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

项目概要 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。这种血液生产主要通过骨髓发生 细胞分化等级由自我更新的造血干细胞 (HSC) 启动和维持， 产生各种祖细胞，并最终产生所有成熟的、终末分化的血液细胞。 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，进行自我更新的细胞 维护 HSC 编号的部门。自我更新和多能分化这两个特征， 代表了 HSC 的关键特征。 HSC 的自我更新尤其引起了双方的极大兴趣 生物机制研究和潜在的转化应用。在临床实践中，HSC 是 造血细胞移植（HCT）的基本单位，用于治疗良性和 恶性血液疾病。尽管尚未应用于临床实践，但基因组编辑方法 血液疾病的治疗需要在移植前对编辑过的 HSC 进行离体扩增，强调 了解 HSC 自我更新的迫切需要。在生物学研究中，小鼠被用作 研究 HSC 自我更新的主要实验模型。许多分子途径已 参与此过程的包括 Wnt/β-catenin、Notch 和 Bmi1，以及我们的团队和其他人 证明粘连蛋白缺乏会导致 HSC 自我更新增加。近期，多项研究 小鼠和人类细胞的研究表明，一个不寻常的同源盒家族成员 Hopx 是一种潜在的 HSC 自我更新的调节因子。 HSC 主要存在于骨髓中，存在于骨髓中 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 (MSC)、内皮细胞等，以及生长因子、细胞因子、粘附分子等 细胞外基质。造血微环境的性质，特别是小鼠 HSC 生态位 一直是一个深入研究的领域，但对人类 HSC 生态位的了解却少之又少。总体而言，我们的 目前对人类造血干细胞自我更新的了解远不如小鼠。在这里，我们 提议通过多种方式识别和研究人类 HSC 自我更新的新调控决定因素 假设和方法。首先，我们将研究对基因表达至关重要的表观遗传 DNA 调控元件。 通过研究粘连蛋白缺陷型 HSC 中染色质的可及性来研究人类 HSC 的自我更新。其次，我们将 研究 Hopx 作为人类 HSC 自我更新调节剂的作用和作用机制，包括 与细胞周期和 Wnt 通路的可能联系。最后，我们将使用一种新型的人源化小骨异种移植物 模型与谱系追踪相结合，以可视化与人类 HSC 相关的骨髓生态位。 总之，这些目标将极大地扩展我们对人类造血干细胞自我更新的理解。

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.