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Title: Modelling of arterial and haemogenic endothelium development from mouse and human pluripotent stem cells
Author: Ruiz Villalobos, Juan Pablo
ISNI:       0000 0004 8507 2272
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2019
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One of the most promising objectives of clinical haematology is to derive transplantable autologous haematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cell (PSC) sources in vitro, but efficient and clinically relevant methodologies remain unavailable. Observations in the developing vertebrate embryo indicate that the haematopoietic stem cells (HSCs) capable of long-term engraftment are produced by the third and last wave of haematopoietic development. This occurs in the developing dorsal aorta (DA) from haemogenic endothelium (HE) in close association with an arterial vascular endothelial niche. Notch and Vascular endothelial growth factor A (VEGFA) signalling pathways are critical regulators of arterial identity and HSPC development. To better understand the lack of engrafting HSCs in in vitro PSC differentiation protocols, we further characterised a previously published, serum-free murine embryonic stem cell (mESC) embryoid body (EB) differentiation system. Results showed lack of a robust arterial niche and Notch signalling, and production of HSPCs resembling the erythro-myeloid progenitors (EMP) normally detected during one of the earlier, extra-embryonic waves of haematopoietic development. We then showed that transiently culturing cells with high concentrations of VEGFA led to activation of an arterial programme in cultured cells, though this was insufficient to drive HSC differentiation. Importantly, we showed the haematopoietic potential of arterial delta-like 4 (DLL4) expressing cells depends on VEGFA concentrations in the culture, demonstrating the potential of arterial cells to become HE. To adapt these findings to a more clinically-translatable setting, we also characterised a serum-free, monolayer-based system for the differentiation of human HSPCs previously developed in the lab, commercially available as STEMdiff™ Hematopoietic Kit. Similar to results in the murine system, the supporting niche showed limited arterial vascular endothelium differentiation and was instead primarily composed of mesenchymal stromal-like cells. Culturing cells in high concentrations of VEGFA markedly enhanced arterial endothelium formation throughout culture, and concurrently decreased mesenchymal stroma. Likewise, we observed the presence of immunophenotypic DLL4+ HE at early stages in cultures treated with high VEGFA concentrations, confirming data obtained from the mESC system. Despite lack of engraftment capacity of HSPCs, our studies contribute potential insights to the question of the haematopoietic potential of arterial endothelium. This will guide further improvement of currently available protocols to support in vitro HSPC differentiation.
Supervisor: Porcher, Catherine ; Patient, Roger Sponsor: Howard Hughes Medical Institute
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Stem Cell Biology ; Developmental Biology ; Haematopoiesis