Extrinsic regulation of primitive, myeloid-biased hematopoietic stem cells by Semaphorin 4a

摘要 尽管大量实验证据表明偏髓系造血干细胞(MyHSC)在 应激、炎症和衰老，控制我的HSC静止和自我更新的机制不是 为人所知。这个项目的长期目标是了解MyHSC自我更新如何通过 骨髓微环境以确定可被激活以增强的新的信号通路 造血再生，如果它们在髓系恶性肿瘤中变得失调，就会被阻断。整体而言 这个应用的目的是在机械水平上理解Sema4a，一个新定义的生态位 选择性地促进MyHSC的静止和自我更新，并测试其潜在的治疗作用 在增殖性应激和肝星状细胞衰竭的临床相关模型中的促再生作用。中环 假设Sema4a是从骨髓中的骨孕激素和内皮细胞中释放出来的 微环境，并与myHSC上的丛蛋白D1结合，从而触发一系列抑制信号，从而屏蔽 MyHSC因耗竭、过早分化和DNA损伤所致。这个项目的基本原理是 Sema4a诱导的MyHSC特异性通路的鉴定及其对MyHSC的保护作用 增殖性应激的临床前模型可能为未来的治疗提供强有力的科学框架 促进我的造血干细胞自我更新和造血再生的策略。中心假设将是 测试有两个特定的目的：1)确定Sema4a影响的分子和细胞机制 MyHSC；以及2)定义Sema4a作为myHSC的增殖性应激“保护者”的治疗机会。 在第一个目标下，将使用遗传小鼠模型、单细胞rna测序和活体显微镜。 评价微环境中Sema4a或其可能的受体丛状蛋白D1缺失对MyHSC的影响 并揭示Sema4a作为myHSC特异性造血调节因子的作用。在第二个目标下，我的- Sema4a的HSC保护特性将在临床相关的功能性HSC丢失模型中进行探索，如 AS在基因修饰过程中暴露于细胞毒性化疗和体外HSC培养。这个 在候选人看来，这项申请中提出的研究是创新的，因为它建立在一部小说的基础上 在体内发现Sema4a作为myHSC的有效和不可或缺的调节因子，并提供了实质性的新的 功能专门化的HSC由不同的监管机构控制这一概念的实验证据 电路。这项拟议的研究意义重大，因为它有望成为 促进化疗和干细胞后造血再生的未来治疗策略 移植，增强基因修饰的HSC的植入，防止髓系偏见，年龄相关的HSC 老年人的功能障碍和向髓系恶性肿瘤的演变。

ABSTRACT Despite substantial experimental evidence for the important functional role of myeloid-biased HSC (myHSC) in stress, inflammation and aging, the mechanisms which control myHSC quiescence and self-renewal are not known. The long-term goal of this project is to understand how myHSC self-renewal can be regulated by the bone marrow microenvironment in order to identify novel signaling pathways that can be activated to enhance hematopoietic regeneration, and blocked if they become dysregulated in myeloid malignancy. The overall objective in this application is to understand at the mechanistic level how Sema4a, a newly-defined niche factor, selectively promotes quiescence and self-renewal of myHSC, and to test potential therapeutic utility of its pro-regenerative effect in clinically relevant models of proliferative stress and HSC exhaustion. The central hypothesis is that Sema4a is released from osteoprogentiors and endothelial cells in the bone marrow microenvironment and binds to Plexin D1 on myHSC, which triggers a cascade of inhibitory signals that shield myHSC from exhaustion, premature differentiation and DNA damage. The rationale for this project is that identification of Sema4a-induced myHSC-specific pathways and testing their protective effect on myHSC in pre-clinical models of proliferative stress is likely to offer a strong scientific framework for future therapeutic strategies to improve myHSC self-renewal and hematopoietic regeneration. The central hypothesis will be tested under two specific aims: 1) Determine molecular and cellular mechanisms of Sema4a effects on myHSC; and 2) Define therapeutic opportunities for Sema4a as a myHSC “protector” from proliferative stress. Under the first aim, genetic mouse models, single-cell RNA Sequencing and intravital microscopy will be used to evaluate the effect of microenvironmental deletion of Sema4a or its putative receptor, Plexin D1, on myHSC and to uncover the role of Sema4a as myHSC-specific hematopoietic regulator. Under the second aim, my- HSC protective properties of Sema4a will be explored in clinically relevant models of functional HSC loss, such as exposure to cytotoxic chemotherapy and ex-vivo HSC culture during gene modification procedure. The research proposed in this application is innovative, in candidate’s opinion, because it builds on a novel discovery of Sema4a as a potent and indispensable regulator of myHSC in vivo, and provides substantive new experimental evidence for the concept that functionally specialized HSC are controlled by distinct regulatory circuits. The proposed research is significant because it is expected to serve as a scientific justification for future therapeutic strategies to improve hematopoietic regeneration following chemotherapy and stem cell transplantation, enhance engraftment of gene-modified HSC and prevent myeloid bias, age-related HSC dysfunction and evolution to myeloid malignancy in the elderly.