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

摘要 尽管有大量的实验证据表明，髓系偏向性HSC（myHSC）在 应激、炎症和衰老，控制myHSC静止和自我更新的机制不是 知道的该项目的长期目标是了解myHSC自我更新如何受到 骨髓微环境，以确定新的信号通路，可以激活，以增强 如果它们在骨髓恶性肿瘤中失调，则会被阻断。整体 本申请的目的是在机械层面上了解Sema 4a，一个新定义的利基， 因子，选择性地促进myHSC的静止和自我更新，并测试其潜在的治疗效用。 其在增殖性应激和HSC耗竭的临床相关模型中的促再生作用。中央 假设Sema 4a从骨髓中的骨祖细胞和内皮细胞释放 微环境并结合myHSC上的丛状蛋白D1，这引发了一系列抑制信号， myHSC衰竭、过早分化和DNA损伤。该项目的基本原理是， Sema 4a诱导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。 改善myHSC自我更新和造血再生的策略。核心假设是 在两个特定目标下进行测试：1）确定Sema 4a对 2）定义Sema 4a作为myHSC“保护者”免受增殖应激的治疗机会。 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 评估Sema 4a或其假定受体丛蛋白D1的微环境缺失对myHSC的影响， 并揭示Sema 4a作为myHSC特异性造血调节因子的作用。在第二个目标下，我的- Sema 4a的HSC保护特性将在功能性HSC丧失的临床相关模型中进行探索，例如 如在基因修饰过程中暴露于细胞毒性化疗和离体HSC培养。的 在候选人看来，这项申请中提出的研究是创新的，因为它建立在一个新的基础上。 Sema 4a作为myHSC在体内的有效且不可或缺的调节剂的发现，并提供了实质性的新发现。 功能特化的HSC由不同的调节因子控制， 电路.这项拟议中的研究意义重大，因为它有望为以下方面提供科学依据： 改善化疗后造血再生和干细胞移植的未来治疗策略 移植，增强基因修饰的HSC的植入和防止髓样偏向，年龄相关的HSC 功能障碍和演变为骨髓恶性肿瘤的老年人。

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.