Cellular barcoding of developmental hematopoiesis

项目摘要/摘要 造血干细胞(HSCs)位于血液等级的顶端，能够产生所有的血液 有机体的细胞。因此，它们在治疗血液方面具有巨大的治疗潜力。 再生生物学的中心目标是在体外培养HSCs。尽管有这种临床需要，我们 缺乏允许我们在体外有效地产生能够长期植入和 多谱系输出。一个主要的障碍是我们对造血干细胞是如何在体内制造的不完全的理解。为 例如，尽管已经确定血细胞是从整个 胚胎，我们仍然不知道哪些胚胎部位会产生长期的造血干细胞，也不知道起源部位如何影响 终生干细胞的功能或行为。此外，我们对内在和本质的理解是有限的 驱动血液内皮细胞功能异质性的外在线索。该项目建议使用 强大的下一代条码技术，丰富了我们对造血干细胞胚胎起源的理解 产生血液的血液来源的内皮细胞状态使我们能够在体外利用这一过程。 鲍林博士从事发育生物学的研究生工作，并在博士后培训期间 专注于开发下一代DNA条形码工具，用于执行单细胞、可诱导细胞 活体血统追踪。配备了这一技能，鲍林博士计划使用创新的手机条形码 首次对长寿造血祖细胞的准确内皮起源进行分类的技术 哺乳动物胚胎(目标1)。此外，她将深入刻画异质的 在胚胎中产生不同血细胞的内皮细胞状态(目标2)。从以下方面产生的知识 这些实验有可能回答发展领域中长期存在的重大问题 造血和改变我们对导致血液生成的步骤的基本认识，因此 革新体外培养HSC的方案。 鲍林博士得到了一组导师和顾问的支持，他们都是世界一流的研究人员 血液学、发育生物学和技术发展。她的导师费尔南多·卡马戈博士和 伦纳德·宗在造血领域做出了非凡的贡献，也有出色的业绩- 导师关系的记录。鲍林博士将从她那里获得进一步的科学培训和职业发展支持 科学委员会：杰伊·申杜尔博士、特里斯塔·诺斯和贝托尔德·戈金斯。最后，她将受益于携带 在波士顿儿童基金会科学刺激和资源丰富的环境中制定了她的研究计划 医院和哈佛医学院。这项提案的目标将使鲍林博士能够在她的技能基础上 在测序数据集的计算分析和诱导多能性的使用方面获得专业知识 干细胞，为她向独立的过渡做准备。因此，她将建立一个独特的利基市场 作为一名独立研究人员，解决重要的、与临床相关的造血发展问题。

PROJECT SUMMARY/ ABSTRACT Hematopoietic stem cells (HSCs) lie at the top of the blood hierarchy and are capable of giving rise to all blood cells of an organism. Consequently, their use has enormous therapeutic potential for the treatment of blood diseases, and generation of HSCs in vitro is a central aim in regenerative biology. Despite this clinical need, we lack protocols that allow us to efficiently generate HSCs in vitro that are capable of long-term engraftment and multi-lineage output. A major hindrance is our incomplete understanding of how HSCs are made in vivo. For instance, although it is established that blood cells develop from endothelial cells in multiple sites throughout the embryo, we still do not know which embryonic sites produce long-term HSCs, nor how site of origin impacts on life-long stem cell function or behavior. Furthermore, we are limited in our understanding of the intrinsic and extrinsic cues driving functional heterogeneity in hemogenic endothelial cells. This project proposes to use powerful next-generation barcoding technology to enrich our understanding of the embryonic origin of HSCs and the hemogenic endothelial cell states that give rise to blood to allow us to harness the process in vitro. Dr. Bowling conducted her graduate work in developmental biology and during her postdoctoral training has focused on the development of next-generation DNA barcoding tools for performing single cell, inducible cell lineage tracing in vivo. Equipped with this skillset, Dr. Bowling plans to use innovative cellular barcoding techniques to, for the first time, catalog the precise endothelial origins of long-lived blood progenitors in the mammalian embryo (Aim 1). Furthermore, she will perform in-depth characterization of the heterogeneous endothelial cell states that give rise to distinct blood cells in the embryo (Aim 2). The knowledge generated from these experiments have the potential to answer major, long-standing questions in the field of developmental hematopoiesis and transform our basic understanding of the steps leading to blood generation, and therefore revolutionize protocols for HSC generation in vitro. Dr. Bowling is supported by a panel of mentors and consultants who are world-class researchers in hematology, developmental biology, and technology development. Her mentors Drs. Fernando Camargo and Leonard Zon have made exceptional contributions to the field of hematopoiesis and also have outstanding track- records for mentorship. Dr. Bowling will gain further scientific training and career development support from her scientific committee: Drs Jay Shendure, Trista North and Berthold Gottgens. Finally, she will benefit from carrying out her research program in the scientifically stimulating and resource-rich environment of Boston Children’s Hospital and Harvard Medical School. The aims in this proposal will allow Dr. Bowling to build on her skills to gain expert knowledge in the computational analysis of sequencing datasets and the use of induced pluripotent stem cells, in preparation for her transition to independence. As a result, she will establish a unique niche for resolving important, clinically-relevant questions in hematopoietic development as an independent researcher.