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

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.

So far, in vitro generation of haematopoietic stem cells (HSCs) has only been achieved through enforced expression of transgenes. This makes them inadequate for clinical use. In contrast, transgene-free generation of other cell types has been obtained by mimicking their embryonic development. In order to use this approach to generate HSCs, we need to fully understand the embryonic programming of the HSC lineage. HSCs derive from definitive haemangioblasts (DHs) emerging from the lateral plate mesoderm (LPM). How the LPM acquires the HSC fate is not known. Here, we aim to understand how nascent LPM is programmed to give rise to DH. In Xenopus embryos, blastomere C3 of the 32-cell stage embryo gives rise to DH and HSCs. Therefore, blastomere C3 will be labelled and its descendants will be isolated at several stages of development, from gastrulation (stage 10.5) up to the emergence of DH (stage 26), and subject to single-cell RNA-sequencing. Then, the differentiation pathway taken by nascent LPM to become DHs will be reconstructed through bioinformatic analyses. As blastomere C3 gives rise to other mesodermal lineages, we will investigate when these lineages branch out from the DH fate and identify transcription factors (TFs) with the potential to drive such branching events. The role of these TFs in cell fate determination will then be investigated by genetic perturbation. LPM marker genes will be identified and their specificity validated by in situ hybridisation on Xenopus and mouse embryos. This set of marker genes will then be used to monitor and optimise the induction of LPM from human ES cells. The optimal conditions for LPM induction will be established through combinatorial manipulation of, amongst other pathways, WNT, BMP and retinoic acid signalling. Finally, using the information obtained from our single-cell RNA-seq on Xenopus cells, ES cell-derived LPM will be differentiated into DHs. This will bring us closer to the generation of HSCs in vitro.

The proposed research will directly contribute to two long-term interlinked goals: (1) to understand the mechanism by which HSCs are generated in the embryo and (2) to apply the knowledge generated in embryos into the in vitro generation of HSCs for clinical use. The immediate beneficiaries of this research will be the research community as it will further our knowledge of the cellular and molecular mechanism underpinning cell fate decisions during early embryogenesis. The development and patterning of the lateral plate mesoderm is particularly poorly understood, therefore, this study will shed light into the development of this tissue and tissues derived from it.
Here, we are proposing to develop a protocol for the differentiation of pluripotent stem cells into the HSC lineage based on the natural sequential steps observed in the embryo. We expect that this strategy will allow the successful generation of HSCs in vitro in the long-term as the proposed protocol begins with the generation of the correct type of mesodermal progenitors, the lateral plate mesoderm, whereas current protocols are likely to start with the generation of posterior (tail) or yolk sac-type mesoderm. In vitro generation of HSCs will benefit, in short to medium term, the treatment of patients with haematological malignancies. A reliable source of transplantable HSCs will have an immediate impact in public heath as new cases of leukaemia (9,534), for example, represented 3% of new cancer cases diagnosed in the UK in 2014 (Cancer Research UK). In vitro generation of HSCs together with the development of early, accurate, and rapid diagnosis of haematological diseases will significantly benefit the quality of life of patients in medium/long term.
Definitive haemangioblasts are at the foundation of the vascular system as they give rise to venous, arterial and lymphatic vessels during embryogenesis. So their availability in vitro could serve as the resource for the differentiation of endothelial lineages for clinical application. Therefore, this research could also benefit patients with vascular diseases.
Through the realisation of this project, the co-applicant will become highly skilled on single-cell RNA-seq, pluripotent stem cell culture, FACS, bioinformatics and statistical techniques. This will be achieved by attending specialised training courses within the University of Oxford and other institutions as well as through direct experimental experience under the supervision of experts in those fields. The training timeline will be in accordance with the projects necessities and progress of the project will be regularly assessed through lab meetings and interaction with colleagues.