No description available.

The thymus is a complex cellular structure made up of several interdependent cell types and is the primary site for T cell development. A population of fetal thymic epithelial cells (TEC), marked by MTS20 and MTS24, when grafted in vivo can generate a functional thymus containing all thymic epithelial cells and is capab,e of supporting T cell differentiation. Further analysis using in vivo grafting experiments have determined the endoderm as the sole origin for all major thymic epithelial subsets. These findings suggest the possibility that a bipotent thmic epithelial progenitor cell (TEPC) gives rise to both cortical and medullary epithelial compartments. The first ai of this study was to address whether bipotent mouse TEPC give rise to both medullary and cortical epithelial cell populations and to begin to establish a model of TEC differentiation through ontogeny. Its second aim was to start to define condidtions for maintaining functionally undifferentiated RTEPC in vitro. Finally, as little is knowth about the genetic regulation of human thymic development and TEC differentiation, I hve used comparative analysis to investigate the similaarities in expression of key regulations of thymus development between human and mouse, which will aid in the translation of mouse thmic research to human. the main findings of this thesis are i) that a bipotent thymic epithelial progenitor cell population that contribute to both medullary and cortical epithelial cell compartments exists in vivo, but is at low frequency even by E12.5. ii) That a unipotent progenitor population committed to a cortical epithelial cell fate is also present as early as E11.5. iii) That E11.5 TEC can be propagated in vitro in semi defined conditions, but appear to revert ro an early endodermal phenotype on prolonged culture and iv) that the genetic program regulating thymus organogenesis appears to be conserved between mouse and human. In addition I have defined the exact time of haematopoietic cell entry into the human thymus, and the time of entry of mesenchymal cells and the mesenchymal distribution pattern throughout human thymic ontogeny. I also establish the time of onset of differentiation and maturation of hTEC, and using known mouse TEPC markets, show the existence of a population of Claudin4+Ueal+ hTEC at week 8, presumed equivalent to the precursor cells for the AIRE1+ subpopulation of medullary TEC in the mouse.

The thymus is the principal site of T-cell development and maturation. Failure to develop a functional thymus leads to severe immunodeficiency, while partially incorrect function of the organ can lead to a variety of autoimmune diseases as well as higher risk for infections and cancer. The thymus is organized into cortical and medullary regions, which are functionally distinct. The diverse array of thymic epithelial cells (TEC) are the key components of the thymic stroma, both the cortical and medullary TEC subsets are responsible for the establishment of a self-tolerant and self-restricted T-cell repertoire. The thymus is most active in young individuals, and undergoes a progressive naturally occurring involution from birth, which accelerates after puberty. Thymic involution is characterized by loss of thymus organization and function, including an overall reduction in the amount of functional thymic tissue. This results in decreased production of new naïve T-cells, and contributes to the diminished capacity of the aged immune system to adequately respond to new antigenic challenge. Involution of the thymus, both natural and in response to different therapies such as chemotherapy, raises interest in developing cell based treatment methods that will allow the restoration of the thymic architecture and so elevate immune reconstitution in vivo. The cellular mechanisms by which the postnatal thymus is maintained during homeostasis and involution are currently unknown. The earliest thymic progenitors in the thymus express Plet1; it has been established that from E12.5 to E15.5 these cells when purified are able to generate all thymic epithelial cell types and initiate thymus organogenesis. However, at least the latter capacity is reported to be lost from E18.5. A number of papers published provide evidence for the existence of both bipotent and unipotent TEC progenitors in the adult thymus. However the identity of these cells remains unknown, nor has the relationship between the mature and immature postnatal TEC compartments been established. The aim of my research was to investigate the cellular mechanism(s) that maintain the postnatal thymus. Specifically, I aimed to determine whether the thymus is maintained by a stem cell mechanism or by division of terminally differentiated thymic epithelial cells, and whether or not postnatal thymic epithelial stem/progenitor cells express functionally relevant levels of the transcription factor Foxn1. To address these aims, I used two approaches: in vivo genetically heritable lineage tracing and a novel grafting assay to assess the contribution of different lineages of TEC. This thesis describes the characterization of a novel mouse strain, the Foxn1CreERt2 line, which was predicted to allow conditional inducible manipulation of gene expression in TEC. I show that this deletor strain, while thymic epithelial cellspecific, could induce cre-mediated recombination in only in a low proportion of TEC and thus could not be used to address the initial aim of this work as described above. However, lineage tracing experiments using this line have provided evidence for a persistent cortical thymic epithelial progenitor/stem cell type, that was capable of rapid expansion within the cortical compartment over time. In parallel with characterisation of the Foxn1CreERt2 strain, I investigated the potential of various defined epithelial populations to contribute to the thymic environment in an assay of TEC potency. Using this technique I have established the potential of defined TEC subpopulations isolated from postnatal mice to generate cortical and medullary TEC. Among the populations analysed I have identified a minor TEC subset that can robustly contribute to both cortical and medullary TEC that coexpress Ly51 and Plet1. I have further shown, using a limiting dilution approach, that this population contains a postnatal common thymic epithelial stem/progenitor cells, present at a frequency of between 87.5 and 92.5 within this population. I have also produced evidence of a unipotent cortical progenitor population that is capable of long term expansion in vivo.

Cardiac allograft vasculopathy (CAV) limits survival after heart transplantation. CAV is caused by damage to the allograft endothelium, resulting in occlusive intimal lesions. Administration of ex vivo cultured early endothelial progenitor cells (eEPCs) enhances endothelial repair and inhibits intimal hyperplasia. However, engraftment rates of eEPCs remain low. We examined changes in eEPC adhesion molecule expression during ex vivo cultivation, and how these changes affect their ability to adhere. Compared to their parent cell population (freshly isolated peripheral blood mononuclear cells, PBMCs), eEPCs had decreased expression of integrins necessary to form firm adhesions with endothelial cells. Despite this eEPCs showed an enhanced ability to adhere under static conditions compared to PBMCs. However, under conditions of physiological flow, eEPC rolling adhesion was reduced compared to PBMCs. We hypothesize that low eEPC retention rates observed in vivo may be due to impaired eEPC rolling resulting from ex vivo culture.

Cardiac allograft vasculopathy (CAV) limits survival after heart transplantation. CAV is caused by damage to the allograft endothelium, resulting in occlusive intimal lesions. Administration of ex vivo cultured early endothelial progenitor cells (eEPCs) enhances endothelial repair and inhibits intimal hyperplasia. However, engraftment rates of eEPCs remain low. We examined changes in eEPC adhesion molecule expression during ex vivo cultivation, and how these changes affect their ability to adhere. Compared to their parent cell population (freshly isolated peripheral blood mononuclear cells, PBMCs), eEPCs had decreased expression of integrins necessary to form firm adhesions with endothelial cells. Despite this eEPCs showed an enhanced ability to adhere under static conditions compared to PBMCs. However, under conditions of physiological flow, eEPC rolling adhesion was reduced compared to PBMCs. We hypothesize that low eEPC retention rates observed in vivo may be due to impaired eEPC rolling resulting from ex vivo culture.

No description available.

Liver progenitor cells (LPCs) respond to hepatic injury when hepatocyte division is impaired in chronic or severe injury. The LPCs are intimately surrounded by myofibroblasts, macrophages and laminin, thus constituting a potential progenitor cell niche. Laminin has been proposed to maintain LPCs in an undifferentiated state within the LPC niche. LPCs differentiate once they leave the laminin niche. However, mechanisms regulating this process have not been completely investigated. I hypothesized that cell membrane proteins which are implicated in intergin activation and mediation of cell adhesion to laminin such as galectin-3 and CD98 may be involved in this mechanism. Galectin-3 is a carbohydrate-binding protein which plays an important role in various cell functions, including cell growth, proliferation, adhesion, and differentiation. Galectin-3 has been reported to bind integrins and regulates β1 mediated adhesion to ECM. In addition, galectin-3 may also indirectly mediate β1 integrin activation by binding to and activating the heterodimeric transmembrane amino acid transporter CD98. However a role for galectin-3 in regulating LPC behavior has not been demonstrated. In this thesis, the mechanisms of galectin-3 mediating LPC proliferation and differentiation were investigated in an experimental model of LPC induction, the CDE diet, by using mutant mice lacking the gene encoding galectin-3. I have found galectin-3 is important for LPC induction and proliferation in vivo. In addition, galectin-3 is crucial for the LPC proliferation but is a negative regulator of LPC differentiation in vitro in a laminin dependent manner, suggesting that galectin-3 is required for LPC to maintain in an undifferentiated state on laminin. Moreover, the 2 extracellular binding activity of galectin-3 is important for LPC proliferation and adhesion to laminin. Furthermore, in the absence of galectin-3, LPCs down-regulate cyclin-D1 and the cyclin inhibitors p21 and p16 are elevated. Finally I suggest that integrin-β1 and CD98 are involved in regulating LPC proliferation. There is an increasing literature examining the role of LPC niche in regulating LPC behavior. My work suggests that galectin-3 is required for the expansion of LPCs in the injured adult liver. Galectin-3 enhances LPC adhesion to laminin. Galectin-3 is a crucial factor for LPCs to maintain in an undifferentiated state on laminin. My findings not only emphasize the requirement of LPCs to interact with their extracellular environment to expand but also propose that galectin-3 is a key signalling intermediary in the LPC niche, regulating homeostatic balance between proliferation and differentiation of LPCs, thus controlling regeneration.

During hepatic disease the liver has the unrivalled ability to regenerate, by activating mature hepatocytes which can divide and thereby reconstitute the functional liver mass. However in the context of chronic hepatocellular disease the liver can regenerate from an endogenous population of hepatic progenitor cells (HPCs). The mechanisms which are involved in the activation and differentiation of these HPCs is not fully understood. To investigate whether there is a differential signalling requirement in HPCs acquiring a biliary versus hepatocellular fate we established in the laboratory two models of chronic liver damage and regeneration, one of which causes hepatocellular death, and results in infiltrating HPCs regenerating hepatocytes, and a second which causes biliary blockage and death, resulting in biliary regeneration. Here we describe how during biliary regeneration the Notch signalling pathway is highly expressed and activated. HPCs cells are consistently associated with a myofibroblast niche which expressed the ligand Jagged-1 at high levels. We have modulated the Notch signalling pathway in both a co-culture system and our models in vivo to demonstrate that Notch signalling is important in the specification of biliary cells, and that inhibition of this pathway both in vitro and in vivo results in the abrogation of biliary commitment. During hepatocellular regeneration we have found that the negative repressor of Notch signalling Numb is highly expressed in tandem with a low expression of the Notch pathway. We suggest that Wnt signalling maintains Numb within these HPCs at a high level and that this, along with stimulation of a hepatocellular programme allows HPCs to exit from a biliary fate and assume a hepatocellular phenotype. Finally we have found that macrophage ingestion of debris promotes the expression of Wnt, and that ablation of these cells results in a phenotypic switch between HPCs assuming a hepatocellular fate and a biliary one.

Percutaneous coronary intervention (PCI) acutely improves coronary blood flow and myocardial perfusion but at the expense of endovascular laceration and endothelial denudation. PCI associated vascular injury is associated with intense inflammation and a loss of vascular function that may lead to significant in-stent restenosis (ISR), and the potentially catastrophic, acute stent thrombosis. Reendothelialisation is essential to the restoration of normal homeostasis and facilitating vascular healing. Attention has recently focused on a novel mechanism of reendothelialisation mediated by bone marrow-derived precursor or stem cells: endothelial progenitor cells (EPC). EPC are thought to home to, and reendothelialise sites of endothelial denudation, and therefore offer the potential to provide exciting new developments in the management of cardiovascular disease. Understanding the role of EPC following vascular injury may help us to enhance vascular repair following PCI. The following studies were performed to clarify the relationships between putative EPC and vascular injury associated with PCI. In studies of patients undergoing elective PCI for stable anginal symptoms I found that concentrations of traditional circulating phenotypic EPC expressing CD34+VEGFR-2+ were unaffected, unlike CD34+CD45- cell concentrations, which were transiently increased six hours following PCI, subsequently returning to normal by 24 hours, notably without an increase in CD34+ adhesion molecule expression or VEGF-A production. However, the purported progeny of CD34+VEGFR-2+ cells, endothelial cell-colony forming units (EC-CFU), were mobilised at 24 hours, commensurate with a systemic inflammatory response. Interestingly the concentration of circulating CD34+VEGFR-2+ cells and EC-CFU were unrelated to each other, emphasising the distinction between these two cell populations. Although EC-CFU contained proliferating cells and exhibited some endothelial characteristics, EC-CFU predominantly expressed the leukocyte antigen CD45 in addition to the lymphocyte markers CD4 and CD8, and most intensely, the surface markers CD68 and CD105, epitopes commonly expressed on macrophages. Notably, EC-CFU were a potent stimulus for the migration of mononuclear cells. However, despite being mobilised in the context of an acute systemic inflammatory response and being composed of leukocytes, isolated systemic inflammation in healthy volunteers (induced by Salmonella Typhus vaccination) in the absence of vascular injury did not cause selective mobilisation of EC-CFU or indeed of putative phenotypic EPC. It is therefore likely that EC-CFU mobilisation is a relatively specific inflammatory response to cardiovascular injury. In a cohort of 201 patients undergoing coronary angiography, traditional circulating phenotypic EPC (CD34+VEGFR-2+ and CD34+VEGFR-2+CD133+) were very rare indeed and were not increased in response to an acute coronary syndrome (ACS). Furthermore traditional EPC concentrations bore no relation to atheroma burden or clinical outcome. In contrast, concentrations of CD34+CD45- cells were increased in patients with coronary artery disease compared to those with normal coronary arteries and were increased in association with more severe coronary disease. Increased concentrations of circulating CD34+CD45- cells were also associated with a shorter cumulative event-free survival. Both EC-CFU and angiogenic monocytes expressing Tie-2 and VEGFR-2 were increased following acute myocardial infarction but did not relate to coronary atheroma or clinical outcome. These studies examine the behavior of putative EPC in response to both discrete vascular injury and myocardial infarction, and isolated inflammation in the absence of vascular injury. I have identified novel characteristics of the EC-CFU assay and determined that specific factors associated with cardiovascular injury likely trigger EC-CFU mobilisation. The clinical relevance of the traditional phenotypic EPC population is uncertain, but a novel CD34+CD45- population is mobilised acutely following discrete vascular injury and is significantly associated with coronary atheroma and clinical events. It is probable that the circulating CD34+CD45- concentration reflects vascular injury and atheroma burden, and I suggest that CD34+CD45- cells are released directly from the vessel wall following PCI, and do not reflect a reparatory response. In order to determine the impact of EPC populations on vascular healing, prospective studies examining the impact of periprocedural EPC concentrations on vascular healing following PCI are required.

No description available.