Sox2 is the most frequently amplified oncogene in lung squamous cell carcinoma (SCC). Lung SCC arises in the proximal to central airways and is thought to originate from the p63-positive basal progenitor cells. Since Sox2 amplification occurs early in SCC pathogenesis, we investigated the oncogenic role of Sox2 using normal primary human lung basal progenitor cells. Although Sox2 is highly expressed in normal basal progenitors in a quiescent tracheal epithelium in vivo, we found that Sox2 expression decreases substantially during in vitro proliferation. When Sox2 expression is elevated in the proliferating basal cells in vitro to a level clinically observed in lung SCCs, Sox2 causes hyperplasia and promotes both squamous and Mucin16-positive glandular lineages at the expense of ciliated cell differentiation. Furthermore, our data suggest that the squamous and glandular-differentiating activity of Sox2 is differentially modulated by Receptor tyrosine kinase (RTK) and/or PI3-kinase signaling to promote squamous metaplasia of basal progenitor cells during SCC development.

Endothelial Progenitor Cells (EPCs) are thought to participate in the healing of injured vascular endothelium by incorporating into the defect sites to mediate endothelial recovery. Recently, osteopontin (OPN) was shown to be fundamental in accelerating estrogen-dependent healing of injured blood vessels. Here, we are investigating the effect OPN has on EPC behavior. Late outgrowth human EPCs (LEPCs) were derived from circulating monocytes isolated by leukophoresis, and grown in culture until passage six. L-EPCs were then assayed for adhesion, spreading, chemotaxis, and haptotaxis, as well as resistance to detachment by flow electric cellsubstrate impedance sensing (ECIS). The results of standard and ECIS methods showed both dose and time dependent responses in cell adhesion and spreading. In addition, OPN promoted haptotactic migration of EPCs in Boyden chamber assays. LEPCs seeded onto 10μM OPN substrates and exposed to laminar flow had grater survival and higher resistance to detachment than OPN/static and flow only conditions. CD44 and !1 integrins were only responsible for approximately 50% of LEPCs adhesion to OPN compared to the unblocked condition. Western blots showed that Rho GTPases were activated in L-EPCs seeded on OPN. However, this activation could not be completely blocked by either CD44 or !1 integrin antagonists. These data confirm the direct effects of OPN on EPCs adhesion, and suggest that OPN works by mediating cell adhesion during vascular injury.

Mitofusin 1 and 2 are outer-mitochondrial membrane proteins that have been shown to be involved in fusion. Mitofusin 2 has also been associated with apoptosis and development. When Mfn1 and Mfn2 were each conditionally knocked out from the cerebellum, Purkinje cells in Mfn2 deficient cerebellum during development had undergone neurodegeneration. Mutations in Mfn2 have also been associated with the Charcot Marie Tooth Type 2A (CMT2A). We want to asses the effect Mfn2 and Mfn1 might have on the development of other regions of the brain such as the telencephalon. We generated Mfn1 and Mfn2 conditional knockouts in the telencephalon by crossing them with Foxg1 Cre - a cre expressed in the telencephalon. We found that Mfn1 deficient mice have lost their corpus callosum at the midline, but survive over 6 months with a decrease in progenitor cells postnatally. Mfn2 deficient mice die between P9 and P12 with a decrease in progenitor cells postnatally and a decrease in number of neurons in the cortex. Therefore, our results suggest that Mfn1 and Mfn2 play a significant role in the development of the telencephalon.

Endothelial Progenitor Cells (EPCs) were first identified from human blood samples as a population of circulating mononuclear cells capable of displaying a mature endothelial cell phenotype in culture. Subsequent studies have established that EPCs arise from the bone marrow (BM) and incorporate into the endothelium at sites of blood vessel growth, suggesting a potential role for these cells in neovascularization. Furthermore, a decline in EPC count has been correlated to multiple vascular pathologies, indicating that EPC number could serve as a biomarker of cardiovascular disease. Unfortunately, due to the variability in techniques used for EPC isolation and identification, considerable heterogeneity exists within the population of cells commonly defined as EPCs. In order for the clinical potential of EPCs to be fully realized, thorough characterization of the BM-derived cell populations involved in neovascularization is required. The objective of our study was to determine the functional significance of circulating EPCs in postnatal vascular growth and repair. Two separate strategies were employed to achieve this objective. In the first, we attempted to generate a novel mouse model where the pool of bone marrow-derived endothelial precursors was drastically reduced or eliminated. Our overall approach was to deliver a "suicide" gene, under control of an endothelial cell-specific promoter, to bone marrow cells for use in bone marrow transplantation (BMT) experiments. Mice receiving BMTs would therefore lack the ability to deliver viable BM-derived EPCs to sites of neovascularization. Our central hypothesis for this study was that a reduction in EPC viability would hinder endogenous vascular repair mechanisms, thereby exacerbating cardiovascular disease. In the second strategy, we attempted to identify novel progenitor cell populations based on the transcriptional regulation of pro-angiogenic genes. Our overall approach was to transduce BM with a retrovirus containing a fluorescent reporter gene under control of pro-angiogenic promoters for use in transplantation experiments. Our central hypothesis for this study was that unique populations of BM-derived cells could be identified by expression of the fluorescent reporter gene directed by the Vascular Endothelial Growth Factor (VEGF), endothelial Nitric Oxide Synthase (eNOS) and Vascular Endothelial (VE) Cadherin promoters. The BMT strategy utilized to address our first hypothesis was unsuccessful due to the use of a truncated form of the pro-apoptotic Bax as our suicide gene target. A plasmid encoding GFP fused to the truncated Bax fragment (ΔN-Bax, consisting of amino acids 112-192 of the full length protein) was used in transfection experiments to assess ΔN-Bax function. The GFP:ΔN-Bax fusion protein formed distinct extranuclear aggregates (presumably due to mitochondrial translocation) but did not induce apoptosis in transfected cells. The ΔN-Bax fragment also did not induce cell death when targeted to endothelial cells with retoviral-mediated gene delivery or in a transgenic mouse setting. To address our second hypothesis, we generated retroviral vectors containing the fluorescent tdTomato reporter under control of the VEGF, eNOS and VE Cadherin promoters. Significant fluorescence was detected in cultured endothelial cells and ex vivo-expanded BM cells. Following transplantation of transduced BM cells into lethally irradiated recipient mice, we were able to identify circulating populations of tdTomato-positive cells using flow cytometry. With these results we have identified novel subpopulations of circulating BM-derived cells which may play a significant role in post-natal neovascularization in mice. Therefore, results acquired from these studies could lead to improved cell therapy techniques for treatment of vascular disease.

Spinal cord injury is a devastating source of suffering in the spectrum of human pathophysiology; advancement for clinical therapy in this area has been stagnant in comparison to modern medical development. Current treatments are palliative, and functional recovery is minimal. During the first two weeks after injury, dense glial scar forms that is impenetrable by regenerating axons. Intervention is imperative to minimize scar formation and provide a supportive environment for axonal regeneration. Oligodendrocytes are critical to maintain the health of growing axons during development and after injury. Obtaining these cells through differentiation of neural progenitor cells (NPCs) is a viable option, but current clinical trials involving stem cells are plagued by poor cell survival and undirected differentiation. Research indicates that local extracellular matrix (ECM) is vital to progenitor differentiation and tissue regeneration. During development, spinal cord ECM is comprised of high concentrations of laminin and hyaluronic acid (HA), which provide essential cues to direct NPC migration and differentiation. The purpose of this research is to create a biomaterial optimized to direct NPC differentiation to oligodendrocytes. Natural biomaterials were optimized from distinct combinations of collagen I, HA, and laminin I to model the native ECM signals found during oligodendrocyte maturation. Four material combinations (collagen, collagen-HA-laminin, collagen-HA, and collagen-laminin) were fabricated into injectable hydrogels to mimic the range of compressive and shear mechanical properties present in neonatal central nervous system (CNS) tissue. Differentiation was assessed by culturing rodent fetal NPCs in these materials without specific soluble factors to direct cellular behavior. The three-component hydrogel performed optimally and achieved a 66% oligodendrocyte differentiation rate compared to approximately 15% in the collagen alone hydrogel. An in vivo study was then conducted using a rat contusion model of spinal cord injury with intervention using the injectable, three-component hydrogel seeded with rat NPCs. Functional recovery was assessed using six behavioral tests. Significant recovery was observed using two behavioral tests six weeks post-treatment. Lesion size was measured and correlated well with behavioral outcomes. The data obtained in this research indicate that a multi-component hydrogel mimicking native, developmental CNS tissue may address problems associated with current clinical practice. / text

The process of cell fate determination, which establishes the vastly diverse set of neural cell types found in the central nervous system, remains poorly understood. During retinal development, multipotent retinal progenitor cells generate seven major cell types, including photoreceptors, interneurons, and glia, in an ordered temporal sequence. The behavior of these progenitor cells is influenced by the Notch pathway, a widely utilized signal during embryogenesis which can regulate proliferation and cell fate decisions. To examine the underlying genetic changes that occur when Notch1 is removed from individual retinal cells, microarray analysis of single cells from wild type or Notch1 conditional knockout retinas was performed. Notch1 deficient cells downregulated progenitor and cell cycle marker genes, while robustly upregulating genes associated with rod genesis. Single wild type cells expressed markers of both rod photoreceptors and interneurons, suggesting that these cells were in a transitional state. In order to examine the role of Notch signaling in cell fate specification separate from its role in proliferation, Notch1 was genetically removed specifically from newly postmitotic cells. Notch1 deficient cells preferentially became cone photoreceptors at embryonic stages, and rod photoreceptors at postnatal stages. In both cases, this cell fate change occurred at the expense of the other cell types normally produced at that time. In addition, single cell profiling revealed that Inhibitor of differentiation 1 and 3 genes were robustly downregulated in Notch1 deficient cells. Ectopic expression of these genes during postnatal development in wild type retinas was sufficient to drive production of progenitor/Müller glial cells. Moreover, Id1 and 3 partially rescued the production of Müller glial cells and bipolar cells in the absence of Notch1, even in newly postmitotic cells. We propose that after cell cycle exit, retinal precursor cells transition through a period in which they express marker genes of several different cell types as they commit to a fate, likely endowed by their progenitor cell. Specifically, cells that will become bipolars or Müller glia depend on Id-mediated Notch signaling during this transitional state to take on their respective fates.

The mouse mammary gland has emerged as a model system for studying processes involved in the development of epithelial tissues. Current evidence suggests the existence of a differentiation hierarchy in the mammary gland, consisting of a stem cell capable of reconstituting the tissue, progenitors with the capacity to produce specific functional cell types, and differentiated cells with limited or no repopulation potential. Although markers for mammary stem cells and progenitors have been identified, these populations have not been isolated to purity and our understanding of how they function in different stages of mammary development remains incomplete. Many adult stem cells are mitotically quiescent and can therefore retain a DNA or histone label significantly longer than differentiated cells. In an attempt to identify mammary stem cells/progenitors by histone label retention, I crossed a mouse carrying the tetracycline-inducible histone 2b/eGFP (H2BGFP) gene with tetracycline transactivator strains expected to induce H2BGFP in the mammary gland. H2BGFP expression was induced in the mammary gland until puberty and then chased for 6-8 weeks; \(H2BGFP^+\) label retaining cells were isolated and assayed. Transplantation experiments comparing MMTVrtTA/H2BGFP MECs isolated after induction to MMTVrtTA/H2BGFP MECs retaining label post-chase failed to prove that label retention enriches for stem cells/progenitors in the MMTVrtTA/H2BGFP system. During the course of these experiments, I unexpectedly discovered that MMTVrtTA induced H2BGFP expression exclusively in the \(CD24^+/CD29^+\) and \(CD24^+/CD29^{lo}\) populations, which contain stem cells and progenitors, respectively. Interestingly, I also discovered that H2BGFP+/CD24+/CD29lo MECs developed limited mammary outgrowths in vivo and that pregnancy increased the repopulation ability of these cells by 5-10-fold. H2BGFP+/CD24+/CD29lo outgrowths contained all mammary lineages and produced milk, but were unable to self-renew in serial transplant assays. Furthermore, \(H2BGFP^+/CD24^+/CD29^{lo}\) and \(H2BGFP^-/CD24^+/CD29^{lo}\) MECs had distinct gene expression profiles, with H2BGFP+/CD24+/CD29lo MECs expressing lower levels of transcripts involved in mammary development and differentiation. These data provide evidence for the existence of a multipotent, pregnancy-activated mammary progenitor and suggests that different progenitor populations are responsible for mammary expansion during puberty and pregnancy. Future studies may identify FACS markers for purification of pregnancy-activated progenitors and further elucidate the role of different mammary cell types during pregnancy.

Myocardial fibrosis is a common pathological finding in patients with cardiovascular disease and is believed to be a major contributing factor in the development of end stage organ failure. Early events that promote the development of myocardial fibrosis are not well understood. Rapid cellular infiltration into the cardiac tissue is evident in fibrosis but the infiltrating populations and their functions have yet to be completely elucidated. The aim of this thesis was to characterize the phenotype and function of this cellular population in a model of hypertension mediated myocardial fibrosis. Furthermore, we intended to explore therapies that target this population and ameliorate fibrosis. We characterized a novel population of infiltrating cells as circulating fibroblast progenitor cells, termed fibrocytes. We determined that this population does not appear to specifically migrate in response to previously established chemotactic signals (CCL2 or CXCL12). We found that fibrocytes respond to fibrogenic stimuli (AngII and CTGF) by increasing the expression of collagen and CTGF, an early molecular mediator of fibrosis, while also promoting fibrocyte differentiation. Using an anti-hypertension treatment, we found that hypertension as a physiologic stimulus likely promotes cellular infiltration and corresponding fibrosis. We also established that treatment with activated protein C (aPC) conferred protection against the development of myocardial fibrosis, potentially by inhibiting fibrocyte recruitment and/or activation. Lastly, to assess fibrocyte involvement in the progression of human myocardial fibrosis we assessed fibrocytes in levels in the circulation of patients with ischemic heart disease compared to healthy controls. We found that patients with ischemic heart disease had an increase of circulating cells that have the potential to become fibrocytes compared to healthy controls and therefore likely contribute to myocardial fibrosis. From this data, we propose that fibrocytes are a key effector cell that directly promotes pathologic fibrosis within the injured myocardium. Understanding their migration and function is therefore essential to the development of future therapies targeting this cell type to inhibit their role in fibrosis.

Cardiac progenitor cells (CPCs) are abundant in the embryonic heart and have hallmark features which include a rapid rate of cell division and the ability to differentiate into mature heart muscle cells (cardiomyocytes). Based on these features, CPCs are considered an attractive candidate cell type for transplantation therapies which aim to replenish the diseased heart muscle tissue (myocardium) with new muscle forming cells. A better understanding of how pharmacological drugs and endogenous hormones/signalling molecules modulate the balance between proliferation and differentiation of CPCs could be used to develop more effective cell based therapies for myocardial repair. Furthermore, this information could provide valuable new insight into molecular mechanisms regulating normal cardiogenesis during the embryonic period. The specific aims of the present study were to characterize the effects of the Ca2+ channel blocking drug nifedipine and the endogenous hormone/paracrine factor atrial natriuretic peptide (ANP) on CPC proliferation and differentiation. Results showed that primary cultured CPCs, isolated from the ventricles of embryonic day (E) 11.5 mouse embryos, underwent a reduction in cell cycle activity following exposure to nifedipine. Furthermore, systemic administration of nifedipine to adult mice receiving transplanted E11.5 ventricular cells (containing CPCs) was associated with smaller graft sizes compared to control animals that did not receive the drug. Results from the present study also demonstrated that ANP receptor mediated signalling systems are biologically active in E11.5 ventricular cells and have an antiproliferative effect on cultured E11.5 CPCs. Moreover, preliminary data provided evidence that genetic ablation of the ANP high affinity receptor (NPRA) may be associated with impaired development of the ventricular cardiac conduction system. Collectively, work from this thesis provides evidence that interactions between transplanted cells and pharmacological drugs could have a significant impact on the effectiveness of cell based therapies and that ANP signalling systems may play a critical role in cardiac ontogeny by regulating the balance between CPC proliferation and differentiation.

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