Instructive signals, whether external or internal, play critical roles in regulating the developmental potency or ability to self-renew of stem cells. External signals may range from secreted growth factors to extracellular matrix proteins found in the stem cell niche. Internal signals include activated signalling cascades and the eventual transcriptional mechanisms they initiate. In either fashion, stem cells are regulated in a complex temporal and context specific manner in order to maintain or maximise their unique characteristics. Previous experiments suggest that Wnt3a plays a role in maintaining the pluripotent state of mouse embryonic stem (mES) cells. However, in the absence of leukemia inhibitory factor (LIF), Wnt signalling is unable to maintain ES cells in the undifferentiated state. This implies that maintaining the pluripotent state of mES cells is not the primary function of canonical Wnt signalling. To further characterize the role of Wnt3a in pluripotency and lineage specification undifferentiated and differentiated mES cells were induced with Wnt3a. Wnt3a induced the formation of a metastable primitive endoderm state and upon subsequent differentiation, the induction of large quantities of visceral endoderm. Furthermore, we determined that the ability of Wnt3a to induce a metastable primitive endoderm state was mediated by the T-box transcription factor Tbx3. Our data demonstrates a novel role for Wnt3a in promoting the interconversion of undifferentiated mES cells into a pluripotent primitive endoderm state. Aging of skeletal muscle tissue is accompanied by fibrosis, atrophy and remodeling all of which negatively affect muscle performance. Whether this reduction in skeletal muscle competency is directly attributed to a resident adult stem cell population called satellite cells remains largely unknown. Here, we undertook an investigation into how age affects the transcriptional profile of satellite cells and their repopulating ability following transplantation. We determined that as satellite cells age, both their regenerative capacity and ability to colonize the satellite cell niche is reduced. Additionally, we identified satellite cell specific transcriptional profiles that differed with respect to age. Therefore, we conclude that intrinsic factors are an important determinant of satellite cell regenerative capacity during the aging process.

Embryonic stem cells (ESCs) possess hypo-immunogenic properties and have the capacity to modulate allogeneic immune response. ESCs have been shown to reduce immune activation in response to third party antigen presenting cells (APCs) in vitro and have the capacity to promote allograft survival in vivo. Clinical use of live ESCs to treat immunological disorders, however, risks teratoma or ectopic tissue formation. Accordingly, the way lab is studying the immune modulatory potentials of ESC-derived factors and recently, found that dendritic cells (DCs) treated with human ESC extracts are poor stimulators of purified allogeneic T cells compared to those DCs treated with vehicle or fibroblast extracts. In the present study, I found that ESC-derived extracts directly inhibit T cell proliferation and suppress their activation without inducing cell death. Furthermore, ESC extracts are able to suppress Th1 polarization while increasing the numbers of Foxp3+ CD4+ CD25+ regulatory T cells. Moreover, I found that a protein called Milk fat globule-EGF factor 8 (MFG-E8) appears to be highly expressed in ESCs. Importantly, neutralizing MFG-E8 substantially abrogated the immune suppressive effects of ESC extracts on T cell activation. These findings lead to future studies to further define specific immunomodulatory factors derived from ESCs for potential applications.

The incidence of heart failure (HF) continues to grow despite advances to current therapies which are effective insofar as slowing disease progression. Cardiac stem cell (CSC) therapy is an emerging treatment for the reversal of HF. We sought to examine the effect of genetically engineering CSCs to over-express stromal cell-derived factor 1α (SDF1α) on myocardial repair. SDF1α over-expressing CSCs exhibited a broader paracrine signature resulting in increased stimulation of capillary network formation and chemotaxis under in vitro conditions. Using a murine model of myocardial ischemia, we demonstrated over-expression of SDF1α increased myocardial SDF1α content, reduced scar burden and increased activation of PI3K/AKT signaling as compared to non-transduced CSC and vehicle controls. These effects improved cardiac function without increasing cell engraftment suggesting that the mechanisms driving these benefits are largely paracrine mediated. Taken together this data suggests that transplantation of CSCs genetically programmed to over-express SDF1α provides superior cardiac repair by boosting the content of cardio-protective cytokines during the critical healing phase after myocardial infarction.

It is well established that the Wnt pathway is associated with tumorigenesis in a wide range of human cancers, including a majority of breast cancers. However, due to diverse roles of Wnt signalling, therapeutic targeting has not yielded consistent results and underlying mechanisms remain unclear. Here, I show that breast cancer cell lines with high E-cadherin expression are resistant to TCF4 inhibitors and develop cancer stem cell characteristics. Conversely, cells with low levels of E-cadherin are very susceptible to cell death with the same treatment. My results suggest that breast cancer cells in an epithelial-like state, but not mesenchymal-like state, will be more responsive to therapeutic targeting of the Wnt/TCF pathway. Importantly, E-cadherin high cells show robust Akt activation, whereas E-cadherin low cells do not. Thus, combinational inhibition of both Wnt and Akt signalling is needed to effectively target breast cancer cells in both the epithelial and mesenchymal states.

The incidence of melanoma and non‐melanoma skin cancer is continuing to increase worldwide. Melanoma is the sixth most common cancer in the United States, making skin cancer a significant public health issue. Photo chemoprevention with natural products is an effective strategy for the control of cutaneous neoplastic. Polyphenols from fruits have been shown to protect the skin from the adverse effects of solar UVR, cancer, and the growth of cancer stem cells. In particular, blueberries are known for their high concentration of phenolic compounds that have the high antioxidant capacity, and their effectiveness in reducing UV damage and, therefore, skin cancer. In Matar's lab, we have shown that Polyphenol-Enriched Blueberry Preparation (PEBP), derived from biotransformation of blueberry juice through fermentation, is effective for targeting skin cancer stem cell proliferation in different skin cancer cell lines. We predicted that PEBP affects melanoma skin cancer stem cells (MCSCs) epigenetically by targeting miRNA pathways. We observed the effects of PEBP on sphere growth and cell motility in vitro. We performed RT2-qPCR analyses to determine PEBP influence on miRNA in B16F10 spheres. We transfected B16F10 cells with miR-200b and performed western blotting analyses. Our results demonstrated that PEBP reduced sphere growth and cell migration, and up regulated miR-200b expression in different biological settings. Inhibition of miR-200b increased Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression. Consequently, PEBP may influence MCSCs through miRNA pathways. Elucidating the mechanisms by which PEBP modulates CSCs biological behavior by controlling miRNAs will enhance our understanding of the molecular mechanisms in skin cancer chemoprevention and might result in their use as natural photo-protectants in skin cancer.

In the normal adult human, hemopoiesis appears to be maintained by the simultaneous activity of many stem cell-derived clones. Conversely, most examples of human myeloid malignancies have been shown to represent clonal populations arising as a result of the unregulated expansion of a single transformed hemopoietic stem cell. The limits of the proliferative capacity of normal hemopoietic stem cells in humans and their persistence in hemopoietic malignancies have, however, not been extensively Investigated. One of the most likely reasons for this is the lack, until very recently, of a widely applicable method to analyze the clonality status of human cell populations. Methylation analysis of two polymorphic genes. HPRT and PGK, now allows such studies to be performed in approximately 50 % of females. The possibility that normal human hemopoietic stem cells might have the capacity to mimic the behaviour of some transformed stem cells by generating clones of progeny that could dominate the entire hemopoietic system was then examined. Such a phenomenon has been well documented in animal models of marrow cell transplantation. I therefore undertook an analysis of all allogeneic marrow transplants performed over a 1 to 1-1/2 year period where the genotype of the donor made clonality analysis using the HPRT or PGK systems possible. Using this approach, I obtained evidence in two patients suggesting that a single or, at most, a very small number of normal primitive hemopoietic stem cells were able to reconstitute the hemopoietic system. In one case the data suggested that such reconstitution was likely to have derived from a stem cell with both lymphopoietic and myelopoietic potential. However, in all other cases hemopoiesis in the transplant recipient was found to be polyclonal. Such findings indicate that clonal dominance in the hemopoietic system is not sufficient to infer that a genetically determined neoplastic change has occurred. In addition, these findings have implications for the design of future gene therapy protocols. The same methodology was also applied to investigate the clonality of different hemopoietic cell populations in patients with chronic myelogenous leukemia (CML) and essential thrombocytosis (ET). In both of these myeloproliferative disorders, the neoplastic clone produces terminally differentiated progeny that appear minimally different from normal. Data from the CML studies confirmed the non-clonal nature of the cells emerging in long-term CML marrow cultures. Similarly, patients transplanted with cultured autologous marrow were shown to undergo polyclonal and bcr-negative reconstitution of their hemopoietic system. Analysis of a series of patients with a clinical diagnosis of ET showed that polyclonal hemopoiesis in the presence of an amplified neoplastic clone is not a rare event in this disorder, and that clonality results do not always correlate with other neoplastic markers associated with myeloproliferative diseases in general. Another example of polyclonal hemopoiesis in the presence of an amplified neoplastic clone was demonstrated in a patient with Ph¹-positive ALL whose disease appeared to have originated in a lymphoid-restricted stem cell. The studies described in this thesis reveal a level of complexity of normal and neoplastic stem cell dynamics not previously documented. They highlight the need for more precise information about the molecular basis of regulatory mechanisms that govern hemopoietic cell proliferation and survival at every level of differentiation. Finally they support the accumulating evidence that acquisition of full malignant potential requires several additive genetic changes first postulated many years ago as the somatic mutation theory of carcinogenesis. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Blood-derived circulatory angiogenic cells (CACs) and resident cardiac stem cells (CSCs) have both been shown to improve cardiac function after myocardial infarction (MI) but the superiority of either cell type has long been an area of speculation with no definitive head-to-head trial. In this study, we compared the paracrine profile of human CACs and CSCs, alone or in combination. We characterized the therapeutic ability of these cells to salvage myocardial function in an immunodeficient mouse model of MI by transplanting these cells as both single and dual cell therapies seven days after experimental anterior wall MI. CACs and CSCs demonstrated unique paracrine repertoires with equivalent effects on angiogenesis, stem cell migration and myocardial repair. Combination therapy with both cell types synergistically improves post infarct myocardial function greater than either therapy alone. This synergy is likely mediated by the complementary paracrine signatures that promote revascularization and the growth of new myocardium.

Brain injury, either surgically induced or as a result of trauma or stroke, is one of the leading causes of death and disability worldwide. Since transplantable stem cell sources are showing a great deal of promise and are actively being pursued to provide neuroprotection post-injury, in this body of work, we set out to characterize and examine the therapeutic potential of amniotic fluid derived (AF) cells as a potential cell source for cell-based therapies in mediating neuroprotection post-injury. Despite their heterogeneity, we found that AF cells are mainly epithelial in origin and express various genes involved in stem cell maintenance and neural commitment. A very small subset of AF cells also express pluripotency markers OCT4a, SOX2 and NANOG, which can be enriched for by single cell cloning. SOX2 positive clones have the capacity to give rise to a neuronal phenotype, in neural induction conditions, which can be used to examine the neural differentiation capabilities of AF cells. Subsequently, we examined the ability of AF cells to mediate a neuroprotective effect in a surgically induced brain injury model through gap junctional-mediated direct cell-cell communication and as a vehicle for GDNF delivery post-injury. AF cells express high levels of CX43 and are able to establish functional gap junctional intercellular communication (GJIC) with cortical astrocytes. We report an induction of Cx43 expression in astrocytes following injury and demonstrate, for the first time, CX43 expression at the interface between implanted AF cells and host astrocytes. In an effort to boost host endogenous neuroprotective mechanisms post-injury, via neurotrophic factor delivery, we engineered AF cells to secrete GDNF (AF-GDNF). GDNF pre-treatment significantly increased AF cell and cortical neuron survival rates following exposure to hydrogen peroxide. AF-GDNF cells, seeded on polyglycolic acid (PGA) scaffolds, survived longer in serum-free conditions and continued to secrete GDNF post-implantation activating the MAPK/ERK signaling pathway in host neural cells in the peri-lesion area. Despite some promising trends, we did not observe significant behavioural improvements following AF-GDNF/PGA implantation nor reduced lesion volume during the 7 day time-frame. In conclusion, through GJIC with cortical astrocytes and delivery of exogenous neurotrophic factors, AF cells hold great promise in mediating neuroprotection post-injury.

Les cellules souches spermatogoniales (SSCs) constituent la population de cellules germinales initiales support de la production des spermatozoïdes tout le long de la vie d’un individu. Ces cellules caractérisées par leur capacité d’auto-renouvellement et de différenciation maintiennent ainsi une réserve et garantissent la production continue de cellules germinales différenciées. Chez les mammifères, plusieurs marqueurs permettant de reconnaitre cette population cellulaire au sein du testicule ont été identifiés. De plus, parmi ces marqueurs, certains permettent d’isoler et de purifier les SSCs. Ils ont ainsi permis d’aborder les mécanismes de régulation du devenir des SSCs par des expériences menées in vitro et in vivo. En revanche, la biologie de ces cellules est beaucoup moins connue chez les vertébrés non mammaliens, en particulier chez les poissons téléostéens. Notre modèle d’étude, la truite arc-en-ciel (Oncorynchus mykiss) est caractérisée par une spermatogenèse cyclique et fortement saisonnée. La croissance du testicule immature, la prolifération active des spermatogonies à la puberté, et la quiescence de ces dernières en fin de cycle semblent être des étapes clés de régulation du devenir des SSCs. Grâce à l’analyse des profils d’expression au cours du cycle spermatogénétique et dans des fractions de cellules testiculaires isolées, nous montrons la conservation d’expression de gènes décrit comme marqueurs de SSCs chez les mammifères (pou2, plzf, nanos2 et 3, gfra1) dans les populations de spermatogonies A indifférenciées de truite. En particulier, gfra1 et nanos2 identifient tous deux une sous-population de cellules au sein des spermatogonies. Nous proposons donc que les cellules gfra1+ et/ou nanos2+ sont des SSCs au sein du testicule de truite. Par ailleurs, nous montrons que l’orthologue truite de gdnf, ligand de gfra1 et régulateur majeur du maintien des SSCs chez la souris, est exprimé très fortement juste avant la fin de cycle spermatogénétique. Cette expression corrélée avec un pic de sécrétion plasmatique de Fsh suggérait une régulation positive de gdnf par cette hormone. Notre étude in vitro n’a pas permis d’aboutir aux mêmes conclusions, mais cette technique ne reflète pas toutes les régulations réciproques ni le rôle des autres facteurs in vivo. En conclusion, nous avons découvert des marqueurs probables de SSCs chez la truite. En particulier, gfra1 et nanos2 qui permettront une analyse plus approfondie de la biologie des SSCs chez les téléostéens. De plus, l’expression de gdnf et de son récepteur dans le testicule, régulée en fonction du stade du cycle spermatogénétique, nous permet d’envisager cette voie comme régulateur du devenir des SSCs chez la truite. / Spermatozoa production throughout life requires the presence of the initial germ cells, the spermatogonial stem cells (SSCs). Their self-renewal and their ability to differentiate assure to keep a reserve and to produce continuously differentiated germ cell. In mammals, several markers of the SSCs have been identified. Interestingly, some of them allow us to sort the SSCs population and further to analyze their fate in vitro and in vivo. By contrast, only scarce information has been obtained in non-mammalian vertebrates including the teleost fishes. Our model of study, the rainbow trout (Oncorynchus mykiss), presents a seasonal spermatogenesis. The growth of the immature testes, the active spermatogonial proliferation starting at puberty, and their quiescent state at the end of the cycle represent interesting stages to study the regulation on the SSCs fate. Using various testicular stages and purified testicular cell fractions we show that pou2, plzf, nanos2 and 3 and gfra1, all expressed by spermatogonial stem cells in mammals, are specifically expressed in the undifferentiated A spermatogonia population. In particular, gfra1 and nanos2 are expressed in a sub-population of these cells. Thus, we propose that the nanos2+ and/or gfra1+ cells are SSCs. Furthermore, in our study, gdnf, ligand of gfra1 regulating the SSCs fate in mouse, is highly expressed just before the end of the spermatogenetic cycle. Such expression correlates with Fsh peak of secretion. However a stimulation of gdnf expression by Fsh was not observed in our in vitro experiments, but this technique doesn’t represent reciprocal regulations nor the roles of all factors in vivo. To conclude, we discovered potent marker of SSCs in trout. In particular, gfra1 and nanos2 will allow us to investigate further the SSCs biology in teleosts. Moreover, gdnf and its receptor expression in the testis in a spermatogenetic-dependent way lead us to propose this pathway as a potent regulator of the SSCs fate in trout.

The differentiation of mesenchymal stem cells (MSCs) to endothelium is a critical but poorly understood feature of tissue vascularisation and considerable scepticism still remains surrounding this important differentiation event. Defining features of endothelial cells (ECs) are their ability to exist as contact-inhibited polarised monolayers that are stabilised by intercellular junctions, and the expression and activity of endothelial markers. During vasculogenesis, communication between MSCs and differentiated ECs or vascular smooth muscle cells, or between MSCs themselves is likely to influence MSC differentiation. In this study, the possibility that cell density can influence MSC differentiation along the EC lineage was examined. High density plating of human bone marrow-derived MSCs induced prominent endothelial characteristics including cobblestone-like morphology, enhanced endothelial networks, acetylated-low density lipoprotein uptake, vascular growth and stimulated expression of characteristic endothelial markers. Mechanistically, this density-dependent process has been defined. Cell-cell contact-induced Notch signalling was a key initiating step regulating commitment towards an EC lineage, whilst VEGF-A stimulation was required to consolidate the EC fate. Thus, this study not only provides evidence that MSC density is an essential microenvironmental factor stimulating the in vitro differentiation of MSCs to ECs but also demonstrates that MSCs can be differentiated to a functional EC. Taken together, defining how these crucial MSC differentiation events are regulated in vitro, provides an insight into how MSCs differentiate to ECs during postnatal neovascularisation and an opportunity for the therapeutic manipulation of MSCs in vivo, enabling targeted modulation of neovascularisation in ischaemia, wound healing and tumourigenesis.