Immunomodulatory drugs (IMiDs) could enhance both direct anti-tumour and graft-versus-tumour effects after allogeneic haematopoietic stem cell transplantation (AHSCT). However, clinical experience with IMiDs after AHSCT using adult peripheral blood (APB) as a stem-cell source has been limited by graft-versus-host disease. Characterization of the mechanisms by which IMIDs modulate alloresponses of T cells and identification of differential effects on T cells from different cell sources could facilitate more effective use of these drugs in the setting of AHSCT. Using in vitro modelling, multi-parameter flow cytometry and gene expression analysis, I have determined the impact of the widely used IMiD lenalidomide on alloresponses of APB and umbilical cord blood (UCB)-derived T cells. Lenalidomide-treatment potentiates net alloproliferation of APB-derived T cells by selectively enhancing proliferation of CD8+ T cells. These CD8+ T cells have enhanced effector memory differentiation, are enriched for polyfunctional effectors, have enhanced direct-cytotoxicity against heamatopoietic target-cells and have a distinct gene expression profile with altered expression of key immunoregulatory-genes and depletion of cellular ikaros. Importantly, while effects on CD8+ T cells derived from UCB are similar, lenalidomide has contrasting effects on allospecific proliferation of APB and UCB-derived CD4+ T cells. While lenalidomide-treatment has no effect on alloproliferation of APB-derived CD4+ T cells, it reduces alloproliferation of UCB-derived CD4+ T cells. The reduction in UCB-derived CD4+ T cell alloproliferation is accompanied by selective expansion of CD4+CD25+FOXP3+ regulatory T cells (Treg), resulting in an overall reduction in UCB-derived T cell alloproliferation. These findings demonstrate that lenalidomide has a differential impact on alloresponses of T cells from different cell sources; alloresponses of APB-derived T cells are increased via selective expansion of polyfunctional CD8+ effectors, while alloresponses of UCB-derived T cells are limited by expansion of tolerogenic Treg. These findings have important implications for the future use of IMiDs in the setting of AHSCT.

The production of fully functional haematopoietic stem cells (HSCs) for clinical transplantation is a highly sought after goal in the field of regenerative medicine. Given their capacity for extensive self-renewal and differentiation into any cell type, human pluripotent stem cells (hPSCs) provide a potentially limitless source of haematopoietic cells in vitro for clinical application. However, to date, fully functional HSCs have not been produced from hPSCs without the overexpression of transcription factors. In this study I first investigated the production of HSCs and haematopoietic progenitor cells (HPCs) in an established clinical-grade haematopoietic differentiation protocol. I demonstrated the efficient and reproducible production of HPCs but showed that the strategy did not produce fully functional HSCs that could repopulate the haematopoietic system of immune-deficient mice. Modification of the protocol by manipulation of the hedgehog signalling pathway and co-aggregation with OP9 stromal cells did not provide any significant enhancement of HPC production. To gain the required knowledge with which to improve our current protocol, I therefore switched my focus towards studying the development of HSCs in the early human embryo. It has been shown that HSCs first emerge from the ventral wall of the dorsal aorta in the aorta-gonad-mesonephros (AGM) region of the human embryo but the precise location and the mechanisms underpinning this process remain unknown. In this study, I established a culture system to map the spatio-temporal distribution of HSCs and to investigate the presence of HSC precursors. I showed that embryonic HSCs emerge predominantly around and above the vitelline artery entry point in the dorsal aorta and can be maintained in our explant culture system. I then performed RNA-sequencing of cells derived from AGM sub-regions, and this identified molecular signatures which could potentially underlie the ventral polarity of HSC emergence in the AGM. To elucidate the role of the stromal compartment in this unique haematopoietic niche, I derived stromal cell lines from the human AGM region and showed they were capable of supporting haematopoiesis in vitro. This work has provided some important insights into the mechanisms regulating HSC development in the human AGM region and identified interesting candidate molecules for future testing in differentiation protocols. This knowledge brings us a step closer to the successful in vitro production of HSCs for clinical use.

In the mouse embryo, the first definitive haematopoietic stem cells (HSCs), capable of repopulating adult irradiated mice, emerge at mid-gestation by embryonic day E11. At this stage, the aorta-gonad-mesonephros (AGM) region is able to initiate and expand HSCs. Recently, it has been shown that the development of HSC in the AGM region results from the maturation of haematopoietic precursors called pre-HSCs. Mounting evidence points at an endothelial origin for these cells, the haematogenic endothelium. Analysis of VEGFs mutants, a critical pathway for endothelial developement, suggested that it also plays a role during early haematopoiesis. The main receptor of the pathway, FLK-1 (also known as VEGRR2 or KDR), is expressed in early hematopoietic and endothelial cells in the mouse embryo. Knock-out mutants for Flk-1 showed a decrease of endothelial and intra-embryonic haematopoietic progenitors. Although Flk-1 has been identified as an essential gene for HSC emergence, its exact point of action in HSC development remains unknown. In this thesis, I investigated the role of FLK-1 signalling in haematopoietic development and defined precise stages and cell types during HSC emergence in which FLK-1 is critically involved. by using a reporter line and antibody staining, I demonstrated that FLK-1 is expressed in the pre-HSCs/HSC lineage. Germ-line Flk-1 knockout results in embryonic lethality at around E9.0, before HSC emergence, mainly due to defects in vasculogenesis. Since arterial specification precedes HSC formation, it has never been elucidated whether the haematopoietic defects found in the knockouts are a secondary effect of the loss of vasculature or it FLK-1 is directly involved in haematopoietic specification. Therefore, to determine the role of the receptor in HSC development, I used a conditional inducible mutagenesis approach that allowed the deletion of Flk-1 precisely when pre-HSCs mature into HSCs at E10.5 and E11.5. My data showed that Flk-1 deletion at these stages affects both endothelial and haematopoietic progenitors, as well as HSCs. This suggests that the VEGF pathway is not only essential in early stages of haematopoietic development, as previously demonstrated, but it may be also involved in the maturation of pre HSC into HSCs at later stages.

Haematopoietic stem cells (HSCs) emerge during embryogenesis and maintain hematopoiesis in the adult organism. Qualitative and quantitative assessment of HSCs can only be performed functionally using the in vivo long-term repopulation assay. Due to the lack of such data, little is known about the development of HSCs in the human embryo, which is a prerequisite for the development of new therapeutic strategies. Employing the xenotransplantation assay, I have performed here the spatio-temporal mapping of HSC activity within the human embryo and have shown that human HSCs emerge first in the aorta-gonad-mesonephros (AGM) region, specifically in the ventral wall of the dorsal aorta, and only later appear in the yolk sac, liver and placenta. Human AGM region HSCs transplanted into immunodeficient mice provide long-term high-level multilineage haematopoietic repopulation. These cells, although present in the AGM region in low numbers, exhibit a very high self-renewal potential. A single HSC derived from the AGM region generates around 600 daughter HSCs in primary recipient mice, which disseminate throughout the entire recipient bone marrow and are retransplantable. These findings highlight the vast regenerative potential of the earliest human HSCs and set a new standard for in vitro generation of HSCs from pluripotent stem cells for the purpose of regenerative medicine. I have also established a preliminary immunophenotype of the earliest human HSC. These data will be useful for my future studies on the mechanisms underlying the high potency of human embryonic HSCs and on the characterisation of embryonic HSC niche.

Regenerative medicine (RM) represents a promising enabling technology to revolutionize healthcare. This said there are still major gaps between the commercial promise and the reality of the cell therapy sector of regenerative medicine. There is consensus to develop high through-put, automated technologies for the manufacture of RM products. Imaging methods will have the capacity to contribute to this technological gap for cell therapies and are particularly attractive to provide non-destructive monitoring with high spatial and temporal resolution. This work applied an automated, non-invasive phase contrast imaging platform (Cell-IQ) to measure, analyse and ultimately quantify image derived metrics for human embryonic stem cells (hESCs) and haematopoietic stem cells (HSCs) as part of the colony forming unit (CFU) assay. This work has shown through thresholding and machine vision identification technology, imaging has the ability to improve the precision of current evaluation methods for cell culture, providing novel information regarding culture state and show image derived metrics to be predictive of future culture state. Building on this, differentiation through the addition of a growth factor cocktail highlighted how in-process monitoring enables protocol optimisation. After equilibrating the Cell-IQ incubator to a standard incubator, the progress of the CFU assay was monitored and image metrics representative of colony phenotype were analysed. Cell count, distance between cells and cell migration within individual colonies were identified to be informative and provide a degree of colony phenotype separation. Quantitative, novel, image derived metrics were identified that improve reliability through computer automation, cost by removing user verification and time by reducing the assay time from 14 days to 7 days. Non-invasive imaging provides a fantastic opportunity to create bespoke sampling frequencies to achieve desired precision for manufacturing cell therapies, this work has developed and shown improvement and a level of control to current culture process for ESCs and HSCs.

Human haematopoiesis resembles a complex hierarchy, however most intermediate stages are only poorly defined. Efforts to characterise human progenitors have been inconsistent and failed to integrate previous knowledge. Furthermore, characterisation of normal progenitors has important implications in acute myeloid leukaemia (AML) biology. We previously established that leukaemic stem cells (LSCs) resemble the immunophenotypic progenitor compartments more closely than the stem cell fraction. Therefore, I set out to characterise human stem and progenitor cells (HSCPs) on phenotypic, molecular and functional level to complete the picture of human haematopoiesis. I purified HSPCs based on their immunophenotype from adult bone marrow (BM) and umbilical cord blood (CB) to investigate steady state and neonatal haematopoiesis. To define differentiation potentials, HSPCs were subjected to functional in vitro assays on bulk and clonal level. Limit dilution assays were used to determine the frequency of cells with multiple differentiation potentials. RNA sequencing revealed underlying lineage priming and specific gene expression signatures. I successfully characterized the incompletely defined Lin^-CD34⁺CD38^-CD45RA⁺ fraction in BM and CB, containing a CD10^lo lymphoid-primed multipotent progenitor (LMPP) with T cell, B cell, NK cell, granulocytic and monocytic differentiation potential, and succeeded in placing it in the haematopoietic hierarchy with relation to similar lympho-myeloid progenitors defined by other groups. This research lays the foundation to characterise early human progenitors with a comprehensive toolkit on a phenotypic, molecular and functional level. Findings from this thesis might provide knowledge about potential targets in LSCs.

The production of adult blood initiates from the haematopoietic stem cell (HSC). This clinically important cell has the capacity to maintain all blood lineages throughout the lifetime of an organism. HSCs emerge de novo from the haemogenic endothelium in the ventral wall of the embryonic dorsal aorta, from where they go on to seed adult sites of haematopoiesis. We have shown that Lmo4a is required for the emergence of HSCs in the zebrafish, and go on to demonstrate that Lmo4a regulates expression of the critical transcription factor, gata2a. Strikingly, both over- and under-expression of gata2a in the dorsal aorta severely diminishes HSC production. The LIM-only domain protein Lmo4 has previously been shown to interact with the known haematopoietic regulator, Ldb1. Together with our collaborators, we have identified novel binding partners of Lmo4 in mouse erythroleukaemic cells. Our functional analysis shows that many of these partners are also necessary for HSC emergence, thus revealing several new potential regulators of HSC formation. Given that these proteins were identified in an in vitro model of definitive erythropoiesis, it is remarkable that they also appear to act together in vivo at the level of HSC formation, and our data suggests that a transcriptional complex containing Lmo4 and these partners may directly repress gata2a. The related protein Lmo2 is also known to bind Ldb1. Together with Scl, Lmo2 is a master regulator of the haemangioblast programme. We have been utilising this activity, together with recent structural studies, to identify functionally important residues in the Lmo2 molecule. As a cell’s transcriptional programme drives both normal and pathological development, and misexpression of both Lmo2 and Lmo4 is involved in a variety of oncogenic states, the work presented in this thesis is likely to inform efforts to develop therapeutically relevant reagents.

Researches on Hematopoietic Stem Cell (HSC) have been expanding that leads to an increase in our understanding of HSC normal behaviors and abnormal alterations. One of the most important issues in the research on HSCs is to understand the mechanism of the homing process of these cells to settle in their niche in the bone marrow and establish the production of various blood cell types after bone marrow transplantation. The cells first must come in contact with the endothelial cells. This contact is known as adhesion and occurs through a multi-step paradigm ending with transmigration to the bone marrow niche. The initial step of the homing, tethering and rolling of HSC, is mediated by P- and E-Selectins present on endothelial cell surface through their interactions with the ligands expressed on the surface of HSC. Thus, understanding the adhesion process and its contribution for efficient HSCs homing will have great impact on HSC therapy. The selectin – ligands interaction has been intensively studied using in vivo and in vitro approaches. However, the molecular mechanism involved by HSCs at single molecule level is poorly understood. Here in this study, a novel experimental method to unravel the molecular mechanisms of the Selectin-ligands interactions in vitro at the single molecule level is developed by combining microfluidics, epi-fluorescence microscopy and live cells. In this work, the new single-molecule imaging technique enabled us to directly visualize the nanoscale spatiotemporal dynamics of the membrane protein-ligand interactions under conditions of shear stress acting on the cells at the molecular level in real time. Using this method, we revealed that selectin ligands on membrane-tethers and slings show unique spatiotemporal dynamics that is distinct from those on the cell body. We demonstrated that the membrane tethers are formed from single microvilli on the cells, which provides a mechanism to spatially localize selectin ligands, PSGL-1 and CD44 on the tethers and slings. We also demonstrated that the selectin ligands show fast diffusional motion along the tethers and slings compared with that on the cell body due to the detachment of cell membranes from actin cytoskeleton during the formation of the tethers. Our results suggest that the spatial confinement of the selectin ligands together with the fast scanning of a large area by the selectin ligands increase the efficiency of selectin-ligands interaction during the rolling, resulting in slow and stable rolling of the cell on selectin. Our findings contribute significantly to molecular level understanding of the initial step of HSCs. This single-molecule imaging technique that we developed in this study will find wide applications in the molecular-level studies on cell-cell interactions including cancer cell metastasis.

Haematopoietic stem cells (HSC) are invaluable, due to their potential to treat malignant and non-malignant diseases. Modern medicine requires a reliable source of human HSCs (hHSCs) for efficient transplantations, which in many cases cannot be obtained from a single donor. Therefore, the ability to amplify donor hHSCs ex vivo would be an ideal alternative. Past attempts to expand hHSCs in vitro, demonstrated that the protocols developed so far have limited success. My research studied the factors which can affect the optimal culture of transplantable HSCs using a 3D culture system that had previously been used to culture HSCs derived from the aorta-gonad-mesonephros (AGM) region of the mouse embryo. This system involved cell culturing at the gas-liquid interface which is particularly sensitive to mechanical disturbances. To overcome this problem, floating Polypropylene support (rings) were designed and tested and I demonstrated that this was able to prolong aggregate culturing for up to 21 days. Further optimisation tests included altering factors such as oxygen levels, and the presence of antioxidants and apoptosis inhibitors in mouse HSCs culture. I have shown that moderate hypoxia (6% O2) did not affect HSCs in culture, while 2% of O2 led to a significant decrease of HSCs activity. Normoxia resulted in higher reactive oxygen species generation, which would likely be detrimental to cells. However, unexpectedly no improvement in repopulation efficiency of cultured HSCs was achieved by the addition of antioxidant. I also found that when the AGM region was dissociated and co-aggregated in the presence of Rho kinase inhibitor a higher level of repopulation was achieved. In addition, troloxpifitrin-a and p38 inhibitor blocked HSC development without affecting progenitor frequency or the total number of live cells. Subclones of mouse stromal cell line (OP9) were used to create a defined haematopoietic niche for hHSC. Functional screening of these lines in co-aggregate culture re- vealed that 3 of the 34 subclones tested were able to maintain hHSC in culture and repopulate immunodeficient mice at a comparable level to uncultured CD34+ cells. The repopulation in engrafted recipients persisted for over 6 months and showed both myeloid and lymphoid potential. These 3 subclones therefore appeared to create a functional niche for hHSCs and were subsequently used to study the impact of a number of factors including SCF, rock inhibitor, TGFb inhibitor, StemRegenin1 (SR), and prolonged culture technique on hHSC expansion. A significant level of fluctuation between experiments was observed and no definitive conclusions could be drawn. I also attempted to establish stromal cell lines from the human AGM region, more specifically from the ventral (AoV) and dorsal (AoD) regions of the dorsal aorta. Despite attempts to immortalise primary stromal cells, all lines went through a growth crisis. Nevertheless, 30 lines were screened for their ability to support haematopoietic cells in co-aggregate culture with results suggesting that lines derived from AoV expanded haematopoietic precursors more efficiently than AoD lines and OP9 control. Many of the tested lines were able to maintain long-term repopulating human HSCs but the level of repopulation was not as high as that achieved from uncultured CD34+ cells. Unfortunately, these human stromal cell lines have an unstable karyotype which may have an impact on their functional characteristics and they may not represent the nature of the primary cells.

Haematopoietic stem cells (HSCs) are capable of self-renewing and multi-lineage reconstitution of the haematopoietic system of irradiated recipient mice. In the mouse embryo, HSCs originate in a step-wise manner from the haematogenic endothelium. The first HSC precursor has been detected at E9.5 in the dorsal aorta, while HSCs emerge in the aorta-gonad-mesonephros (AGM) region around E11. To date, the molecular mechanisms regulating these events are poorly characterized. Through the activating role of Angiopoietin1 (Ang1) on Tie2 receptor, the Ang-Tie signalling pathway plays a critical role in HSC maintenance in the adult bone marrow niche. Tie2 ligand Angiopoietin2 (Ang2) is described as being a Tie2 inhibitor, however its role is unknown. The aim of this thesis was to characterise the role of Ang-Tie signalling pathway in HSC formation in the mouse embryo. First, I used an ex vivo aggregate system to culture with angiopoietins cells derived from the AGM region at stages of development preceding HSC formation (E9.5-E11). Ang2- treated cells were able to reconstitute the peripheral blood of recipient mice to a higher extent compared to control, indicating a role for Ang2 in promoting HSC maturation. Then, I characterized the expression pattern of Ang-Tie molecules in the AGM region. Ang2-expressing cells were identified as perivascular and sub-aortic mesenchymal cells located in the ventral side of the aorta and in proximity of intra-aortic haematopoietic clusters. Finally, I performed an RNA-seq analysis with the aim of unravelling the molecular mechanisms involved in Ang2-mediated HSC maturation. Pre-HSC-I were cultured in presence or absence of Ang2 and their transcriptional profiles were compared, revealing a number of genes and pathways up-regulated or down-regulated in presence of Ang2, which might indicate a role for Ang2 in increasing cell proliferation, favouring cell migration, and regulation of other signalling pathways involved in HSC development. All together, these data support Ang2 as a novel regulator for HSC formation.