Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region

A CD45-negative population of pre-HSCs develops into definitive HSCs in the AGM region of the embryo

Understanding the origins of haematopoietic stem cells in the E11.5 AGM region using a novel reaggregate culture system

Identifying the sites and mechanisms involved in haematopoietic stem cells (HSCs) during development would improve our understanding of how to induce HSCs from alternative sources like embryonic stem cells, while offering insight into pathways involved in HSC-related diseases such as leukaemia. Adult-type HSC, or long-term reconstituting HSCs (LTR-HSCs), are widely defined as cells capable of reconstituting the entire haematopoietic system of a lethally irradiated adult recipient. The first LTR-HSCs emerge and expand in the aorta-gonad-mesonephros (AGM) region of the mid-gestation mouse embryo. Recently, the development of a novel reaggregate culture system has provided a valuable tool to identify key cell populations involved in LTR-HSC development. This system allows the mechanical dissociation of the E11.5 AGM region prior to culture whilst maintaining its ability to autonomously expand LTR-HSCs. Here, I show that reaggregate LTR-HSCs are CD45+Sca1+c-kit+CD31med and that IL-3, SCF, and Flt3l are required in order to achieve an optimal 150 fold LTR-HSC expansion. I also characterise the pattern of Runx1 expression in the adult and E11.5 AGM region of our novel Runx1EGFP reporter mouse and identify a population of EGFP+CD45-VE-cadherin- cells in the E11.5 AGM region that disappears during reaggregate culture. Finally, using the E11.5 AGM reaggregate culture, I show that while uro-genital ridges are potentially required for optimal LTR-HSC expansion, most LTR-HSCs are derived from the dorsal aorta (Ao) region, and that the dorsal aspect of the dorsal aorta (AoD) can contribute to the reaggregate LTR-HSCs compartment.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Analysis and manipulation of hematopoietic progenitor and stem cells from murine embryonic tissues

Hematopoietic development begins in several locations in the mammalian embryo: yolk sac, aorta-gonad-mesonephros region (AGM), and the chorio-allantoic placenta. Generation of the most potent cells, adult definitive hematopoietic stem cells (HSCs), occurs within the body of the mouse embryo at midgestation in the AGM region. Similarly, at the equivalent developmental time in the human embryo, the AGM region has been shown to contain multipotent progenitors. Hence, the mouse embryo serves as an excellent model to study hematopoietic development. To further studies on the ontogeny of the adult hematopoietic system, the focus of this unit is on the experimental methods used in analysis of the AGM region