colonies. Robust miR-126 expression in these cells predicted the generation of late EPCs. Overall, our results suggest that miR-146a in early EPCs likely contributes to repair by suppressing inflammation during cardiovascular injury; while in late EPCs, miR-126 directly promotes angiogenesis and vascular repair. Finally, we highlight a unique method for the efficient generation of late EPCs by using MCAM selection and screening for miR-126.

The unique pluripotent character of human embryonic stem cells (hESCs) places them in the forefront of scientific research, especially as they hold great promise for application in regenerative medicine, as well as drug discovery and toxicity analyses. Conventionally hESCs are cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) that are derived from E13.5 mouse embryos. One of the biggest challenges in the hESC field is the development of a reproducible and defined hESC culture system that would eliminate batch-to-batch variability of the MEFs as well as exposure to feeder cells that makes hESCs less applicable for clinical use. Previous studies have shown that maintenance of pluripotency can be achieved using Matrigel, a mixture of ECM components, or ECM derived from MEFs or human fibroblasts (Xu, et al., 2001, Klimanskaya, et al., 2005). Other groups have succeeded in culturing feeder-free hESCs by using extracellular matrix (ECM) proteins, such as fibronectin, vitronectin or laminin, as substrates for hESC culture in the absence of feeders, confirming that ECM plays a key role in maintaining hESC growth (Amit, et al., 2004, Braam, et al., 2008, Baxter, et al., 2009, Rodin, et al., 2010).The aim of this work was to investigate the ECM deposited by MEF feeder cells and to isolate and identify proteins in the ECM that support undifferentiated growth of hESCs in the absence of feeders. We have investigated whether matrices derived from different passage feeders differ in their ability to support pluripotency. I also assessed the integrin receptor profile of hESCs in order to define the mechanisms of ECM engagement. ECM was extracted from two strains of feeder cells, CD1 x CD1 and MF1 x CD1, at passages 4 (early passage), 9 and 14 (late passage), and assessed for its ability to support hESC self-renewal over at least 3 passages. Tandem mass spectrometry was used to analyse the ECM composition of each MEF line, thereby allowing a comparison between different passages and different cell lines. More than 100 proteins were identified for each sample, the majority of which were ECM proteins and shared between different passage feeders. As predicted, fibronectin, which is known to support hESC self-renewal was the most prevalent species in all MEF-derived matrices. Furthermore a proteomic analysis of matrix derived from hESCs cultured in feeder-free conditions on fibronectin coating substratum revealed a number of proteins shared between supportive MEF populations and hESC, suggesting other potential candidates that may either assist or interfere with the maintenance of pluripotent hESCs. Of the proteins identified fibrillin-1, perlecan, fibulin-2 were tested as substrates for culturing hESCs in the absence of feeders, with the prospect of developing an optimised feeder-free culturing system that uses a combination defined animal-free substrates. Finally this study sought to dissect the interaction between ECM and growth factors and how these extrinsic factors may affect self-renewal and maintenance of pluripotency-associated gene expression. Interruption of hESC attachment, as well as removal of growth factors appeared to affect transcript levels of pluripotency genes, OCT4 and NANOG, suggesting that the microenvironment can influence hESC fate.

Aromatic peptide amphiphiles that self-assemble in response to a trigger, such as pH or enzymes, have the ability to support the culture of somatic cell types, in both two-dimensional (2D) and three-dimensional (3D) culture. Although a fully defined synthetic substrata is required for the successful clinical applications of Embryonic Stem (ES) cells hydrogels of SA aromatic peptide amphiphiles have not been investigated for this purpose. The aim of this investigation is to produce alkaline phosphatase (AP) triggered hydrogels as a substratum for ES cell culture. This SA trigger was chosen as it utilizes inherent biological processes, through the enhanced AP activity of ES cells, with SA occurring in otherwise constant conditions. We also sought to overcome the current inability to consistently control ES cell behaviour in vitro through two different routes that have previously been demonstrated to effect stem cell culture. Firstly, control of the hydrogels mechanical properties and secondly through the incorporation of biological function, principally through the addition of glycosaminoglycans (GAGs). Firstly AP triggered hydrogels of 9-fluorenylmethoxycarbonyl-Tyrosine-OH (Fmoc-Y-OH) were studied and compared with those formed by pH trigger. An unexpected relationship between AP concentration and molecular order was detected. It was observed that the hydrogels stiffness was controlled through the AP concentration; ideal for ES cell culture as Engler et al. (2006) has previously demonstrated the effect material stiffness had on the differentiation pathways chosen by mesenchymal stem cells. Differences between the SA trigger were detected with the hydrogels formed by pH trigger exhibiting significantly lower mechanical properties. This was attributed to the SA process and the disorder that arises from forming all of the hydrogelators instantaneously. The SA process of AP triggered Fmoc-Y-OH showed a 4 stage process. The first stage, dephosphorylation, occurred in a time and AP concentration dependent manner. The second stage transpired due to the spontaneous SA of Fmoc-Y-OH providing a temporary change in fluorenyl environment. The final stages, formation of chiral one dimensional (1D) fibres through proposed -β interactions and gelation through the entanglement of fibres are closely linked. Secondly we investigated AP triggered Fmoc-Phenylalanine-Tyrosine-OH (Fmoc-FY-OH) hydrogels in two distinct physiological environments. When formed in buffer (0.15M, pH 7) an optimum AP concentration was observed in terms of molecular interactions, -β interactions, which translated to the hydrogels mechanical properties. In these conditions helical fibres were imaged by AFM. The second medium for SA was KnockOUT™ DMEM, developed for ES cell culture. Although similarities in the molecular interactions were detected it appears the SA environment effects the structures formed with non-helical fibres imaged. GAGs were successfully incorporated into the hydrogels at biologically relevant levels at the extremes of sulphation. The sulphation patterns of the GAGs secreted by ES cells changes during differentiation and may provide a way to guide cell behaviour through growth factor binding. However the GAGs were not entrapped in the fibre network and leached out into solution limiting their ability to guide ES cell behaviour. Unfortunately both of the hydrogels produced in this study were deemed unsuitable as ES cell substrata due to their instability (Fmoc-Y-OH) or low biocompatibility (Fmoc-FY-OH). However we have demonstrated that it was possible for endogenous AP to trigger SA, indicating that in the future ES cells may be able to form their own substrata preventing the need for exogenous AP.

Although the key role played by stem cells in maintaining hemopoiesis is well recognized, mechanisms that determine stem cell behaviour (e.g. self-renewal) have remained poorly defined. Historical precedent has illustrated the usefulness of in viitro colony assays in facilitating the investigation of various primitive hemopoietic progenitor classes with restricted differentiation and proliferative potential. In particular, such assays have made possible the definition of the sequential nature of a series of events that early erythropoietic cells undergo and suggested properties that might be anticipated to characterize colonies derived from stem cells proliferating and differentiating in vitro. The present studies were undertaken to test the hypothesis that very large, late maturing erythroid colonies previously noted on occasion in routine erythroid colony assays were, in fact, derived from progenitors with the self-renewal and multiple myeloid differentiation properties associated with stem cells. Initial experiments showed that cells capable of yielding macroscopic sized erythroid colonies were present at low frequency in normal marrow but became the predominant erythropoietic cell type after 2 to 3 weeks in flask culture. Macroscopic erythroid colony formation in assays of cells from either source were shown to have: 1) identical very late maturation kinetics (onset of hemoglobinization after 1 week of initial colony growth), 2) the same high erythropoietin requirements, and 3) similar responsiveness to factors present in media conditioned by mitogen stimulated spleen cells. Optimization of these 2 classes of stimulant yielded an assay that was linear down to very low cell concentrations (less than 5 x 103 cells/ml). This made possible the cytological analysis of macroscopic erythroid colonies under conditions where overlap with other colony types was minimal. Such studies revealed that macroscopic colonies contained, in addition to erythroid cells, megakaryocytes ( > 90% of colonies) and granulocyte cells (30% of colonies) including cells of the eosinophil lineage. Such colonies were also found to contain cells capable of macroscopic spleen colony formation in irradiated mice, the conventional assay for mouse hemopoietic stem cells (on average 1 CFU-S per colony of flask culture origin, and 0.3 CFU-S per colony in assays of fresh marrow). Direct evidence for self-renewal was obtained from replating experiments using irradiated feeders to optimize plating efficiency. Mixed colonies of macroscopic size were regularly demonstrable in replating assays after 1, and even 2, generations of mixed colony formation indicating up to 6 self-renewal divisions during colony formation. By comparison to flask culture cells, the extent of self-renewal exhibited by cells in fresh marrow yielding macroscopic erythroid colonies was found to be 5-fold lower. Finally, the in vitro expression of stem cell self-renewal behaviour was investigated in individual colonies. The number of stem cells generated, when assessed either by CFU-S or replating assays was found to vary markedly. This variation was not accounted for by errors of detection or by variations in colony size. Such findings are similar to previous data on the CFU-S content of individual spleen colonies and provide the first evidence that the type of variation in stem cell self-renewal observed in vivo also occurs in vitro where microenvironmental factors are unlikely to be contributing factors. These results are consistent with a model of stem cell self-renewal in which intrinsic cellular factors play the key role in influencing the decision of individual cells to self-renew. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The respiratory system represents a major interface between the body and the external environment. Its design includes a tree-like network of conducting tubules (airways) that carries air to millions of alveoli, where gas exchange occurs. The conducting airways are characterized by their great diversity in epithelial cell types with multiple populations of secretory, multiciliated, and neuroendocrine cells. How these different cell types arise and how these populations are balanced are questions still not well understood. Aberrant patterns of airway epithelial differentiation have been described in various human pulmonary diseases, chronic bronchitis, asthma, neuroendocrine hyperplasia of infancy, and others. The goal of this thesis is to investigate mechanisms of regulation of airway epithelial cell fate in the developing lung epithelium. More specifically, these studies focus on Notch signaling and address a long unresolved issue whether the different Notch ligands (Jagged and Delta) have distinct roles in the epithelial differentiation program of the extrapulmonary and intrapulmonary airways. Moreover, these studies investigate the ontogeny of the bHLH transcription factor Ascl1 and identify its targets in the developing airways as potential regulators of neuroepithelial body (NEB) size and maturation. My studies provide evidence that the Notch ligand families Jag and Dll are required for the specification and formation of different cell lineages in the developing airway epithelia. Jag ligands regulate multiciliated versus secretory (club) cell fates but also controls abundance of basal cell progenitors in extrapulmonary airways. Dll ligands regulate pulmonary neuroendocrine versus club cell fates in intrapulmonary airways. Analysis of mouse mutants showed that loss of Jag ligands has minimal impact on the size or abundance of NEBs and their associated secretory cells while loss of Dll ligands results in an expansion of NEB size and associated cells. To gain additional insights into the potential mechanisms of how neuroendocrine cells develop and undergo aberrant hyperplasia, I characterized the global transcriptional profile of embryonic lungs from mice deficient in Ascl1, which lack NEBs and neuroendocrine cells and identified a number of genes associated with neuroendocrine cell development, maturation, and the NEB microenvironment. Among these genes, components of the catecholamine biosynthesis pathway, such as tyrosine hydroxylase (Th), a key enzyme for catecholamine production, were downregulated in Ascl1 null lungs. Subsequent functional analysis using a pharmacological inhibitor of this pathway in lung organ cultures showed expansion of pulmonary neuroendocrine cells and NEB size, an observation of potential relevance in human diseases in which neuroendocrine cells are aberrantly expanded. Together these studies highlight the distinct role of Notch ligands and further implicate Ascl1 targets, as illustrated by catecholamine pathway components, in regulating epithelial cell fate. Further examination of these pathways may provide insights into the pathogenesis and ultimately therapeutic approaches for airway diseases.