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

项目摘要造血干细胞(HSCs)存在于骨骼内的调节微环境中 骨髓(BM)，称为利基。尽管在过去的几十年里进行了广泛的研究，但细胞 HSC生态位的组成和位置仍然存在争议。虽然早期的研究表明 造血干细胞在骨表面与成骨细胞相邻，越来越多的最新证据表明 HSCs与中央骨髓中的血窦血管相当接近；其他一些显示HSCs靠近骨髓。 动脉。这些相互矛盾的观察结果可以用造血干细胞和生态位的异质性来解释。 在组织学分析中缺乏识别HSC亚群的标记物。该项目的重点是提出 其中最原始的HSC，以及它们的利基。需要检验的总体假设是一氧化氮(NO) 确认史无前例的HSC具有明显的高度自我更新和重建潜力。我们进一步 假设这些NO+HSCs受连接动脉和HSCs的独特过渡血管调节 正弦血管，而非肝星状干细胞具有相对有限的自我更新能力，邻近正弦血管 静脉。这一假设是基于以下初步数据。我们证明了，在 CD150+CD48+cKit+Sca1+LIN-HSCs，有一个静止群体(5-10%)高表达 NO和CD200受体(CD200R)。这些NO+CD200RHigh HSCs显示出很高的血液重建潜力 经连续移植后进一步增加，而NO-HSCs的重建潜能相对有限 并通过连续移植废除了。这一观察结果表明，NO+HSC具有明显的高度自我更新能力 和重建潜力。此外，NO+HSCs与移行血管相邻，而NO-HSCs与移行血管相邻 邻近不表达纤毛或CD200的血窦。进一步将机械洞察力带入NO+HSC 和他们的利基，并促进我们的临床翻译的基本发现，我们将追求以下目标。在AIM 1，我们将寻求表征NO+HSCs的自我更新、重组潜力和分子特征，以及 结合单细胞RNA阐明其对NO+HSCs的内在调控机制 对称性或不对称性的测序、移植试验、基因缺失和体内显微镜追踪 光标记单个肝星状细胞的分裂。在目标2中，我们将阐明NO+HSCs的位置和外部调节 机械装置。我们将通过进行深部整装来验证NO+HSCs对移行血管的定位 新型HSC报告小鼠的双光子骨髓成像。我们将进一步阐明移行血管CD200的作用 和纤毛在HSC的调节和辐射应激防护中的作用。我们将最终调查治疗潜力 CD200R激动剂治疗减轻照射后造血功能衰竭。该项目的完工时间可能 通过提出史无前例的原始HSC及其利基来解决关于利基位置的争议。 成功的研究将进一步确定cilia-CD200/CD200R-NO轴为新的HSC调节器，导致新的 造血衰竭的治疗。

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.