Dissertations / Theses on the topic 'Haematopoiesis'

The cellular integrity of the blood is maintained by the cellular output of the haematopoietic stem cell population which produces the specialized precursors and differentiated cells which constitute the blood. The investigation of haematopoietic stem cell behaviour and regulation has been hampered by both the difficulty in their identification and the development of relevant assay systems. The purpose of this investigation was to study the behaviour and regulation of the haematopoietic stem cell population in normal and leukaemic haematopoiesis using an in vitro assay of a primitive haematopoietic precursor. The use of a combination of haematopoietic colony-stimulating factors [interleukin 3 (IL3)/multi-CSF and macrophage colony-stimulating factor (M-CSF/CSF-1)] in semi-solid agar culture of murine haematopoietic tissue, stimulated the proliferation of a haematopoietic colony-forming cell, defined as the "HPP-CFCIL3+CSF-1" population, which was characterized by a high proliferative potential, a multipotency and behavioural and regulatory properties consistent with its being a primitive haematopoietic precursor and possibly a component of the haematopoietic stem cell population. The proportion of the in vitro HPP-CFCIL3+csf-1 population in S-phase in normal murine marrow, was determined to be relatively low at approximately 10%, increasing to approximately 40% in sublethally X-irradiated, regenerating murine marrow and the respective presence of the haematopoietic stem cell proliferation inhibitor and stimulator was demonstrable by the induction of appropriate kinetic changes in the in vitro HPP-CFCIL3+CSF-1 population. In leukaemic haematopoiesis, leukaemic proliferation often occurs at the expense of apparently suppressed normal haematopoiesis. In vitro HPP-CFCIL3+CSF-1 assay of the haematopoietic stem cell proliferation regulators in a number of murine, myeloid leukaemic cell lines, failed to demonstrate either increased levels of the haematopoietic stem cell proliferation inhibitor, or evidence of a direct-acting, leukaemia- associated proliferation inhibitor, however, evidence of a leukaemia- associated impairment of inhibitor and stimulator production was observed and this may be a possible mechanism by which the leukaemic population develops a proliferative advantage over normal haematopoietic tissue. The identification of a possible mechanism of leukaemic progression and suppression of normal haematopoiesis may subsequently allow the development of potentially more effective disease treatment and management regimes. The endogenous haemoregulatory tetrapeptide: Acetyl-N-Ser- Asp-Lys-Pro [AcSDKP, Mr=487 amu] is reported to prevent the G0-G1 transition of haematopoietic stem cells into S-phase. The mechanism of action of AcSDKP and a number of related peptides, was investigated in relation to the stem cell proliferation stimulator and inhibitor. AcSDKP demonstrated no direct haemoregulatory role against the in vitro HPP-CFCIL3+CSF-1 population, which is consistent with reports that AcSDKP is not active against cells already in late G1, or S-phase, rather it appeared to act indirectly by impairing the capacity of the haematopoietic stem cell proliferation stimulator to increase the proportion of the in vitro HPP-CFCIL3+CSF-1 population in S-phase. An apparent impairment of stimulator action may explain the reported AcSDKP-associated 'block' of haematopoietic stem cell recruitment. A putative endogenous AcSDKP precursor and synthetic and degradative enzyme systems have been reported and the possible physiopathological role of AcSDKP in a number of myeloproliferative disorders has been implicated. The potential application of AcSDKP as a 'haemoprotective' agent administered prior to the use of S-phase- specific chemotherapy may be of clinical significance. The in vitro HPP-CFCIL3+CSF-1 assay of a primitive haematopoietic precursor cell population, which may be a component of the haematopoietic stem cell population, should play a significant role in the investigation of haematopoietic stem cell behaviour and regulation in both normal and aberrant haematopoiesis. With the characterization of the mechanism(s) of action of the haematopoietic stem cell proliferation inhibitor and stimulator and the haemoregulatory tetrapeptide AcSDKP, the manipulation of the haematopoietic system to clinical advantage can be envisaged, while the identification of the aberrant regulatory mechanism(s) in haematopoietic dysfunction may allow, the development of more effective disease treatment and management regimes.

Acute myeloid leukaemia (AML) is characterised by an accumulation of immature blasts that fail to fully differentiate. Leukaemia is organised as a hierarchy, which is maintained by leukaemic stem cells (LSC). To identify molecular differences between normal haematopoietic stem cells (HSC) and LSC, we performed microarray analysis and found that haematopoietic cell kinase (HCK) is overexpressed in LSC. Thus, by knocking-down HCK in leukaemic cells or overexpressing it in stem cell enriched fractions, we should be able to evaluate its role in leukaemogenesis. Since LSC are difficult to culture in vitro, we started to validate HCK silencing in leukaemic cell lines, Mono-mac-6 (MM6), U937 and Fujioka/P31, which highly express HCK. In all cell lines studied, no more than 50% silencing could be achieved, even when a short-hairpin anti-HCK was cloned into a lentiviral backbone to follow the long-term effect of HCK silencing. Decrease in kinase activity was confirmed using kinase assay and phospho-specific antibody recognising the activated HCK kinase. We show that HCK silencing does not affect the cell cycle, proliferation, differentiation or apoptosis of the cell lines. However, using methylcellulose assay, we observe a significant change in MM6 colony morphology caused by a decrease in their migration properties. Moreover, using phospho-flow cytometry, G-CSF and GM-CSF signal transduction towards STAT5 could be proven to occur via HCK in MM6, but not in Fujioka/P31 or U937 cells. HSC enriched umbilical cord blood cells were also transduced with a lentivirus encoding p59HCK. Overexpression of p59HCK in these cells led to their enhanced erythroid differentiation at the expense of myeloid differentiation underlined by the activation of c-Raf, ERK and STATS. Overall, this study can be used as a preliminary set up for further investigation of the role of HCK in normal human stem cells and in primary AML samples in vivo.

The Hedgehog (Hh) family proteins and their signalling pathway are key mediators of, and important in, many mammalian developmental processes. Malfunction of the Hh signalling pathway contributes to developmental disorders and birth defects. This project aims to investigate the role of the Hh signalling pathway in murine haematopoiesis. In our study, we found that Dhh plays a negative regulatory role in normal erythropoiesis and under stress conditions. However, it is not required for regulating erythropoiesis in the fetal liver during embryo development. In contrast, analysis of conditional deletion of Smo in haematopoietic cells revealed that Smo controls early haematopoietic differentiation in the fetal liver but is dispensable for regulating haematopoiesis in adult bone marrow and spleen. Furthermore, pervious studies have demonstrated that Hh signalling is involved in T-cell development throughout maturation. We tested the hypothesis that Foxa2, a downstream target gene of Hh during pre-TCR signalling, is also required for late T-cell development and activation. Analysis of mice conditionally Foxa2-deficient in mature T-cells revealed that Foxa2 is important in the process of maturation in late thymocyte development. In addition, Foxa2 is also involved in regulation of T-cell activation, and the differentiation of T helper cells. Gene expression experiments confirmed that Foxa2 is also a Hh target gene in the thymus. Taken together, our findings revealed that the Hh signalling pathway and its target genes play critical roles in haematopoiesis during embryogenesis and in adult mice.

Haematopoietic stem cells (HSC) are strictly regulated by intrinsic regulators and extrinsic signals from the microenvironment. Nov (CCN3), a matricellular protein of the CCN family, has been reported as a suppressor gene in solid tumours and chronic myeloid leukaemia (CML). Recent study identified Nov as a positive regulator in human cord blood CD34+ stem cells. However, the functions of Nov in haematopoiesis and adult HSC remain largely unknown.

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.

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.

GATA-1 is a key haematopoietic transcription factor which plays a pivotal role in differentiation of the erythroid, megakaryocytic, eosinophilic, mast cell and dendritic cell lineages. Since its initial cloning and characterisation in 1989 a huge amount of information has been gathered on the molecular mechanisms of action of GATA-1. This knowledge has helped understanding of the processes by which cells enact differentiation programmes and suppress alternative lineage choices. GATA-1 produces at least two protein isoforms – the well characterised GATA-1 full-length (GATA-1FL) isoform and a truncated isoform – GATA-1 short (GATA-1s). GATA-1FL comprises two conserved Zinc fingers (which interact with DNA and essential co-factors), a C-terminal tail (of mostly unknown function) and an N-terminal domain (thought to confer activation properties to the molecule, but which may also be involved in transcriptional repression). GATA-1s lacks the N-terminal domain but is otherwise identical. The biological role of GATA-1s is unknown and this isoform received scant attention until the discovery that GATA-1FL mutations were linked to a rare, but highly informative, acute megakaryoblastic leukaemia seen in children with Down syndrome (constitutional trisomy 21). This discovery was particularly interesting, not only because the association between trisomy 21 and the X-linked GATA-1 mutation was extremely tight (being seen in 100% of the cases examined), but also because the GATA-1FL mutations were not randomly located, but rather clustered within the N-terminus, allowing unhindered production of the GATA-1s isoform. This finding led to interest in the pathological and physiological role of GATA-1s in haematopoiesis. Some insight has been gained into the pathological role of GATA-1s by creation of a GATA-1s knock-in transgenic mouse and by experiments looking at the ability of GATA-1s to rescue GATA-1 deficient embryonic stem (ES) cell lines. GATA-1s produces hyper-proliferation of fetal liver meg-erythroid progenitors but allows at least partial differentiation of these cells. However, a number of key questions remain. In particular what is the physiological role of GATA-1s and the reason for the tight association between trisomy 21 and GATA-1s mutations? Given this background, this thesis describes experiments designed to address the physiological role of GATA-1s, to establish whether additional GATA-1 isoforms exist, and to investigate the association between GATA-1 isoform expression and trisomy 21. Firstly a comprehensive expression analysis was performed in murine and human primary tissues and cell lines. This aimed to identify whether GATA-1s had a unique expression profile, either in particular lineages, or at distinct stages of haematological ontogeny. Reverse-transcriptase polymerase chain reaction (RT-PCR) and western blot analyses showed that the expression patterns of GATA-1s and GATA-1FL were virtually identical, with the possible exception of one human primary monocytic cell preparation which appeared to preferentially express GATA-1s. Before proceeding to further analysis of GATA-1s a search was made for additional GATA-1 isoforms using in silico analysis, RT-PCR and western blotting. This led to identification of a clone carrying a GATA-1 mutation involving the C-terminal tail, derived from a patient with chronic myeloid leukaemia. An analysis of the properties of this clone was performed, confirming its altered C-terminus and demonstrating that this conferred increased transactivation properties on the molecule as measured by luciferase assays. This observation suggests that the C-terminal tail may be an important, and previously under-recognised, functional region of the GATA-1 molecule. The discovery of this potentially hyper-functioning GATA-1 mutation led to investigation of whether GATA-1 mutations could be a widespread phenomenon in CML. However, GATA-1 mutational analysis in 21 patient samples from CML blast crisis did not reveal any additional coding mutations. To address the physiological role of GATA-1s, attempts were made to perform gene targeting in murine embryonic stem cells to produce isoform specific knock-out cells i.e. ES cells engineered so that they exclusively express the GATA-1FL isoform (a GATA-1s knock-out) or the GATA-1s isoform (a GATA-1FL knockout). These cells could then be used in in vitro haematopoietic differentiation assays and for transcriptional profiling. In this way it was hoped to establish whether GATA-1s fulfilled any unique roles in primitive or definitive haematopoiesis that could not be compensated for by the presence of the GATA-1FL isoform. Unfortunately, despite evidence of apparently successful targeting from PCR screening of ES cell clones, it was impossible to confirm the existence of endogenously targeted alleles on Southern blotting. Following exhaustive attempts at screening further clones and subclones (more than 1000 clones in total), this approach was abandoned in favour of transgenic expression of GATA-1 isoforms in cell lines. Transgenic expression studies in murine ES cells showed that whilst GATA-1FL expression led to an expansion in numbers and maturity of erythroid and non-erythroid haematopoietic colonies in vitro, GATA-1s was incapable of supporting colony formation in this assay. Studies then moved on to human cell lines. Two cell lines were identified, both capable of in vitro haematopoietic differentiation into megakaryocytic and erythroid cells, but one carrying trisomy 21 (Meg-01) and the other disomic for chromosome 21 (K562). GATA-1FL expression in these cells generally drove differentiation along the megakaryocytic or erythroid lineage as measured by DNA ploidy analysis, haemoglobinisation, upregulation of erythroid or megakaryocytic gene expression (by quantitative PCR) and suppression of alternative lineage genes (PU.1 and Ikaros) and genes associated with progenitor proliferation (cyclin D2 and c-myb). GATA-1s, in contrast, produced less evidence of differentiation with lower DNA ploidy, less up-regulation of erythroid genes and failure to repress other lineage and haematopoietic progenitor associated genes. Examination of the link with trisomy 21 confirmed that that the chromosome 21 candidate gene Erg3 was upregulated in trisomic cells and that expression of GATA-1s appeared to confer a selective advantage in the presence of trisomy 21. However, no clear mechanistic reasons for the selective advantage could be identified. Overall, these studies show widespread GATA-1s expression in haematopoietic cells, confirm the association with inadequate repression of genes associated with primitive progenitors, and suggest that the C-terminal tail of GATA-1 may be an important functional part of the molecule. Finally, these observations have generated a number of testable hypotheses which could form the basis for future work.

High turnover in the haematopoietic system is sustained by stem and progenitor cells, which divide and mature to produce the range of cell types present in the blood. This complex system has long served as a model of differentiation in adult stem cell systems and its study has important clinical relevance. Maintaining a healthy blood system requires regulation of haematopoietic cell fate decisions, with severe dysregulation of these fate choices observed in diseases such as leukaemia. As transcriptional regulation is known to play a role in this regulation, the gene expression of many haematopoietic progenitors has been measured. However, many of the classic populations are actually extremely heterogeneous in both expression and function, highlighting the need for characterising the haematopoietic progenitor compartment at the level of individual cells. The first aim of this work was to chart the single-cell transcriptional landscape of the haematopoietic stem and progenitor cell (HSPC) compartment. To build a comprehensive map of this landscape, 1,654 HSPCs from mouse bone marrow were profiled using single-cell RNA-sequencing. Analysis of these data generated a useful resource, and reconstructed changes in gene expression, cell cycle and RNA content along differentiation trajectories to three blood lineages. To investigate how single-cell gene expression can be used to learn about regulatory relationships, data measuring the expression of 41 genes (including 31 transcription factors) in 2,167 stem and progenitor cells were used to construct Boolean gene regulatory network models describing the regulation of differentiation from stem cells to two different progenitor populations. The inferred relationships revealed positive regulation of Nfe2 and Cbfa2t3h by Gata2 that was unique to differentiation towards megakaryocyte-erythroid progenitors, which was subsequently experimentally validated. The next study focused on investigating the link between transcriptional and functional heterogeneity within blood progenitor populations. Single-cell profiles of human cord blood progenitors revealed a continuum of lympho-myeloid gene expression. Culture assays performed to assess the functional output of single cells found both unilineage and bilineage output and, by investigating the link between surface marker expression and function, a new sorting strategy was devised that was able to enrich for function within conventional lympho-myeloid progenitor sorting gates. The final project aimed to study changes to the HSPC compartment in a perturbed state. A droplet-based single-cell RNA-sequencing dataset of 44,802 cells was analysed to identify entry points to eight blood lineages and to characterise gene expression changes in this transcriptional landscape. Mapping single-cell data from W41/W41 Kit mutant mice highlighted quantitative shifts in progenitor populations such as a reduction in mast cell progenitors and an increase towards more mature progenitors along the erythroid trajectory. Differential gene expression identified upregulation of stress response and a reduction of apoptosis during erythropoiesis as potential compensatory mechanisms in the Kit mutant progenitors. Together this body of work characterises the HSPC compartment at single-cell level and provides methods for how single-cell data can be used to discover regulatory relationships, link expression heterogeneity to function, and investigate changes in the transcriptional landscape in a perturbed environment.

Several congenital disorders of human haematopoiesis including Diamond- Blackfan anaemia result from heterozygous loss of genes involved in ribosome biogenesis. Further, hemizygosity for ribosomal protein gene RPS14 has been implicated in the pathogenesis of myelodysplastic syndrome with loss of 5q, suggesting that genes involved in ribosome biogenesis may act as both haploinsufficient tumour suppressors and regulators of normal haematopoiesis. Ribosome biogenesis is highly conserved through evolution and readily studied in simple organisms such as yeasts. However the zebrafish provides a wellestablished genetic model system which is ideally suited to rapid assessment of vertebrate haematopoiesis. I have therefore used the zebrafish to study genes involved in ribosome biogenesis and their effects on developmental haematopoiesis relevant to human disease. Presented in this work is investigation of the effect of disruption of 4 genes known to be involved in ribosome biogenesis on zebrafish haematopoiesis. Firstly, I describe a gene, Dead-box 18 (ddx18), identified in a forward genetic screen, whose disruption results in defective haematopoiesis and embryonic lethality. Secondly, I have studied the effects of loss of zebrafish orthologues of the human nucleophosmin gene (NPM1), the most frequently mutated gene in human acute myeloid leukaemia. Loss of Npm1 resulted in aberrant numbers of myeloid cells. Heterologous overexpression of mutated NPM1(NPMc+) resulted in increased production of haematopoietic stem cells suggesting a role for NPMc+ in pathogenesis of AML. Finally, I have shown that loss of Rps14 and Rps19 result in anaemia in developing zebrafish and have investigated p53-independent mechanisms for this effect. The findings described herein demonstrate that disruption of normal ribosome biogenesis frequently results in abnormal developmental haematopoiesis. Further genetic assessment of these tissue-specific pathways deregulated by loss of normal ribosome function may represent an important common mechanism underlying the pathogenesis of congenital and acquired disorders of haematopoiesis, and may provide novel pathways for therapeutic targeting.

The finely tuned regulation of haematopoietic stem and progenitor cells (HSPCs) is crucial to sustain normal haematopoiesis. The disruption of the balance between the quiescence state of haematopoietic stem cells (HSCs) and the proliferation/differentiation programs that are necessary to meet daily haematopoietic demands and respond to external insults, can lead to malignant transformation, such as acute myeloid leukaemia (AML). Therefore, it is essential to investigate the players that are responsible to maintain haematopoietic homeostasis, so that novel therapeutic targets can be identified. HSCs reside in a hypoxic environment that is crucial for their maintenance, as it protects them from over-proliferation and exhaustion. The response to a limited availability of oxygen is critically mediated by a transcription factor - hypoxia inducible factor (HIF). HIF is predominantly regulated by prolyl hydroxylases (PHDs) that are 2-oxoglutarate (2OG) dependent oxygenases. This superfamily of oxygen-sensing enzymes has been assigned important roles ranging from hypoxia signaling, DNA repair, chromatin modifications and oncogenesis Following the data published by our group attesting that HIF is dispensable for HSC survival and maintenance, we focused our investigation on HIF-independent pathways. This manuscript describes the study of the role of an oxygen-sensor enzyme, member of the 2OG oxygenases and HIF negative regulator, jumonji domain-containing protein 6 (Jmjd6), in normal and malignant haematopoiesis. Our knockout studies deleting Jmjd6 specifically within the haematopoietic system (Jmjd6fl/fl;Vav-iCre) demonstrate that the homeostasis of HSPC pool was compromised and lymphopoiesis was attenuated in Jmjd6-deficient cohorts. Upon transplantation, HSCs lacking Jmjd6 exhibited a defective chimerism and impaired capacity to fully reconstitute haematopoiesis of recipient mice. Thus, Jmjd6 is essential for HSC self-renewal and maintenance. Our assessment of the impact of Jmjd6 deletion in the context of inflammatory response and recovery from treatment with a myelotoxic agent treatment revealed that Jmjd6 is a positive regulator of HSC homeostasis and recovery from cytotoxic stress. There are accumulating data on the importance of epigenetics in the development of haematological malignancies. Being an epigenetic regulator, clearly involved in RNA splicing, we investigated Jmjd6 as possible player in leukaemogenesis. The results from our leukaemic studies unravelled a new biological function for Jmjd6 as a tumour suppressor in Meis1/Hoxa9 murine model. Altogether, our findings offer important novel insights into the biological functions of Jmjd6 and pave the way for further studies to discover on the mechanism of action of this complex enzyme. Our observations add value to the idea that Jmjd6 might constitute a good candidate for cancer diagnosis, that can be use to ameliorate patient’s prognosis and that it can be used to help patient prognosis in the future.

Blood erythrocytes and leukocytes were serially sampled over many months from female mice that were heterozygous at the X-chromosomal locusencoding the glycolytic enzyme phosphoglycerate kinase (PGK-1). PGK-1A andPGK-1B alloenzymes were identified and quantified electrophoretically. There was little variation in PGK-1 phenotype between serial samples from individual mice. This small amount of variation was discussed in terms of the number of clones participating in haematopoiesis and the contribution of technical factors. Similar studies were performed using radiation chimaeras, repopulated with either a high dose (107 cells) or a low dose (105 cells) of PGK-1AB bonemarrow. The variation in PGK-1 phenotype between serial samples taken fromthe animals repopulated with a high dose of bone marrow was comparable to that seen in normal animals. In contrast, the variation observed in the low- dose chimaeras was. relatively large. These animals were used to study the clonal organisation of the haematopoietic system. The development of B lymphocytes carrying the X-linked immunodeficiency mutation (xid) was studied in mice that were heterozygous at both the x[d and the Pgk-1 loci. An abnormallly large population of B lymphocytes, possessing an characteristic membrane phenotype, was observed in the peripheral blood of a group of experimental mice. This behaved as a transplantable neoplasia. Subsequently, similar populations were found in several aged (>2 years) CBA/Ca mice. A preliminary characterisation of these cells was carried out and their possible relevance to human chronic lymphocytic leukaemia (CLL) was discussed.

The Homeobox (Hox) gene network in conjunction with their Tale cofactors act as master regulators of embryogenesis and haematopoiesis. Co-ordinated and overlapping expression of the genes has been reported in several developmental processes including malignant haematopoiesis, indicative of a 'Hox code.' Hox codes may have value as prognostic indicators in disease conditions, particularly in leukaemia subtypes. Gene expression profiling in normal embryonic stem (ES) cells and a Tel- PDGFRbeta conditional leukaemic ES model indicated a core subset of Hox-Tale members involved in normal and malignant haematopoiesis. The combination of Hox genes a7, a9, and b2-b4, along with Meisl were identified as a candidate 'Hox code' based on their differential expression at key stages of development. Investigations of two of these genes, Hoxa9 and Meisl, in factor- dependent BaF3 cells revealed cytokine independent growth consistent with cellular transformation. Altered expression of the HOX-MEIS axis is associated with leukaemia, where it plays a role in increased proliferation and impaired differentiation. A re- transplantable murine model using co-overexpression of Hoxa9-Meisl was developed to evaluate downstream targets and potential therapeutic intervention. The immature myeloid progenitor phenotype leukaemia showed highly infiltrative pathology with observable splenomegaly. Genes profiled within transcription factor, immune and pluripotency signature platforms showed decreased expression between leukaemic and normal bone marrow samples. miRNAs also showed measurable changes in expression with many showing increased expression in the leukaemic setting. predictive target analysis demonstrated potential overlap with candidate genes identified as being down- regulated in the same setting. Protein arrays indicated enhanced angiogenesis related expression with reduced phospho-receptor tyrosine kinase protein levels in the leukaemia. Connectivity mapping of the deregulated gene expression profiles compared to human leukaemia profiles identified potential drug therapies. Disruption of colony growth was observed following candidate drug treatment of the leukaemic cells, suggesting potential clinical application of these compounds in HOXA9 and MEISl associated leukaemia. The data confirm this mouse leukaemia as an appropriate and relevant model of human disease, and its usefulness for profiling downstream events and the identification of candidate drugs for future investigations.

Cell states in haematopoiesis are controlled by complex circuits, involving master regulators transcription factors and a growing family of RNA species, shaping cell phenotype, its maintenance and plasticity. Amongst RNA species, circular RNAs (circRNAs) are rapidly gaining the status of particularly stable transcriptome members with distinctive qualities. Regarding molecular functions, circRNAs modulate host gene expression, compete for binding of microRNAs, RNA-binding proteins and translation initiation, and participate in regulatory circuits. RNA-seq studies identified thousands of circRNAs with developmental stage- and tissue-specific expression corroborating earlier suggestions that circular isoforms are a natural feature of the cell expression program. circRNAs are abundantly expressed and highly regulated also in the haematopoietic compartment, as described by recent and preliminary studies on circRNAs in blood cells. In my PhD project we focused on the development of a bioinformatics pipeline to detect, quantify and characterize circRNAs from RNA-seq data, by combining both publicly available tools and custom scripts. Aiming to increase the discovery power of the pipeline as well as results robustness, we combined four programs for circRNA detection in parallel. The pipeline was tested on a publicly available dataset of haematopoietic lineage cells such as Haematopoietic Stem Cells, Lymphoid progenitors, Myeloid Progenitors and Megakaryocyte–Erythroblast Progenitors. This pilot analysis allowed to retrieve a great number of circRNAs despite features of the data that were not optimal for circRNA detection. Major results from the pilot study were the identification of distinct sets of circRNAs specifically expressed in different cell types, and the feasibility and convenience of circRNA detection in published datasets to complement the original studies. In parallel, we studied circRNA and linear RNA expression in differentiated cells of the haematopoietic compartment, specifically B cells T cells and Monocytes. We produced RNA-seq data of 12 samples, obtained by cell sorting from peripheral blood of healthy donors and using ribosomal RNA depletion for the library construction. Out of the over 115000 detected backsplices supported by at least two reads, we selected putative circRNAs found by at least two methods, gaining also indirect support that most of them are truly circular forms thanks to independent evidence. This subset consists of 26211 circRNAs expressed by 7307 different genes, with 38.6% of genes expressing one circRNA each, and 40.7% of genes producing from 2 to 5 different circular isoforms and the remaining genes expressing a higher number of circRNAs. The large majority of circRNAs are exonic, 11.5% have backsplice ends falling into intronic regions and only a few (2.5%) probably derive from genomic regions annotated as intergenic. Comparison with the analysis of the linear transcriptome pointed out that the expression levels of linear and circular RNAs expressed from the same gene have only a very slight tendency toward positive correlation, with most of the pairs showing scarce or even negative correlations, suggesting specific regulatory mechanisms underlying the expression of circRNAs. The comparison between B cells T cells and Monocytes indicated groups of circRNAs expressed in all the cell types and specific of each cell type. Unsupervised analyses of expression profiles showed for the first time specificities of circRNA expression associated to different blood cells. B cells and T cells circRNAomes are similar from quantitative and qualitative points of view, whereas Monocytes express a lower number of circRNAs and have a more specific circRNAome. Indeed, differential expression tests outlined sets of circRNAs with significantly variable expression in B cells compared to Monocytes (2589), B cells compared to T cells (168) and Monocytes compared to T cells (977). Differentially expressed circRNAs are associated to genes enriched in protein products involved in key blood processes and pathways. Finally, we focused on 74 circRNAs upregulated in B cells compared to both Monocytes and T cells, 40 upregulated in T cells and 159 upregulated in Monocytes, for a total of 273 circRNAs with differential expression and cell specificity. Additional criteria for circRNA prioritization selected circRNAs associated to genes with key functions in haematopoiesis, or altered/deregulated in haematologic malignancies. Prioritized circRNAs will undergo experimental validations. The sequence analyses for in silico prediction of possible circRNAs functions, as presence of multiple miRNA binding sites, protein binding motifs, or open reading frames, will be the starting point for experimental studies to better elucidate the functions of more promising circRNAs. In conclusion we performed the first study of circRNAs in normal B cells T cells and Monocytes grounding on several biological replicates of each cell type being informative on circRNA differential expression. The integration of circular and linear RNA expression profiles with gene annotations and functions, in conjunction with differential expression data, produced new and original results. We showed that taking into account circRNA expression might add definition to the representation of transcriptome variations in normal haematopoiesis, posing the basis to better comprehend the role of circRNAs in the regulatory circuits of blood cells differentiation, which is a prerequisite for transferring this knowledge to research on haematological malignancies.
Il differenziamento cellulare durante l’ematopoiesi è controllato da circuiti complessi, che coinvolgono fattori di trascrizione e diverse specie di RNA che concorrono a stabilire il fenotipo delle cellule e a mantenerlo, e ne assicurano anche la plasticità. Negli ultimi anni sono emerse chiaramente la diversificazione e l’importanza di varie classi di RNA non codificanti. Tra questi, gli RNA circolari (circRNA), prodotti mediante backsplicing di trascritti primari, si stanno rapidamente affermando come membri del trascrittoma particolarmente stabili e con ruoli biologici rilevanti, prevalentemente regolativi. Per quanto riguarda le loro funzioni molecolari i circRNA sono in grado di modulare l’espressione del gene da cui derivano, possono competere per il legame di microRNA, regolando quindi l’espressione dei loro target, ma anche interagire con proteine che legano l’RNA modulandone le funzioni. Diversi circRNA la cui funzione è stata chiarita recentemente partecipano ad importanti assi o circuiti regolatori, intervenendo in processi chiave, di grande rilevanza anche in ambito oncologico, quali la regolazione del ciclo cellulare, il controllo dell’espressione di oncogeni e l’attivazione di specifiche vie di segnale. Studi di RNA-seq hanno identificato migliaia di circRNA con espressione specifica per lo stato di sviluppo o per il tipo di tessuto, corroborando precedenti indicazioni che le isoforme circolari siano una sfaccettatura del programma cellulare, tanto interessante quanto precedentemente sottovalutata. I circRNA sono molto espressi e fortemente regolati anche nel comparto ematopoietico, come mostrato da alcuni studi preliminari sulla loro presenza nelle cellule del sangue. L’identificazione dei circRNA mediante RNA-seq si basa sulla ricerca di backplice, ovvero di sequenze che non mappano linearmente sul genoma ma che sono formate dalla fusione di due sequenze in maniera non colineare, e ciò richiede specifici metodi computazionali. In questo progetto di dottorato è stata sviluppata una pipeline bioinformatica che consente di identificare, quantificare e caratterizzare i circRNA a partire da dati di RNA-seq, mediante quattro metodi computazionali già disponibili utilizzati in parallelo, e di combinare ed elaborare i risultati grazie a una serie di programmi scritti appositamente. La pipeline è stata testata su un dataset di cellule del lineage ematopoietico disponibile nei database pubblici, che contiene dati di sequenziamento di cellule staminali ematopoietiche, di progenitori linfoidi, di progenitori mieloidi e di progenitori di megacariociti ed eritroblasti. Questa analisi pilota ci ha consentito di identificare molti circRNA nonostante le caratteristiche dei dati non fossero ottimali per questo tipo di analisi. I principali risultati di questo studio pilota sono stati l’identificazione di sottogruppi distinti di circRNA specificamente espressi in diversi tipi cellulari, e l’indicazione di fattibilità e convenienza dell’applicazione di questo approccio anche su dati già pubblicati per ampliare e complementare gli studi originali che non avessero preso in considerazione i circRNA. Il progetto principale si è quindi focalizzato sull’analisi dell’espressione di circRNA e RNA lineari in cellule differenziate del comparto ematopoietico. Sono stati prodotti dati RNA-seq di linfociti B, linfociti T e monociti ottenuti tramite sorting da sangue periferico di donatori sani, per un totale di 12 campioni ad alta profondità di sequenziamento e processati mediante un protocollo di sottrazione dell’RNA ribosomale particolarmente adatto per lo studio dei circRNA. Degli oltre 115.000 backsplice identificati da almeno 2 reads di sequenziamento considerando l’insieme dei 12 campioni analizzati, sono stati selezionati 26.211 circRNA identificati da almeno due metodi computazionali. Considerato che studi precedenti basati sull’arricchimento di circRNA in seguito al trattamento con RNAsi R hanno chiarito che i backsplice identificati da almeno due metodi indipendenti sono più affidabili, questo insieme di 26.211 circRNA selezionati dovrebbe risultare robusto. Essi risultano espressi da 7.307 geni diversi, di cui il 38,6% esprime un solo circRNA per gene, il 40,7% produce da 2 a 5 isoforme circolari e i restanti geni ne esprimono 6 o più. La maggioranza dei circRNA identificati è esonica, l’11,5% ha gli estremi della giunzione che mappano su regioni annotate come introniche nel genoma, e solo il 2,5% probabilmente deriva da regioni genomiche annotate come intergeniche. I livelli di espressione dei circRNA e degli RNA lineari espressi dallo stesso gene hanno una leggere tendenza a correlare positivamente, mentre la gran parte delle coppie mostrano scarsa o negativa correlazione, suggerendo che ci siano dei meccanismi di regolazione specifici che sottendono all’espressione di circRNA. Questo dato è in linea con studi recentissimi che presentano lo splicing alternativo delle isoforme circolari come un ulteriore meccanismo che genera complessità nello splicing dei trascritti eucariotici. L’analisi non supervisionata dei profili d’espressione dei circRNAs in linfociti B, linfociti T e monociti ha mostrato per la prima volta la specificità dell’espressione di circRNA associata ai tipi cellulari considerati. I circRNAomi di linfociti B e T risultano simili sia dal punto di vista qualitativo che quantitativo, mentre invece i monociti esprimono un numero minore di circRNA e hanno un circRNAoma più specifico. Il confronto tra tipi cellulari ha indicato gruppi di circRNA espressi in tutti e tre i tipi cellulari, e altri specificamente espressi in un solo tipo. L’analisi statistica dell’espressione differenziale ha evidenziato dei gruppi di circRNA con espressione significativamente diversa nei linfociti B confrontati con monociti (2589), linfociti B confrontati con linfociti T (168) e monociti confrontati con linfociti T (977). CircRNA differenzialmente espressi sono associati a geni le cui proteine sono coinvolte in processi e pathway chiave nel comparto ematopoietico. Infine sono stati evidenziati circRNA differenzialmente espressi e specificamente up-regolati in un solo tipo cellulare: 74 circRNA risultano up-regolati nei linfociti B in confronto a monociti e linfociti T, 40 nei linfociti T e 159 nei monociti, per un totale di 273 circRNA con espressione differenziale e specificità cellulare. I circRNA differenzialmente espressi, altri con altissima espressione o derivati da geni particolarmente importanti nell’ematopoiesi normale o maligna, sono stati selezionati per ulteriori analisi in silico e alcuni verranno validati sperimentalmente. L’analisi della sequenza dei circRNA per la predizione in silico di siti di legame multipli per miRNA, motivi di legame per proteine oppure open reading frames, fornirà utili predizioni funzionali e sarà anche punto di partenza per studi sperimentali focalizzati su alcuni circRNA particolarmente promettenti. In conclusione, questa tesi costituisce il primo studio sui circRNA in linfociti B, linfociti T e monociti sani, fondato su replicati biologici di ogni tipo cellulare. L’integrazione dei profili d’espressione di circRNA e RNA lineari con l’annotazione dei geni e le funzione, congiuntamente all’espressione differenziale, ha prodotto risultati nuovi e originali. Lo studio è molto informativo sull’abbondanza e la diversificazione dei circRNA espressi e fornisce numerosi nuovi dati sui circRNA, nonché robuste indicazioni sull’espressione differenziale dei circRNA nelle cellule considerate. Abbiamo dimostrato che considerare l’espressione dei circRNA aggiunge definizione alla rappresentazione delle variazioni del trascrittoma nell’ematopoiesi normale, ponendo le basi per ampliare la comprensione del ruolo dei circRNA nei circuiti regolatori del differenziamento delle cellule del sangue, prerequisito per trasferire queste conoscenze alla ricerca nell’ambito delle patologie ematopoietiche.

In normal human somatic cells, the length of telomeres (chromosomal end structures) decreases with each cell division until reaching a critically short length, which halts cell proliferation and induces senescence. The enzyme telomerase, which functions to maintain telomeres at a length that is permissive for cell division, is expressed in approximately 85% of cancer cells and some stem and progenitor cells, including haematopoietic progenitor cells (HPCs), but not most other normal somatic cells. Previous investigations have demonstrated that ectopic expression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity, resulting in telomere elongation in some normal human cell types. However, similar experiments performed in HPCs and endothelial cells have demonstrated a dissociation between the expression of telomerase activity and telomere lengthening. This thesis is focussed on further investigating telomerase-mediated telomere length regulation in HPCs and endothelial cells. Short telomeres in bone marrow and blood leukocytes are associated with the development of disorders linked to bone marrow failure. However, to date a relationship between telomere length and myeloid cell proliferative potential has not been demonstrated. In the current investigations, the telomere length and proliferative potential of 31 cord blood-derived HPCs was determined. Regression analysis revealed a significant correlation between mean telomere length and erythroid cell expansion, but not expansion of other myeloid lineage cells. Another novel finding was that telomerase activity was upregulated in lineage-committed CD34- erythroid cells that were positive for the erythroid-specific lineage marker glycophorin A. It was also functionally demonstrated that telomerase activity facilitates the maximum expansion of erythroid cells. To address the dissociation between telomerase activity and telomere maintenance in BMECs, a dominant negative mutant of the telomere binding protein TRF1, which functions to regulate telomere accessibility, was over-expressed in hTERT-transduced BMECs. These studies showed that telomere access, as well as oncogene expression and exposure to oxidative stress, contribute to telomere length regulation in BMECs. Overall, the results from these investigations demonstrate for the first time the functional significance of telomere length and telomerase activity in ex vivo expansion of erythroid cells, and provide novel insight to the molecular complexity of telomere length maintenance in endothelial cells.

During homeostatic conditions replenishment of the hematopoietic system occurs through the constitutive release of differentiated cells from the bone marrow to the circulatory system. Depletion of these mature immune cells during an immune response results in a compensatory haematopoietic response that can involve suppression or amplification of haematopoiesis as well as phenotypical and functional variation of progenitor cells. However, in many cases lymphopoiesis is often suppressed and is usually concomitant with amplification of myelo and granulopoiesis in a process known as “emergency granulopoiesis”. Here I investigated the effect of central nervous system (CNS) infection with West Nile Virus (WNV) on the lymphopoietic modulation in the bone marrow and conducted comparative studies using a diverse range of disease models, including ZIKV and lymphocytic choriomeningitis viruses (LCMV), as well as parasitic malaria infection. Our results suggest that both WNV and ZIKV act in a very similar way during late stage lethal infection to deplete the early and intermediate developing B cells, primarily via cell cycle interruption and subsequent proliferative suppression in a TNF dependent manor. I demonstrated that these similarities extend to other lethal disease models with similar phenomena observed in both acute LCMV and cerebral malaria models. In contrast, while there was an overall decline in the number of bone marrow B cells during chronic infection models, I observed an increase in the number of early stage pro B cells, but no evidence of increased proliferation, suggesting lymphopoietic modulation tends towards expansion of these early cell populations at a point prior to the pro B stage of development. It is also possible that the presence of encephalitis or encephalopathy results in a response that abrogates this expansion. In contrast to previous influenza studies, I found in vivo evidence to suggest that the depletion of early stage B cells is, at least partly, associated with an increase in apoptosis during both WNV and ZIKV infections. In addition, I ruled out mobilisation of transitional B cells and other, less developed cell populations to the periphery during WNV infection as an explanation for these changes. Interestingly, I show evidence to suggest that cells, classically seen as irreversibly locked to the B cell lineage, can be diverted away from the classic B cell development pathway. Finally, I conclude that the depletion of early-stage bone-marrow B cells during WNV and ZIKV infections is primarily associated with cell cycle inhibition and these cells likely undergo apoptosis. I also propose that a small proportion of these cells are subject to phenotypic diversion away from the classic B cell lineage.

Haematopoiesis is the process to produce haematopoietic stem cells (HSCs), haematopoietic progenitors (HPCs) and terminally differentiated cell types. In the adult, HSCs resided in bone marrow while in the embryo, haematopoiesis occurred sequentially in several niches including yolk sac, aorta-gonad-mesonephros (AGM) region, placenta and fetal liver. The AGM region is the first place where HSCs arise in vivo and therefore should provide important factors to induce haematopoiesis. The mouse embryonic stem cells (mESC) system is a powerful platform to mimic the development process in vitro and is widely utilized to study the underlying mechanisms because they are pluripotent and can be genetically manipulated. A novel co-culture system has been established by culturing differentiating mESCs with primary E10.5 AGM explants and a panel of clonal stromal cell lines derived from dorsal aorta and surrounding mesenchyme (AM) in AGM region. Results of these co-culture studies suggested that the AM-derived stromal cell lines could be a potent resource of signals to enhance haematopoiesis. Molecular mechanism involved in haematopoiesis is a key research direction for understanding the regulation network of haematopoiesis and for further clinical research. A series of studies have demonstrated involvement of the Notch signalling pathway in haematopoiesis during development but with controversial conclusions because of the difference of models concerning various time windows and manipulating populations. This project aimed to investigate the role of Notch signalling pathway during haematopoiesis in the AGM environment. We analyzed the expression of Notch ligands in AGM-derived stromal cells with or without haematopoietic enhancing ability. No correlation was observed between ligand expression and haematopoietic enhancing ability in stromal cell lines or between Notch activity in EBs and haematopoietic enhancing ability. We demonstrated that inhibition of the Notch signalling pathway using the γ-secretase inhibitor could abrogate Notch activity in both mES-derived cells and the haematopoietic enhancing AM stromal cell line. To better understand the involvement of the Notch signalling pathway in a more specific spatial-temporal environment, we established a co-culture system of haemangioblast like cells (Flk1+) with one of AM region derived stromal cell lines with haematopoietic enhancing ability . We found that the AM stromal cell line could enhance Flk1+ derived haematopoiesis as assessed by haematopoietic colony formation activity and production of CD41+cKit+ progenitor cells. Based on the issue that the inhibitor could potentially affect both the ES cells and stromal cells, we carried out genetic approaches to overexpress or knock down Notch signalling pathway in this Flk1+/AM co-culture system. Interestingly, it was found that when Notch activity was enhanced in Flk1+ cells, the production of haematopoietic progenitors was inhibited and the number of cells expressing the pan-haematopoietic marker CD45 was reduced. By using the inducible dominant negative MAML1 system to knock down Notch activity, it was found that the haematopoiesis in the Flk1+/AM co-culture system was not affected, which could be accounted for the low Notch activity in this system. These results supported the hypothesis that the Notch signalling pathway plays a role in modulating Flk1+ derived haematopoietic differentiation within the AGM microenvironment.

Extensive efforts have shed light on the identity and biology of cancer stem cells, required and sufficient for the propagation of hematological malignancies and solid tumours. Much less is understood about the closely related issue as to the identity and properties of the normal stem and progenitor cells targeted by oncogenic lesions, and how the nature of the targeted cell might impact on the biology and clinical picture of the resulting cancer. To address this, we developed a mouse model allowing targeted inactivation of Ezh2 and Runx1 to different haematopoietic compartments. Inactivating mutations of EZH2 and RUNX1 frequently co-occur in haematological malignancies with markedly different phenotypes including myelodysplastic syndrome (MDS) and early thymic progenitor (ETP) leukaemia. Inactivation of Ezh2 and Runx1 in adult haematopoietic stem cells (HSCs) resulted in perturbed haematopoiesis leading to development of an MDS-like disease. Unexpectedly, this MDS phenotype could be fully reproduced when Ezh2 and Runx1 inactivation was targeted to multipotent progenitors (MPPs) using Flt3-Cre. Furthermore, the disease was transplantable by MPPs, but not more committed progenitor populations, demonstrating that MDS tumour propagating potential is not exclusive to intrinsically self-renewing HSCs. Targeting Ezh2 and Runx1 inactivation to early lympho-myeloid progenitors did not result in an MDS phenotype. These mice showed a marked expansion of ETPs within the thymus, combined with a block in thymocyte differentiation. These expanded ETPs displayed transcriptional features characteristic of ETP leukaemia, a treatment-resistant acute leukaemia subtype hypothesised to originate from ETPs. Combination of inactivation of Ezh2 and Runx1 in ETPs with the constitutively activating Flt3-ITD signalling mutation resulted in an aggressive lympho-myeloid acute leukaemia, which could be propagated by the expanded ETP population. These findings demonstrate the potential of lympho-myeloid progenitors such as ETPs to become leukaemia stem cells which propagate a disease retaining lympho-myeloid features. We used this novel ETP leukaemia model to explore therapeutic targeting of Ezh2-inactivated ETP leukaemias using inhibitors of the bromodomain and extra terminal (BET) proteins. Aberrant transcription resulting from epigenetic changes induced by Ezh2 loss could be reversed by BET inhibitors, and these compounds showed therapeutic efficacy against both mouse and human ETP leukaemias in vitro and in vivo.

The Mll gene was originally identified through its role in infant acute myeloid and lymphoid leukaemias. Mll also has a role in normal haematopoiesis as identified using mouse knockout models. Homozygous mutation of Mll is embryonic lethal, which has limited research into its role in haematopoiesis. To overcome this embryonic lethality, a conditional knockout mouse model of Mll was established. In this model, exons 9 and 10 of Mll were flanked by LoxP sites ('floxed'), which recombined to induce deletion of exons 9 and 10 in the presence of the Cre recombinase. Deletion of exons 9 and 10 lead to complete loss of the MLL protein as detected by immunoblotting. By breeding mice homozygous for the floxed Mll allele to mice that carried the Cre recombinase under the control of the Vav promoter, it was possible to delete Mll within the haematopoietic system. Analysis of foetal and adult haematopoiesis in the absence of Mll was carried out using the new model. In embryos lacking Mll, the foetal liver showed a marked reduction in the number of colony forming cells as well as both long and short term haematopoietic stem cells. When transplanted into lethally irradiated recipients with wild type competitors, Mll deficient foetal liver cells were unable to contribute to reconstitution of the haematopoietic system. In adult mice, removal of Mll had no apparent effect on the steady state haematopoietic system. Populations of myeloid, lymphoid and stem cells were unaffected. However, in competitive repopulation assays, Mll deficient bone marrow cells were unable to compete with wild type cells. This work suggests Mll is needed for the correct development of foetal liver haematopoiesis and also to maintain self-renewal potential in adult haematopoietic stem cells. However, it appears that Mll is not needed to maintain adult haematopoiesis under homeostatic conditions.

Visceral leishmaniasis (VL) in humans and in animal models is associated with, among other factors, parasite persistence in the bone marrow (BM) and significant changes in haematological function. However, the mechanisms underlying haematologic dysregulation are largely unknown. Using a panel of stem cell markers, we characterized murine haematopoietic stem and precursor cells in the BM over the course of L. donovani-infection in C57BL/6 (B6) mice. In steady-state, the majority of LT-HSCs (Long-term haematopoietic stem cells) (LSK CD150+ CD34- CD48- cells) were found in a quiescent state, representing cells with the highest degree of reconstitution potential. In contrast, during chronic infection, most LT-HSCs were found to have progressed to cell-cycle and this correlated with a reduced potential to engraft into syngeneic recipients. The loss of quiescent LT-HSCs was associated with expansion of cells displaying a phenotype attributed to early, and uncommitted progenitors. However this increase in uncommitted progenitors did not result in an increase in effective haematopoiesis, but rather chronically infected mice displayed signs of anaemia and thrombocytopenia. The loss of quiescent HSCs and other alterations in the haematopoietic compartment were absent in infected RAG2 KO mice, but adoptive transfer of CD4+ T cells restored this phenotype. In subsequent experiments, we transferred IFNγ-deficient CD4+ T cells into RAG KO recipients and established that this pro-inflammatory cytokines was pivotal for the depletion of the reservoir of LT-HSCs in quiescence, as well as for the establishment of anaemia and thrombocytopenia. Subsequently, using mixed BM chimeras, we established that IFNγ signalling and TNF signalling pathways converge to induce an expansion of BM T cells, suggesting that both cytokines are required to drive the development of CD4+ T cells with the potential to cause alterations in haematopoiesis and haematological dysfunction in the periphery. These data suggest new avenues for clinical research into the pathogenesis of VL and have relevance for the development of new therapeutic strategies and clinical follow-up.

Haematopoietic stem cells (HSCs) are multipotent stem cells that sustain long-life haematopoiesis. External signals delivered from the bone marrow (BM) niche are crucial for HSC homeostasis, alongside a cell intrinsic transcriptional program driven by transcription factors (TFs). Translocations, epigenetic and genetic mutations altering TF activity, subvert HSC homeostasis leading to the development of preleukaemic stem cells (pre-LSCs) and LSCs that drive the development and maintenance of acute myeloid leukaemia (AML). Gata2, a zinc finger TF expressed in the haematopoietic system, is essential for the generation and survival of HSCs in development, however the requirement for Gata2 in adult HSCs is less clear. Perturbation of GATA2 function by loss of function mutations of the GATA2 gene, gives rise to immunodeficiency syndromes with a high risk of transformation to myelodysplastic syndrome and AML. Conversely, overexpression of GATA2 is observed in approximately 40% of AML patients and has been linked with poor prognosis. However, the precise biological impact of GATA2 expression on AML cell fate, including LSCs, has yet to be interrogated.

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.