Epigenetic, Transcriptional, and Microenvironmental Determinants of Human HSC Self-Renewal

项目总结 造血是人类一生中持续活跃的血液生产过程。 成熟的效应细胞不断被耗尽，导致需要每天大量生产 白细胞、红细胞和血小板。这种血液产生主要发生在骨髓中，通过一种 由自我更新的造血干细胞(HSCs)启动和维持的细胞分化层次 产生各种各样的祖细胞，并最终产生血液中所有成熟的、终末分化的细胞。 造血干细胞能够在个体的整个生命过程中产生血细胞后代，经历自我更新的细胞。 维护HSC编号的部门。这两个特征，自我更新和多潜能分化， 代表HSC的主要特征。特别是HSC的自我更新一直是双方非常感兴趣的问题 生物机制研究和潜在的翻译应用。在临床实践中，造血干细胞是 造血细胞移植的基本单位在治疗良性和非霍奇金淋巴瘤中的应用 恶性血液病。虽然还没有在临床实践中使用，但基因组编辑方法 血液疾病的治疗需要在移植前对编辑好的HSC进行体外扩增，强调 了解HSC自我更新的迫切需要。在生物学研究中，老鼠被用作 HSC自我更新研究的初步实验模型。许多分子途径都有 参与了这一过程，包括WNT/β-连环蛋白、Notch和Bmi1，以及我们的团队和其他人 表明粘附素缺乏会导致HSC自我更新增加。最近，一些研究表明， 与小鼠和人类细胞都有关联，可能是一个不寻常的同源盒子家族成员，Hopx HSC自我更新的调节器。HSCs主要存在于骨髓中，在那里它们被包含在 由成骨细胞、间充质基质细胞等细胞成分组成的复杂微环境 (MSCs)、内皮细胞等，以及生长因子、细胞因子、黏附分子和 细胞外基质。造血微环境的性质，特别是小鼠HSC的生态位 一直是一个密集调查的领域，但对人类HSC利基环境的了解要少得多。总的来说，我们的 目前对人类HSC自我更新的理解远不如在小鼠中广泛。在这里，我们 建议通过以下几个途径识别和研究人类HSC自我更新的新的调控决定因素 假设和方法。首先，我们将研究表观遗传DNA调控元件，这些元件对 通过研究凝集素缺乏的HSC中染色质的可及性来研究人类HSC的自我更新。第二，我们将 研究Hopx作为人类HSC自我更新调节因子的作用和机制，包括 可能与细胞周期和Wnt途径有关。最后，我们将使用一种新型的人源化异种小骨。 与血统追踪相结合的模型，以可视化与人类造血干细胞相关的骨髓壁龛。 总而言之，这些目标应该会极大地扩展我们对人类HSC自我更新的理解。

PROJECT SUMMARY Hematopoiesis is the process of blood production that is continuously active in humans for their entire lifespan. Mature effector cells are continually being exhausted leading to a requirement for a massive daily production of leukocytes, erythrocytes, and platelets. This blood production occurs primarily in the bone marrow through a cellular differentiation hierarchy initiated and maintained by self-renewing hematopoietic stem cells (HSCs) that give rise to a variety of progenitor cells and eventually all the mature, terminally differentiated cells of the blood. HSCs are able to produce blood cell progeny for the entire life of an individual, undergoing self-renewing cell divisions that maintain HSC numbers. These two features, self-renewal and multipotent differentiation, represent the key characteristics of HSCs. HSC self-renewal in particular has been of great interest for both biological mechanistic studies and potential translational applications. In clinical practice, HSCs are the fundamental unit of hematopoietic cell transplantation (HCT) utilized in the treatment of both benign and malignant blood disorders. Although not yet utilized in clinical practice, genome editing approaches to the treatment of blood disorders will require ex vivo expansion of edited HSCs prior to transplantation, highlighting the critical need to understand HSC self-renewal. For biological studies, the mouse has been used as the primary experimental model for the investigation of HSC self-renewal. A number of molecular pathways have been implicated in this process including Wnt/beta-catenin, Notch, and Bmi1, and our group and others demonstrated that cohesin-deficiency results in increased HSC self-renewal. Recently, a number of studies with both mouse and human cells have implicated an unusual homeobox family member, Hopx, as a potential regulator of HSC self-renewal. HSCs predominantly reside in the bone marrow where they are contained in a complex microenvironment consisting of cellular components including osteoblasts, mesenchymal stromal cells (MSCs), endothelial cells, and others, as well as growth factors, cytokines, adhesion molecules, and extracellular matrix. The nature of the hematopoietic microenvironment and specifically the mouse HSC niche has been an area of intense investigation, but much less is known about the human HSC niche. Overall, our current understanding of human HSC self-renewal is much less extensive than in the mouse. Here, we propose to identify and investigate novel regulatory determinants of human HSC self-renewal through several hypotheses and approaches. First, we will investigate epigenetic DNA regulatory elements that are critical for human HSC self-renewal by investigating chromatin accessibility in cohesin-deficient HSCs. Second, we will investigate the role and mechanisms of action of Hopx as a regulator of human HSC self-renewal, including possible links to the cell cycle and Wnt pathway. Finally, we will use a novel humanized ossicle xenograft model in conjunction with lineage tracing to visualize bone marrow niches associated with human HSCs. Together, these aims should greatly expand our understanding of human HSC self-renewal.