Identification of unique nitric oxide-expressing hematopoietic stem cells and their special vascular niche

造血干细胞（HSCs）存在于骨内的调控微环境中 骨髓（BM），称为niche。尽管在过去几十年中进行了广泛的研究， HSC利基的组成和位置仍然存在争议。虽然早期的研究表明， HSC与骨表面的成骨细胞相邻，越来越多的最新证据表明， 在中央骨髓中，HSC与窦状血管相邻;其他一些研究显示，HSC与BM相邻 动脉这些相互矛盾的观察结果可以解释的异质性HSC和生态位，以及 因为缺乏在组织学分析中识别HSC亚群的标记物。该项目旨在提出 最原始的HSC和它们的生态位。待检验的总体假设是一氧化氮（NO） 识别出具有明显高自我更新和重建潜力的前所未有的HSC。我们进一步 假设这些NO+ HSC由连接动脉的独特过渡血管调节， 窦状血管，而具有相对有限的自我更新潜力的NO-HSC邻近窦状血管， 静脉这一假设基于以下初步数据。我们发现， CD 150 + CD 48 +cKit+ Sca 1 +Lin-HSC中，存在具有高表达水平的CD 150 + CD 48 +cKit+ Sca 1 +Lin-HSC的静止群体（5-10%）。 NO和CD 200受体（CD 200 R）。这些NO+ CD 200 Rhigh HSC具有较高的血液重建能力 连续移植可进一步提高NO-HSC的再生能力，但NO-HSC的再生能力相对有限 并通过连续移植消除。这一观察结果表明NO+ HSC具有明显的高自我更新能力 和重建潜力。此外，NO+ HSC与移行血管相邻，而NO-HSC与移行血管相邻。 邻近窦状隙，不表达纤毛或CD 200。进一步深入了解NO+ HSC的机制 和他们的利基，并促进我们的基本研究结果的临床转化，我们将追求以下目标。在Aim中 1、我们将寻求表征NO+ HSC的自我更新、重建潜力和分子特征， 通过使用单细胞RNA的组合，阐明NO+ HSC的内在调节机制， 测序、移植测定、遗传缺失和体内显微镜追踪对称或不对称的 光标记的单个HSC的分裂。在目标2中，我们将阐明NO+ HSC的定位和外源性调控， 机制等我们将通过进行深整体包埋， 2-新型HSC报告小鼠的光子BM成像。我们将进一步阐明移行血管CD 200的作用， 和纤毛在HSC调节和辐射应激保护中的作用。我们将最终研究治疗潜力 CD 200 R激动剂治疗以减轻辐射后造血衰竭。该项目的完成可能 通过提出前所未有的原始HSC及其生态位来解决关于生态位位置的争议。 成功的研究将进一步确定纤毛-CD 200/CD 200 R-NO轴作为新的HSC调节因子，从而导致新的HSC调节因子。 造血衰竭的治疗方法

Project Abstract Hematopoietic stem cells (HSCs) reside in the regulatory microenvironment within the bone marrow (BM), termed the niche. Despite extensive research efforts made over the past decades, cellular constituents and locations of the HSC niche have remained controversial. While early studies suggested that HSCs are adjacent to osteoblasts on the bone surface, a growing amount of more recent evidence indicate that HSCs are rather adjacent to sinusoidal vessels in the central marrow; some other showed HSCs are near BM arteries. These conflicting observations may be explained by the heterogeneity of HSCs and the niche, as well as the paucity of markers to identify HSC subsets in the histological analysis. This project focuses on proposing most primitive HSCs among others, and their niche. The overall hypothesis to be tested is that nitric oxide (NO) identifies unprecedented HSCs with distinctly high self-renewing and reconstituting potential. We further hypothesize that these NO+ HSCs are regulated by unique transitional vessels which connect arteries to sinusoidal vessels, while NO- HSCs with a relatively limited self-renewing potential are adjacent to sinusoidal veins. This hypothesis is based on the following preliminary data. We showed that, within CD150+CD48+cKit+Sca1+Lin- HSCs, there was a quiescent population (5-10%) with high expression levels of NO and CD200 receptors (CD200R). These NO+CD200Rhigh HSCs exhibited high blood reconstituting potential which was further increased by serial transplant, while NO- HSCs' reconstituting potential was relatively limited and abolished by serial transplant. This observation suggest NO+ HSCs possess a distinctly high self-renewal and reconstituting potential. Moreover, NO+ HSCs were adjacent to transitional vessels, while NO- HSCs were adjacent to sinusoids which did not express cilia or CD200. To further bring mechanistic insights into NO+ HSCs and their niche and to promote clinical translation of our basic findings, we will pursue the following aims. In Aim 1, we will seek to characterize NO+ HSCs' self-renewal, reconstituting potential and molecular features, and elucidate their intrinsic regulatory mechanisms of NO+ HSCs, by using the combination of single cell RNA sequencing, transplantation assay, genetic deletion, and in vivo microscopy tracking of symmetric or asymmetric division of photolabeled individual HSCs. In Aim 2, we will elucidate NO+ HSCs' locations and extrinsic regulatory mechanisms. We will validate localization of NO+ HSCs to transitional vessels by performing deep whole-mount 2-photon BM imaging of novel HSC-reporter mice. We will further elucidate roles of transitional vessels' CD200 and cilia in HSC regulation and protection from radiation stress. We will finally investigate therapeutic potential of CD200R agonist treatment to mitigate post-irradiation hematopoiesis failure. Completion of the project may address the controversy about the niche location by proposing unprecedented primitive HSCs and its niche. Successful studies will further identify cilia-CD200/CD200R-NO axis as new HSC regulators, leading to novel treatments for hematopoiesis failure.