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

造血干细胞(HSCs)具有在一生中产生所有血细胞的能力。它们在临床上具有很高的相关性，因为它们是骨髓移植的基本组成部分，可以恢复白血病、贫血和淋巴瘤等血液疾病患者的正常造血。然而，尽管经过了半个多世纪的研究，造血干细胞的供应并不充足，而且不能在骨髓外扩增。然而，最近的技术进步开启了通过从多能干细胞(PSCs)中产生HSC来缓解HSC短缺的可能性。然而，造血干细胞的体外生产一直是具有挑战性的，到目前为止，它只能通过强制表达潜在的有害基因来实现，这使得它们不适合在人类身上移植。因此，需要一种在不使用这种基因操作的情况下生产造血干细胞的方法。重要的是，肌肉、肾脏和其他组织的生产是通过模仿它们的胚胎发育实现的。因此，了解指导胚胎中HSCs生成的细胞和分子过程有可能为PSC的高效生成提供基础知识。胚胎中的骨髓HSCs是通过一系列复杂的事件在胚胎中产生的，起始于胚胎主干区域侧板中胚层(LPM)的形成。从历史上看，这些事件最好的研究对象是低等脊椎动物模型生物，如青蛙。由于易于获得外部发育的胚胎，以及产生HSCs的过程的进化保守，对青蛙胚胎血液发育的研究有助于我们理解这一过程中涉及的分化途径。通过一系列的诱导事件，LPM首先产生确定的血管母细胞(DHS)，然后是动脉内皮细胞、血源性内皮细胞，最后是HSCs。在体外生成HSCs将需要准确地重现指导这些瞬时前体细胞产生的外部信号(细胞/分子)。这只有在我们知道所需信号的性质和时间要求的情况下才能实现。因此，充分了解HSCs的胚胎发育是至关重要的。重要的是，LPM最初是如何产生的，它是如何获得DH的命运尚不清楚。如果我们要从PSC生成HSC，这些信息是必不可少的。在非洲爪哇胚胎中，LPM中胚层来自32-细胞期胚胎的一个细胞，即卵裂球C3。因此，通过标记卵裂球C3，中胚层前体及其衍生物可以在它们成为DH之前被分离出来。在这个项目中，我们将在几个阶段分离C3来源的细胞，从LPM的产生到DHS的出现，并对它们进行单细胞RNA测序，目的是确定DH在新生LPM中的命运是如何建立的。这也将允许识别新生的LPM的标记基因，然后可以用于优化其在PSC分化系统中的生成。目前，PSCs的血细胞分化是在生长因子条件下实现的，这种条件有利于尾部中胚层的形成，而不是干LPM。因此，我们将操纵生长因子的量来定义产生LPM的最佳条件。然后，使用C3来源的细胞上的单细胞RNA测序产生的知识，LPM将被指示产生水解酶。从PSCs产生肝干细胞是临床应用HSCs的第一步，也是最重要的步骤之一。

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.