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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 (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.

The mammalian kidney and reproductive systems both derive from a common embryological origin, the intermediate mesoderm. Abnormal intermediate mesoderm development can result in congenital abnormalities of the urogenital system, yet the molecular mechanisms that govern intermediate mesoderm development are incompletely defined. The spatial and temporal expression of the proteins BMP2 and 4 and their receptor ALK3, in urogenital tissue, suggests a function for BMP-ALK3 signaling in the intermediate mesoderm. It was found that Alk3IM null kidneys display renal hypoplasia, associated with a decrease in kidney size and nephron number. The phenotype of renal hypoplasia in Alk3IM nulls was associated with early decreased number of developing nephron structures and secondary defects in branching morphogenesis. While neither apoptosis nor cell proliferation differed in metanephric mesenchyme cells in Alk3IM nulls, markers of renal progenitor cells were decreased in mutant animals. It was observed that Alk3 expression in the intermediate mesoderm also controls mesonephric tubule number. Alk3IM nulls had fewer mesonephric tubules and fewer derivative Leydig cells. The reduction in Leydig cells resulted in decreased levels in serum testosterone and defects in seminal vesicle formation and fertility. Alk3 expression was also required for normal development of the corpus epididymis. The morphological defects in nephrogenesis were associated with decreased phospho-p38 MAPK expression and in the testis with decreased Phospho-SMAD1/5/8. These results elucidated a requirement for Alk3 signaling in controlling progenitor cells derived from the intermediate mesoderm.

The mammalian kidney and reproductive systems both derive from a common embryological origin, the intermediate mesoderm. Abnormal intermediate mesoderm development can result in congenital abnormalities of the urogenital system, yet the molecular mechanisms that govern intermediate mesoderm development are incompletely defined. The spatial and temporal expression of the proteins BMP2 and 4 and their receptor ALK3, in urogenital tissue, suggests a function for BMP-ALK3 signaling in the intermediate mesoderm. It was found that Alk3IM null kidneys display renal hypoplasia, associated with a decrease in kidney size and nephron number. The phenotype of renal hypoplasia in Alk3IM nulls was associated with early decreased number of developing nephron structures and secondary defects in branching morphogenesis. While neither apoptosis nor cell proliferation differed in metanephric mesenchyme cells in Alk3IM nulls, markers of renal progenitor cells were decreased in mutant animals. It was observed that Alk3 expression in the intermediate mesoderm also controls mesonephric tubule number. Alk3IM nulls had fewer mesonephric tubules and fewer derivative Leydig cells. The reduction in Leydig cells resulted in decreased levels in serum testosterone and defects in seminal vesicle formation and fertility. Alk3 expression was also required for normal development of the corpus epididymis. The morphological defects in nephrogenesis were associated with decreased phospho-p38 MAPK expression and in the testis with decreased Phospho-SMAD1/5/8. These results elucidated a requirement for Alk3 signaling in controlling progenitor cells derived from the intermediate mesoderm.