Image guided profiling of the native HSC niche

原生 HSC 利基的图像引导分析

基本信息

批准号：
10212380
负责人：
Fernando Camargo
金额：
$ 30.86万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-17 至 2023-06-30
项目状态：
已结题

项目摘要

The concept of the stem cell niche is central both to the fundamental understanding of how stem cells are regulated by their microenvironment, and to clinical translation that targets the microenvironment for improving therapeutic outcome. The bone marrow (BM), where hematopoietic stem cells (HSCs) reside, is a crowded space packed with a diversity of cell types derived from both hematopoietic and nonhematopoietic precursors. A major challenge in studying the HSC niche has been the difficulty in identifying the rare HSCs and their neighboring cells in the native BM microenvironment. Elegant cell type-specific deletion of molecules critical for HSC maintenance has led to the identification of vascular endothelial cells (ECs) and CXCL12-abundant reticular (CAR) cells as two major cell types of the HSC niche. However, deletion of such factors impacts all ECs and CAR cells that are present throughout the BM, and are therefore not specific in terms of their local impact in the HSC niche. Direct imaging has the potential to uncover which cell types are in close contact with the HSCs, provided that specific markers are available for all cell types involved. As markers for HSCs are now just beginning to emerge, and visualization of minor cell types remains a challenge, the direct imaging approach has not progressed beyond resolving whether HSCs are in proximity to ECs, CAR cells, or bone-lining osteoblasts. Imaging on its own also does not provide the molecular information essential for understanding how the signals from the niche are communicated to the HSCs. We propose that two things are needed for the field to move forward. First, development of an HSC-specific reporter mouse will enable the identification of endogenous stem cells in their native microenvironment without transplantation. Second, development of a method to selectively isolate the cells in close proximity to the HSCs will enable unbiased profiling of cell types and their molecular signatures (for example, by single-cell RNA sequencing) involved in HSC maintenance. We have now taken steps to address both of these needs. First, we have developed (Camargo Lab) a dual genetic strategy in mice that restricts reporter labeling near exclusively to the most quiescent long-term subset of the HSC compartment (LT-HSCs). This reporter line is fully compatible with current intravital imaging approaches in the calvarial BM and enables live animal tracking of native HSCs (Lin Lab) based on the expression of the green fluorescent protein (GFP) alone, without the need for additional markers and without transplantation. In addition, we have developed a technique for micropipette aspiration of single cells and cell clusters directly from the BM under two-photon image guidance, enabling single cell analysis with high spatial definition. Here, we propose to bring the two teams together to work on an integrated approach for marking, isolating and profiling the native HSCs together with their neighboring “niche cells”, whose cell types will be identified retrospectively from the transcriptome profiles.

干细胞生态位的概念对于基本了解干细胞如何存在至关重要受其微环境调节，并针对针对微环境的临床转化改善治疗效果的骨髓 (BM) 是造血干细胞 (HSC) 所在的地方。拥挤的空间充满了来自造血和非造血的多种细胞类型研究 HSC 生态位的一个主要挑战是难以识别稀有的 HSC。及其邻近细胞在天然 BM 微环境中的优雅细胞类型特异性分子删除。血管内皮细胞 (EC) 的鉴定对于 HSC 的维持至关重要，并且 CXCL12 丰富的网状 (CAR) 细胞作为 HSC 生态位的两种主要细胞类型，但删除了此类细胞。因素会影响整个 BM 中存在的所有 EC 和 CAR 细胞，因此在就其对 HSC 生态位的局部影响而言，直接成像有可能揭示哪些细胞类型。与 HSC 密切接触，前提是所有涉及的细胞类型都有特定标记。 HSC 标记现在才刚刚开始出现，小细胞类型的可视化仍然是一个尽管面临挑战，直接成像方法除了解决 HSC 是否在附近之外还没有取得进展 EC、CAR 细胞或骨衬成骨细胞的成像本身也不提供分子信息。对于理解来自利基的信号如何传递到 HSC 至关重要的信息。提出该领域的发展需要两件事：首先，开发特定于 HSC 的项目。报告小鼠将能够识别其天然微环境中的内源干细胞其次，开发一种选择性分离邻近细胞的方法。 HSC 将能够对细胞类型及其分子特征进行公正的分析（例如，通过单细胞 RNA 测序）涉及 HSC 维护，我们现在已采取措施解决这两个问题。这些需求首先，我们（卡马戈实验室）开发了一种限制报告基因的小鼠双重遗传策略。几乎专门标记 HSC 区室 (LT-HSC) 的最静止的长期子集。报告线与颅骨 BM 中当前的活体成像方法完全兼容，并可实现活体成像基于绿色荧光蛋白 (GFP) 表达的天然 HSC 动物追踪（Lin Lab）单独使用，不需要额外的标记，也不需要移植。此外，我们还开发了一种方法。双光子下直接从 BM 微量移液器吸取单细胞和细胞簇的技术图像引导，实现高空间清晰度的单细胞分析在这里，我们建议将两者结合起来。团队共同开发一种用于标记、分离和分析本地 HSC 的综合方法与其邻近的“利基细胞”一起，其细胞类型将从转录组概况。