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

鉴定独特的表达一氧化氮的造血干细胞及其特殊的血管生态位

• 批准号: 10331423
• 负责人: Joji Fujisaki
• 金额: $ 34.48万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-28 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10331423
• 关键词: Address; Agonist; Arteries; Biological Assay; Blood; Blood Vessels; Bone Marrow; Bone Surface; CD 200; CD34 gene; Cells; Chimerism; Cilia; Conflict (Psychology); Data; Discontinuous Capillary; Ensure; Exhibits; Failure; Generations; Genetic; Goals; Hematopoiesis; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cell heterogeneity; Hematopoietic Stem Cell subsets; Hematopoietic stem cells; Heterogeneity; Histologic; Image; Individual; Location; Marrow; Microscopy; Molecular; Molecular Profiling; Mus; Nitric Oxide; Nitric Oxide Synthase; Osteoblasts; Pancytopenia; Patient-Focused Outcomes; Population; Publishing; Radiation; Radiation Protection; Regulation; Reporter; Research; Role; SLAM protein; Sleeping Beauty; Soluble Guanylate Cyclase; Stress; Testing; Therapeutic; Transplantation; Veins; base; clinical translation; hematopoietic hierarchy; hematopoietic stem cell niche; hematopoietic stem cell quiescence; hematopoietic stem cell self-renewal; improved outcome; in vivo; insight; irradiation; microscopic imaging; novel; novel therapeutic intervention; receptor; reconstitution; self-renewal; single-cell RNA sequencing; stem cell division; stem cell function; stem cell population; treatment strategy; two photon microscopy; two-photon; young adult

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.

项目摘要 造血干细胞 (HSC) 存在于骨内的调节微环境中 骨髓（BM），称为利基。尽管过去几十年进行了大量的研究工作，但细胞 HSC 利基的组成部分和位置仍然存在争议。虽然早期研究表明 HSC 与骨表面的成骨细胞相邻，越来越多的最新证据表明 HSC 与中央骨髓中的正弦血管相当相邻；其他一些显示 HSC 靠近 BM 动脉。这些相互矛盾的观察结果也可以用 HSC 和生态位的异质性来解释 由于组织学分析中缺乏识别 HSC 亚群的标记。该项目的重点是提出 其中最原始的 HSC 及其利基。要测试的总体假设是一氧化氮 (NO) 鉴定出前所未有的具有明显高自我更新和重组潜力的 HSC。我们进一步 假设这些 NO+ HSC 受到连接动脉和 正弦血管，而自我更新潜力相对有限的 NO-HSC 与正弦血管相邻 静脉。该假设基于以下初步数据。我们证明，在 CD150+CD48+cKit+Sca1+Lin- HSCs，有一个静止群体（5-10%），高表达水平 NO 和 CD200 受体 (CD200R)。这些NO+CD200Rhigh HSCs表现出高血液重建潜力 通过连续移植进一步增加，而NO-HSCs的重建潜力相对有限 并通过连续移植而废除。这一观察结果表明 NO+ HSC 具有明显高的自我更新能力 并重构潜力。此外，NO+ HSC 邻近移行血管，而 NO- HSC 则邻近移行血管。 邻近不表达纤毛或CD200的正弦波。进一步深入了解 NO+ HSC 的机制 及其利基并促进我们的基本发现的临床转化，我们将追求以下目标。瞄准 1、我们将寻求表征NO+ HSC的自我更新、重构潜力和分子特征，以及 通过使用单细胞 RNA 的组合阐明 NO+ HSC 的内在调节机制 对称或不对称的测序、移植测定、基因缺失和体内显微镜追踪 光标记的单个 HSC 的划分。在目标 2 中，我们将阐明 NO+ HSC 的位置和外在监管 机制。我们将通过进行深度整体安装来验证 NO+ HSC 在过渡船上的定位 新型 HSC 报告小鼠的 2 光子 BM 成像。我们将进一步阐明过渡船CD200的作用 和纤毛在 HSC 调节和辐射应激保护中的作用。我们最终将研究治疗潜力 CD200R激动剂治疗减轻辐射后造血衰竭的研究。项目完成后可 通过提出前所未有的原始 HSC 及其生态位来解决有关生态位位置的争议。 成功的研究将进一步确定纤毛-CD200/CD200R-NO轴作为新的HSC调节剂，从而产生新的 造血功能衰竭的治疗。