UNDERSTANDING MOLECULAR MECHANISMS UNDERLYING DEVELOPMENT OF HIGHLY REGENERATIVE HUMAN HAEMATOPOIETIC STEM CELLS

血液干细胞，也称为造血干细胞（hsc），产生成人血液系统。由于造血干细胞可以自我更新，它们在动物的整个生命周期中维持各种血细胞类型的产生。这些重要的细胞如何在胚胎发育过程中出现是干细胞和发育生物学中最有趣的问题之一。造血干细胞是骨髓和脐带血的重要组成部分，广泛用于血液疾病或癌症患者的临床移植，需要恢复其血液系统。全球每年进行的造血干细胞移植超过5万例，但对造血干细胞的需求大于供应。尽管付出了巨大的努力，但在实验室条件下，寻找从替代细胞来源生产高质量造血干细胞的方法取得了有限的成功。尽管人们寄予厚望，但在培养皿中能够产生血细胞的多能干细胞（hPSCs）为何不能产生真正的造血干细胞，这仍是一个谜。最有可能的原因是，我们对这些细胞在胚胎发育过程中，特别是在人类中，是如何首次出现的理解仍然不足。我们的目标是在这里通过询问人类HSC胚胎发育过程中表达的基因功能来解决这一基础知识的差距。这个项目是基于我们在小鼠和人类成人造血干细胞胚胎发育领域的成就。我们之前发现造血干细胞首先出现在胚胎内部的区域，包括背主动脉，现在称为AGM区域，并通过表面标记物识别这些造血干细胞。此外，我们发现这些在人类AGM区域出现的第一批造血干细胞具有巨大的再生潜力，远远高于目前用于临床移植的脐带血造血干细胞。这种巨大的再生潜力使得在人类AGM区域出现的第一批hsc的特性不仅对基础干细胞生物学而且对临床应用具有很高的吸引力。利用细胞纯化、分子生物学和生物信息学方法，我们揭示了那些在第一批造血干细胞中表达的基因，而不是在培养的造血干细胞衍生的类似细胞群中表达的基因。这些基因中的一些一定对胚胎中高度再生的造血干细胞的发育负责我们这里的主要目标是识别它们并了解它们在造血干细胞发育过程中究竟起什么作用？为了解决这个问题，我们将设计这些通常沉默的基因在造血干细胞中表达。这将使我们能够确定这些基因对造血干细胞重要特征的影响，如自我更新潜力和免疫细胞的产生。最终，我们将测试这些基因的任何组合是否会导致产生临床相关的、高效的可移植造血干细胞。为此，我们将把这些细胞移植到所谓的异种移植物NSG小鼠中，在这种小鼠中可以维持人类血细胞。此外，我们将测试这些基因是否可以增强脐带血移植中造血干细胞的再生能力。这项研究将为人类血液发育的正常和潜在的先天病理过程的基本分子机制提供深入的见解。这也将有助于我们在实验室条件下更好地控制HSC的操作，并长期帮助开发新的基于细胞的疗法来满足临床需求。

Blood stem cells, also called haematopoietic stem cells (HSCs), give rise to the adult blood system. Since HSCs can self-renew, these sustain production of various blood cell types throughout the entire life of an animal. How these important cells emerge during embryo development is one of the most intriguing questions of stem cell and developmental biology. HSCs are important components of the bone marrow and umbilical cord blood, which are broadly used for clinical transplantation into patients who have blood disorders or cancers and require restoration of their blood system. More than 50,000 HSC transplantations are performed yearly worldwide, but demand for HSCs outstrips supply. Despite extensive efforts, the search for methods to produce high quality HSCs from alternative cell sources, in the laboratory conditions has met with limited success. It remains a puzzle, why in spite of great hopes, pluripotent stem cells (hPSCs) that can generate blood cells in the Petri dish, fail to generate true HSCs. The most likely reason for this is that our understanding of how these cells first emerge during embryo development, particularly in humans, remains insufficient. We aim here to address this gap in fundamental knowledge by interrogating functions of genes expressed during human HSC embryonic development. This project is based on our achievements in the field of embryonic development of adult HSCs, both in mouse and human. We previously found that HSCs first emerge in the region inside the embryo, which encompasses the dorsal aorta, called now the AGM region, and identified these HSCs by surface markers. Furthermore, we have found that these first HSCs emerging in the human AGM region possess an enormous regenerative potential, much higher than umbilical cord blood HSCs, which are currently used for transplantation in clinics. This vast regenerative potential makes the properties of the first HSCs emerging in the human AGM region highly attractive not only for fundamental stem cell biology but also for clinical applications. Using cell purification, molecular biology and bioinformatics methods, we have revealed those genes that are expressed in the first HSCs but not in the similar cell population derived from hPSCs in culture. Some of these genes must be responsible for the development of the highly regenerative HSCs in the embryo and our main goal here is to identify them and understand - what exactly they do in the HSCs during development? To address this question, we will engineer hPSCs in which these normally silent genes will be expressed. This will allow us to determine the effects of these genes on important HSC characteristics, such as self-renewal potential and generation of immune cells. Ultimately, we will test whether any combination of these genes will result in the generation of clinically relevant, highly potent transplantable HSCs. To this end, we will transplant the cells into so-called xenograft NSG mice, in which human blood cells can be maintained. Additionally, we will test, whether these genes can enhance the regenerative power of HSCs within umbilical cord blood transplants. This study will provide deep insights into fundamental molecular mechanisms of normal and, potentially, inborne pathological processes underlying human blood development. This will also help us to gain a better control of HSC manipulations in laboratory conditions and in the long-term help with development of new cell-based therapies to meet clinical demands.