Tracking the embryonic origin of the adult haematopoietic system

When a patient who has undergone chemotherapy or radiotherapy receives a bone marrow transplant, it includes blood stem cells also known as haematopoietic stem cells (HSCs) which are the long-term source of normal blood. However, there is a shortage of bone marrow and umbilical cord transplants and there is a major interest to generate HSCs in the laboratory conditions. Although HSCs develop during embryo development, it is not yet clear how to reproduce this process in cultured cells. To understand mechanisms that the embryo uses to generate HSCs, researchers need to first trace their origin in the embryo and track their development, which is a surprisingly poorly understood process. The first blood cells in the early embryo develop outside its body in a membrane called the yolk sac. However, true HSCs which can persist in the adult animal appear later in a specific part of the developing embryo. The main objective of this research proposal is to identify the precise site of origin of adult HSCs. The chick embryo is very useful for this research as we can see its development in a dish or in the egg, without disrupting normal development. To this end we have developed new tools - transgenic chickens that express fluorescent proteins, so that their cells can be easily followed in live embryos under the microscope.

By transplantation of precise transgenic chick embryonic tissue fragments from regions suspected to harbour the ancestors of HSCs into normal chicken embryos we will monitor whether they develop into blood. This will allow us to define the exact location of embryonic cells from which HSCs develop. Novel transgenic chick strains where particular cell types are labelled by different fluorescent reporter proteins will further define more precise localisation of embryonic ancestors of HSCs. The development of these new tools will give us a unique opportunity to identify and characterise HSCs. In the longer terms, this knowledge will inform the development of methods for generation of HSCs from embryonic stem cells for treatment of patients whose blood formation was impaired due to chemo- or radiotherapy.

The embryonic origin of haematopoietic stem cells (HSCs) is a controversial issue and HSC development remains poorly understood. This issue is of potentially high importance for medical treatment of haematopoietic disorders. The analysis of HSC development in the mammalian embryo is hampered by intra-uterine development and the main objective of this research proposal is to identify the HSC origin in amniotes. Chick embryos develop outside the mother and can thus be manipulated and observed without significant disruption of development. Using recently developed transgenic chicken technology, we will perform orthotopic grafting of tissue fragments from ubiquitously expressing GFP-positive embryos to determine the location of embryonic HSC precursors in the primitive streak and lateral mesoderm. We will establish whether the transitory yolk sac and adult (permanent) haematopoietic hierarchies are separate at early stages of embryogenesis. We will also generate two novel transgenic chicken models, Flk1-GFP and Runx1-tdTomato which will allow us to monitor and map progressive development of the mesoderm/ endothelium/ blood in space and time. Well recognisable two-colour pattern evolving in compound [Flk1-GFP;:Runx1-tdTomato] embryos will allow us to perform fine systematic isolation and orthotopic grafting of small groups of cells. By this, the origin of the developing yolk sac haematopoietic wave and of the HSC lineage will be mapped with high definition. In the early embryo the cell fates may not be fixed and remain rather plastic. By heterotopic transplantations we will investigate whether this is the case. Live imaging of transitory and permanent HSC hierarchies will allow us to define a potentially complex series of trajectories of migration of HSC precursors. Observing these intermediate staging-posts and correlating them with the known locations of extracellular signalling molecules is essential to define the niches for HSC development.

This research project, investigating fundamental aspects of origin and development of haematopoietic stem cells (HSCs), has significant translational implications in the longer term. This project aims to identify early embryonic precursors of transplantable HSCs. Although pluripotent EE/iPS cells are an excellent potential source of HSCs which can be used to overcome shortages of bone marrow and umbilical cord transplants in clinics, no reproducible protocols have been developed to generate these cells in the laboratory. The proposed research will allow us to make a step forward in understanding the nature of embryonic precursors of HSCs and in future analyse molecular mechanisms underpinning their specification. This programme is a strong and realistic pathway to translation of our results into medical practice in future with great positive impact on human health. In addition we will develop two novel transgenic chicken lines. These will be of considerable value to research projects of the named collaborators but also to developmental biologists who use the chick model. By the end of this proposed programme the fertile eggs from these lines will be available to the research community. The information obtained during this research may help to develop efficient protocols for production of specialized haematopoietic cells from human embryonic stem cells/iPS cells.