Functional Analysis of the Hematopoietic Niche during Ontogeny

描述(申请人提供)：据认为，在骨髓中，至少有两个在解剖和生理上不同的造血区：成骨细胞和血管。在这些利基中，许多分子和分泌因子参与了严格调控的造血过程。然而，对与每个生态位相互作用的HSC亚群以及每种生态位类型在调节干细胞层次和功能中的生理作用仍然缺乏透彻的了解。在这里，我们将使用骨髓获得支持造血的能力的发育过程作为一个独特的范例，确定每个利基内在表型、功能和转录水平上发生的细胞相互作用。将要进行的实验将使用与生理相关的大型动物模型、高分辨率成像/捕获技术、敏感的转录组测序和功能研究，这些技术结合在一起为我们提供了独特的能力来研究和操纵体内人类HSC和特定的利基细胞的相互作用，从而揭示每个离散的微环境在建立和维持人类造血系统中所起的作用。这些研究的总体假设是，胎儿骨髓获得支持造血能力的发育过程可以作为一个模型，用于理解HSC和构成骨髓壁龛的细胞之间的相互作用，以及这些生态位元素在造血启动/维持中的作用。我们进一步假设，这些知识将提供必要的工具来操纵这些小生境，以促进植入和加速供者HSCTx后的造血恢复。我们提出了以下具体目标：1)定义胎儿BM造血生态位出现和骨髓造血开始的步骤和关键细胞角色，作为一种工具，以了解发生在血管和成骨细胞生态位内和之间的细胞相互作用，并表征每个支持的造血细胞的性质，从而描述每个细胞在维持和扩大干细胞池中所起的作用，以驱动终生造血；2)确定新生骨髓中的血管/血管周围壁龛是否不仅是支持来自胎肝的HSC安放的场所，而且还包含有可能促进造血的细胞；以及3)研究特定的供体来源细胞群体是否可以操纵骨和血管壁龛的组成，以提高移植水平和/或加速供体来源的HSC在异基因移植后的重建。这些研究的成功将对分离和扩增原始HSC和了解恶性血液病的起源具有重要意义，并可能显著提高临床HSC移植的安全性和成功率，并有可能使其应用于影响人类患者的更广泛的疾病。 公共卫生相关性：以胎儿发育为模型，彻底了解造血干细胞和构成骨髓微环境的细胞之间的相互作用，将为操纵这些微环境提供必要的工具，以促进供者HSC的植入，并加速HSC移植后的造血恢复。这将显著提高临床造血干细胞移植的安全性和成功率，并有可能将其应用于影响人类患者的更广泛的疾病。

DESCRIPTION (provided by applicant): It is thought that within the marrow there are at least two anatomically and physiologically distinct hematopoietic niches; the osteoblastic and the vascular. Within these niches, numerous molecules and secreted factors have been implicated in the strictly regulated process of hematopoiesis. Nevertheless, a thorough understanding of the subpopulations of HSC that interact with each niche and the physiological role of each niche type in the regulation of stem cell hierarchy and function is still missing. Here, we will identify the cellular interactions that occur within each of the niches at the phenotypic, functional, and transcriptional levels, using as a distinct paradigm the developmental process whereby the marrow acquires the ability to support hematopoiesis. Experiments to be performed will employ a physiologically relevant large animal model, high resolution imaging/capture technology, sensitive transcriptome sequencing, and functional studies, which combined provide us with the unique ability to study and manipulate the interactions of human HSC and specific niche cells in vivo, thereby unraveling the role played by each discrete microenvironment in the establishment and maintenance of human hematopoiesis. The overall hypothesis of these studies is that the developmental process whereby the fetal marrow acquires the ability to support hematopoiesis can be used as a model for understanding both the interactions that occur between HSC and the cells comprising the marrow niches, and the role of these niche elements in the initiation/maintenance of hematopoiesis. We further hypothesize that this knowledge will provide the necessary tools for manipulating these niches to facilitate the engraftment and accelerate hematopoietic recover of donor HSC after HSCTx. We propose the following Specific Aims:1)Define the steps and key cellular players in the emergence of the fetal BM hematopoietic niche and in the onset of marrow hematopoiesis, as a tool to understand the cellular interactions that occur both within and amongst the vascular and the osteoblastic niches as they arise, and characterize the nature of the hematopoietic cells each supports, thus delineating the role each plays in the maintenance and expansion of the stem cell pool to drive lifelong hematopoiesis;2) Determine whether the vascular/perivascular niches present within the nascent marrow not only serve as a supportive site for lodging of HSC arriving from the fetal liver, but also harbor cells with the potential to contribute to the establishment of hematopoiesis; and 3) Investigate whether the composition of the bone and vascular niches can be manipulated by specific populations of donor-derived cells to increase the levels of engraftment and/or accelerate reconstitution of donor derived HSC following allogeneic transplantation. Success of the proposed studies would have significant implications in the isolation and expansion of primitive HSC and the understanding of the origin of hematologic malignancies, and could significantly improve the safety and success of clinical HSC transplantation and potentially allow its application to a wider range of diseases affecting human patients. PUBLIC HEALTH RELEVANCE: Using fetal development as a model to gain a thorough understanding of the interactions that occur between hematopoietic stem cells and the cells that comprise the bone marrow microenvironmental niches will provide the necessary tools for manipulating these niches to facilitate the engraftment of donor HSC and accelerate hematopoietic recover following HSC transplantation. This would significantly improve the safety and success of clinical HSC transplantation and potentially allow its application to a wider range of diseases affecting human patients.