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.

This thesis demonstrates a pulse-chase assay that describes the turnover of the .r J normal human oesophageal epithelium and Barretr~- metaplasia. to be approximately 11 days. This assay also identified the existence of label-retaining cells after 67 days within the basal layer of the human oesophagus and showed that these cells were epithelial in origin, capable of division yet are not frequently dividing. Furthermore, label-retaining cells were identified within the base of Barrett's glands and were also epithelial and primarily undifferentiated. This thesis suggests that these cells may represent a population of stem cells within the human oesophagus. Using mtDNA mutations as markers of clonal expansion, this thesis demonstrates the presence of a stem cell niche within the normal human oesophagus. Furthermore, clonal patches covering large areas of squamous epithelium were observed. In Barrett's oesophagus, partially-mutated glands were observed indicating that glands are maintained by multiple stem cells. Wholly-mutated Barrett's glands contain all the expected differentiated cell lineages; demonstrating multilineage differentiation from single stem cells. Patches of clonally-related Barrett's glands were also observed; indicating that glands can divide by fission. In one patient the squamous epithelium and the underlying glandular tissue were shown to be derived from a common progenitor cell. TNFa was shown to induce migration of Barrett's cells in vitro suggesting that the inflammatory environment contributes to the expansion of Barrett's lesion. Furthermore, data presented here shows that NSAIDs can act as TNFa inhibitors in the human oesophagus, by decreasing epithelial cell membrane TNFa levels in vivo. This suggests that anti-TNFa therapy may prevent further growth of Barrett's lesions. III

Hematopoietic Stem Cells (HSCs) are rare, self-renewing, and multipotent cells that sustain lifelong production of blood and immune cells. Much of our understanding of hematopoiesis, including the process of divergence and commitment into specific lineages during differentiation, is derived from the analysis of static composition of HSC and progenitor compartments as well as the measurement of their potential using transplantation-based studies. As such, the dynamics of endogenous HSCs, including the kinetics of their differentiation and their interactions with the bone marrow (BM) niche in real-time is poorly understood. The current study aims to characterize HSCs in their native, unperturbed environment by using inducible lineage tracing in combination with high-dimensional flow cytometry and single cell transcriptomics. Our findings provide an unbiased kinetic roadmap of early steps of hematopoietic differentiation and reveal fundamental differences in the sequence of lineage emergence from HSCs. We found a rapid and preferential emergence of megakaryocytic lineage followed by erythroid and myeloid lineages, whereas a substantial delay in lymphopoiesis at steady state. We also used intravital microscopy to visualize endogenous HSCs in the BM of live animals and discovered them to undergo short-range directional movements with extensive morphological changes. Furthermore, our findings revealed profound changes in HSC behavior following treatment with drugs that are used to induce their mobilization into peripheral blood. Overall, the present study offers novel insights into the fundamental features of endogenous HSC differentiation and their in-vivo dynamics during steady state.

No description available.

In the mid-gestation embryo, the first definitive transplantable hematopoietic stem cells (dHSCs) emerge by embryonic day E10.5-E11 in the aorta-gonadomesonephros (AGM) region, as a result of a step-wise maturation of precursors called pre-HSCs. The analysis of several Notch mutants suggests that Notch signalling is essential for the execution of the definitive hematopoietic programme in the AGM. Mouse embryos deficient for Notch1, RBP-Jk or Jagged1 cannot efficiently generate intra-embryonic hematopoeitic progenitors. It has also been reported that knockdown of Notch target genes (Hes1, Hes5) results in hematopoietic impairment. However a clear picture of the role of Notch pathway in HSC development is still missing. In this work we characterised precise stages and cell types during HSC development in which Notch signalling is involved. First we used a Hes1-dEGFP reporter mouse line that allowed us to monitor Notch pathway activity in a narrow window of time. The results suggest that the level of Notch activity fluctuates in HSC lineage in the AGM region and is down-regulated in dHSCs in the foetal liver (where dHSCs migrate after generation in the AGM region). By using transplantation assay, we further showed that fluctuations of Notch activity are essential for HSC development, and that this pattern in the HSC lineage might work as a switch between maturation and proliferation of PreHSC1, PreHSC2 and dHSC, in which temporary decrease might be required to mature from one type to another, both in vitro and in vivo. These findings might need to be taken into consideration for in vitro generation of haematopoietic stem cells, where a fine tuning of Notch signalling activity could greatly improve their emergence.

Haematopoietic stem cells (HSCs) are capable of differentiation into all mature haematopoietic lineages, as well as long-term self-renewal and are consequently able to sustain the adult haematopoietic system throughout life. Currently, in the mouse, HSCs are understood to first appear in the aorta-gonad-mesonephros (AGM) region at embryonic day 11 via a process of maturation from precursors (pre-HSCs). This maturation within the AGM region involves the complex interplay of signalling between cells of the niche and maturing precursor cell populations, but is relatively little understood at a molecular level. Recently our understanding of the AGM region has been refined, identifying the progression from E9.5 to E10.5 and the polarity along the dorso-ventral axis as clear demarcations of the supportive environment for HSC maturation. In this thesis, I investigated the molecular characteristics of these spatio-temporal transitions in the AGM region through the application of RNA-sequencing. This enabled the identification of molecular signatures which may underlie the supportive functionality of the niche. I further compared these expression signatures to the transcriptional profile of an independent cell type, also capable of supporting HSC maturation, the OP9 stromal cell line. By combining this transcriptional information with an ex vivo culture system, I screened a number of molecules for their ability to support HSC maturation from early precursors, leading to the discovery of a novel regulator of HSC maturation: BMPER. Further characterisation of this molecule enabled the identification of its specific cellular source and the proposal that through its action as an inhibitor of BMP signalling it facilitates the maturation of precursors into HSCs. These results lend further detail and support to the role of BMP signalling in the regulation of HSC maturation as well as demonstrating the potential of these transcriptional profiles to yield novel mechanistic insight.

One of the requisites for a growth factor and a biomaterial in tissue engineering, cell therapy and regenerative medicine is the ability to control cell fate. Cells exist in a complex micro-environment consisting of extra-cellular matrix, growth factors, together with adjacent cells. Stem cell culture and modulation remains a challenge due to insufficient, undefined and costly culture systems. This thesis describes synthetic approaches that can modulate stem cell fate by the identification of new synthetic substrates for the growth of cancer, embryonic stem cells and potential short peptide sequences that can mimic the biological functions of the native cytokine used to culture stem cells. Glioma cancers exist as a heterogeneous population of cancer stem cells and cancer progenies. Scale up and spin coating of a polyurethane and polyacrylate polymers was done on agarose for the enrichment of the cancer stem cell population from glioma cells. A polyurethane, synthesised from poly(tetramethylene glycol) and 1,3-bis(isocyanatomethyl)cyclohexane spin (PU10) coated on an agarose surface, was identified to have a higher affinity for the cancer stem cell population over its progenies. By using this polymer to study the mechanism of the cancer stem cell adhesion, two niche components i.e. galectin, transferrin that are enriched by the polymer that contributed to the growth of the cancer stem cells were identified. A synthetic hydrogel (HG21) was identified as substrate for the culture of mouse embryonic stem cells (mESC) as a replacement for gelatin. mESCs were cultured on the hydrogel under undefined and defined conditions. Under both culture conditions, mESC pluripotency and naïve phenotype markers were verified. Marker profiles by immunostaining (Oct-4, Nanog), flow cytometry (SSEA-1) and qPCR (14 gene markers) of mESC grown on the hydrogel were comparable to gelatin, while enabling thermo-detachment for enzyme free passaging of mESC. To identify alternative substances to the cytokines used in stem cell culture, a microarray system was developed. The microarray system was developed initially with adhesion cellulose peptides printed onto polyacrylamide coated microscope slides. These slides were then screened for interaction with human embryonic stem cells (hESCs). After successful development of the cell based cellulose peptide microarray system, overlapping 25-mer peptides based on of basic fibroblast growth factor were synthesised, printed onto the same type of slide and screened with hESC. The screen identified “hit” peptides, which could potentially mimic the biological effects of the native cytokine on hESCs. These “hit” peptides were scaled up and tested in solution with hESC. In the linear form the peptides were not sufficient to sustain pluripotency and further optimisation is needed.

The liver has the ability to regenerate rapidly during acute liver injury by activating mature hepatocytes to divide and restore the damaged liver mass. In contrast, the liver relies on hepatic progenitor cells (HPCs) which have the ability to differentiate into both hepatocytes and biliary cells for regeneration during chronic liver injuries. Whole organ transplant is the most effective treatment for end stage liver diseases. However, there is a constant shortage of donor organs causing the death of many patients while waiting for suitable donor organs. HPC transplant is a potential alternative for whole organ transplant. However the isolation of HPC which is scarce in the liver and the expansion of these cells to a number that is suitable for transplant have been challenging. To investigate the plausibility of using HPCs as an alternative for liver transplant, I developed a protocol to isolate and expand HPC in vitro. Using this system, I investigated the complex hierarchy of HPCs in aid to select a defined population of HPC that is suitable for transplant. I found the EpCAM+ CD24+ population marks a naïve population of HPC that might be suitable for cell therapy. I further investigate the liver repopulating capacities of these cells by isolating EpCAM+CD24+ HPC population by Fluorescence Activated Cell Sorting (FACS) from a hepatocellular injury model. Surprisingly, a subpopulation of the EpCAM+ CD24+ HPCs which are also CD133+ possesses a higher colony forming capacities has been identified. Most importantly, this population can be expanded to a large scale in vitro and able to repopulate the injured liver after transplant. This defined population of HPCs can also be isolated from a mouse model of fatty liver disease and the isolated HPCs can be expanded in vitro. These cells are able to repopulate the liver after cell transplantation. The presence of HPCs that are capable of being isolated from the fatty liver proved the potential of using HPCs for transplant in a clinical setting by using cells isolated human fatty liver that are from rejected for transplant to overcome the shortage of donor organs.

Myocardial infarction is the most common cause of heart failure and remains one of the leading causes of morbidity and mortality in humans. Stem cells are important in the maintenance and repair of adult tissues. Hoechst effluxing cells, termed side population cells are a rare subset of cells found in adult tissues that are highly enriched for stem and progenitor cell activity. Recent studies have suggested that Sca-1+/CD31- cardiac side population cells are capable of differentiation into cardiomyocytes in vitro. However, the response of cardiac side population cells to myocardial injury remains unknown in vivo. In this study, we directly transplanted Sca-1+/CD31- cardiac side population cells into an acutely infarcted mouse heart. After two weeks, the transplanted cells were found to express cardiomyocyte or endothelial cell markers. Importantly, when these cells were transplanted into a remote nonischemic part of the heart after MI, they were able to migrate to the damaged myocardium. Consistent with these cells homing property, we found that SDF-1α, a chemotactic chemokine and its receptor, CXCR4 were up-regulated in the damaged myocardium and on Sca-1+/CD31- cardiac SP cells respectively following an acute myocardial infarction. We further showed that SDF-1α was able to induce migration of Sca-1+/CD31- cardiac side population cells in vitro. Our results have therefore suggested that Sca-1+/CD31- cardiac side population cells are able to migrate to damaged myocardium from non-ischemic myocardium and differentiate into cardiomyocytes as well as endothelial cells in the acutely infarcted mouse heart. We postulate that the SDF-1α/CXCR4 interaction may play an important role in the migration of these cells. Understanding and enhancing these processes may hold enormous potential possibilities for therapeutic myocardial regeneration for the treatment of cardiovascular disease.

Thesis (M. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.