Spatial and molecular characterisation of the cellular niches interacting with intra-embryonic haemato-endothelial progenitors

Haematopoietic stem cells (HSCs) are self-renewing cells sustaining production of the adult blood system throughout life. The entire pool of HSCs is produced during embryonic development, from a specialised arterial endothelium primarily in the dorsal aorta (DA). There is increasing evidence that blood fate is specified during mesoderm patterning, at a restricted anterior-posterior (A/P) embryonic position, before emergence of the first HSCs. Although essential when aiming to produce HSCs in vitro from pluripotent stem cells (PSCs), and for a complete understanding of HSC production, little is known about the signalling pathways and molecular mechanisms instructing a blood cell identity in mesodermal haemato-endothelial progenitors (HEPs). I have studied the microenvironment supporting some of the earliest haematopoietic specification mechanisms in 2 different ways. First, as intra-embryonic HEPs and intermediate mesoderm (IM)- derived mesonephric duct share restricted A/P co-ordinates, I differentiated human PSCs in culture conditions known to support the development of the mesonephric duct and permissive for endothelial differentiation. I report production of KDR+ endothelial progenitors in PAX8+ IM niches and their differentiation towards CD144+DLL4+ arterial endothelium. Testing four haemogenic induction regimes did not reproducibly impart a haematopoietic fate, suggesting our culture conditions were not optimum for initiation or maintenance of a blood cell fate. Secondly, I studied HEPs in their native environment in mouse embryos. After establishing a quantitative, multiplexed single molecule RNA FISH method, I described the complex cellular environment of distinctly located HEPs in the E8.0 mouse embryonic posterior region, including those forming the future DA. To explore how these spatially distinct neighbourhoods might govern blood cell fate acquisition, I generated scRNA-seq data from the same embryonic region and performed cell communication analyses. I identified many classes of signalling critical for HSC emergence, suggesting these signals could function earlier than previously described, and define thrombospondin-CD47 and Cgrp-Calcrl as novel signals for functional validation