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

以单细胞分辨率产生造血干细胞谱系的侧板中胚层的体内表征

• 批准号: BB/S008144/1
• 负责人: Catherine Porcher
• 金额: $ 56.76万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FS008144%2F1
• 关键词: vivo; characterisation; lateral; plate; mesoderm

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.

造血干细胞 (HSC) 具有产生一生中所有血细胞的能力。它们在临床上具有高度相关性，因为它们是骨髓移植的重要组成部分，可帮助患有白血病、贫血和淋巴瘤等血液疾病的患者恢复正常造血功能。然而，尽管经过半个多世纪的研究，造血干细胞的供应仍然不足，并且无法在骨髓外扩增。不过，最近的技术进步使得通过多能干细胞 (PSC) 生成 HSC 来缓解 HSC 短缺成为可能。然而，HSC 的体外生产一直具有挑战性，到目前为止，只能通过强制表达潜在有害基因来实现，这使得它们不足以移植到人体。因此，需要一种不使用此类基因操作来产生HSC的方法。重要的是，肌肉、肾脏和其他组织的产生是通过模仿胚胎发育来实现的。因此，了解指导胚胎中 HSC 生成的细胞和分子过程有可能为 PSC 的有效生成提供基础知识。骨髓 HSC 是通过一系列复杂的事件在胚胎中生成的，这些事件始于胚胎躯干区域中侧板中胚层 (LPM) 的形成。从历史上看，这些事件在低等脊椎动物模型生物（例如青蛙）中得到了最好的研究。由于很容易获得外部发育的胚胎，并且产生 HSC 的过程具有进化保守性，因此对青蛙胚胎血液发育的研究有助于我们了解该过程中涉及的分化途径。通过连续的诱导事件，LPM 首先产生最终的成血管细胞 (DH)，然后产生动脉内皮、造血内皮，最后产生 HSC。体外 HSC 的生成需要精确重现指导这些瞬时前体细胞生成的外在信号（细胞/分子）。只有当我们知道所需信号的性质和时间要求时，才能实现这一点。因此，充分了解 HSC 的胚胎发育至关重要。重要的是，LPM 最初是如何产生的以及它如何获得 DH 命运尚不清楚。如果我们要从 PSC 生成 HSC，此信息至关重要。在爪蟾胚胎中，LPM 中胚层源自 32 细胞阶段胚胎的一个细胞，即卵裂球 C3。因此，通过标记卵裂球 C3，可以在中胚层前体及其衍生物变成 DH 之前将其分离出来。在这个项目中，我们将在从 LPM 的产生到 DH 出现的几个阶段分离 C3 衍生细胞，并对它们进行单细胞 RNA 测序，目的是确定新生 LPM 中 DH 命运是如何确定的。这也将允许鉴定新生 LPM 的标记基因，然后可用于优化其在 PSC 分化系统中的生成。目前，PSC 的血细胞分化是在有利于尾部中胚层形成而不是躯干 LPM 形成的生长因子条件下实现的。因此，我们将操纵生长因子的数量来定义 LPM 生成的最佳条件。然后，利用 C3 衍生细胞的单细胞 RNA 测序产生的知识，LPM 将被指示生成 DH。从 PSC 生成 DH 是生成用于临床应用的 HSC 的第一步，也是最重要的步骤之一。

项目成果

Specification of the haematopoietic stem cell lineage: From blood-fated mesodermal angioblasts to haemogenic endothelium.

• DOI: 10.1016/j.semcdb.2022.01.008
• 发表时间: 2022-02
• 期刊: Seminars in cell & developmental biology
• 影响因子: 7.3
• 作者: V. Ho;D. E. Grainger;H. Chagraoui;C. Porcher