Functional Analysis of the Hematopoietic Niche during Ontogeny

描述（由申请人提供）：据认为，骨髓内至少有两个解剖学和生理学上不同的造血小生境：成骨细胞和血管。在这些小生境中，许多分子和分泌因子与严格调控的造血过程有关。然而，与每个小生境相互作用的HSC亚群以及每个小生境类型在干细胞层次和功能调节中的生理作用的透彻理解仍然缺失。在这里，我们将确定在表型，功能和转录水平上的每个小生境内发生的细胞相互作用，作为一个独特的范例，骨髓获得支持造血的能力的发育过程。将要进行的实验将采用生理相关的大型动物模型，高分辨率成像/捕获技术，灵敏的转录组测序和功能研究，这些结合为我们提供了研究和操纵人HSC和特定的小生境细胞在体内的相互作用的独特能力，从而揭示了每个离散的微环境在建立和维持人造血中所起的作用。这些研究的总体假设是，胎儿骨髓获得支持造血的能力的发育过程可以用作理解HSC和包括骨髓小生境的细胞之间发生的相互作用以及这些小生境元素在造血的启动/维持中的作用的模型。我们进一步假设，这方面的知识将提供必要的工具，操纵这些壁龛，以促进移植和加速造血干细胞移植后供体造血恢复。我们提出了以下具体目标：1）定义胎儿BM造血小生境出现和骨髓造血发生的步骤和关键细胞参与者，作为理解血管和成骨细胞小生境内和之间发生的细胞相互作用的工具，并表征每个支持的造血细胞的性质，从而描绘每个在维持和扩增干细胞库以驱动终身造血中发挥的作用; 2）确定新生骨髓中存在的血管/血管周围小生境是否不仅作为来自胎儿肝脏的HSC寄宿的支持位点，而且还具有有助于建立造血的潜能的细胞;和3）研究骨和血管小生境的组成是否可以通过特定的供体来源的细胞群来操纵，以增加移植水平和/或加速同种异体移植后供体来源的HSC的重建。所提出的研究的成功将对原始HSC的分离和扩增以及对血液恶性肿瘤起源的理解具有重要意义，并且可以显着提高临床HSC移植的安全性和成功率，并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：使用胎儿发育作为模型，以获得造血干细胞和细胞之间发生的相互作用，包括骨髓微环境的小生境将提供必要的工具，操纵这些小生境，以促进供体HSC的植入和加速造血恢复造血干细胞移植后。这将显著提高临床HSC移植的安全性和成功率，并可能使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.