In vivo characterisation of the lateral plate mesoderm giving rise to the haematopoietic stem cell lineage at a single cell resolution

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Haematopoietic stem cells (HSCs) have the capacity to produce all blood cells throughout life. They are highly relevant in the clinic as they are the essential components in bone marrow transplants that restore normal haemopoiesis in patients with blood diseases such as leukaemias, anaemias and lymphomas. Yet, despite over half a century of research, HSCs are not in sufficient supply and cannot be expanded outside the bone marrow. Recent technological advances, though, have opened the possibility of alleviating the HSC shortage through their generation from pluripotent stem cells (PSCs). However, in vitro production of HSCs has been challenging and, so far, it has only been achieved through forced expression of potentially harmful genes which makes them inadequate for transplantation in humans. Therefore, there is a need for a method to produce HSCs without the use of such genetic manipulation. Importantly, production of muscle, kidney and other tissues has been achieved by mimicking their embryonic development. Thus, understanding the cellular and molecular processes guiding the generation of HSCs in embryos has the potential to provide fundamental knowledge for their efficient generation from PSCs.Bone marrow HSCs are generated in the embryo through a complex sequence of events initiating with the formation of lateral plate mesoderm (LPM) in the trunk region of the embryo. Historically, these events have been best studied in lower vertebrate model organisms, such as frogs. Because of easy access to externally developing embryos and the evolutionary conservation of the processes giving rise to HSCs, studies of blood development in frog embryos have been instrumental for our understanding of the differentiation pathways involved in this process. Through sequential inductive events, LPM first produces definitive haemangioblasts (DHs) and then arterial endothelium, haemogenic endothelium and, finally, HSCs. Generation of HSCs in vitro will require the exact recapitulation of the extrinsic signals (cells/molecules) directing production of these transient precursor cells. This can only be achieved if we know the nature and temporal requirement of the signals needed. Full understanding of embryonic development of HSCs is therefore essential.Importantly, how the LPM is generated in the first place and how it acquires the DH fate is not known. This information is essential if we are to generate HSCs from PSCs. In Xenopus embryos, the LPM mesoderm derives from one cell, blastomere C3, of the 32-cell stage embryo. Therefore, by marking blastomere C3, mesodermal precursors and their derivatives can be isolated before they become DH. In this project, we will isolate C3-derived cells at several stages, from the generation of LPM up to the emergence of DHs, and subject them to single-cell RNA-sequencing, with the goal of establishing how the DH fate is established in nascent LPM. This will also allow the identification of marker genes for nascent LPM which can then be used to optimise its generation in PSC differentiation systems. Currently, blood cell differentiation from PSCs is achieved under growth factor conditions that favour tail mesoderm formation rather than trunk LPM. Therefore, we will manipulate the amount of growth factors to define optimal conditions for the generation of LPM. Then, using the knowledge generated from single-cell RNA-sequencing on C3-derived cells, LPM will be instructed to generate DH. The generation of DH from PSCs represents the first, and one of the most important steps in the generation of HSCs for clinical applications.