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既是对干细胞的基本理解的核心由其微环境调节，并针对以微环境为目标的临床翻译改善治疗结果。造血干细胞（HSC）的骨髓（BM）是一个拥挤的空间充满了来自造血和非hematopoietic的各种细胞类型的多样性前体。研究HSC利基市场的主要挑战是很难识别罕见的HSC 及其在本地BM微环境中的相邻细胞。优雅的细胞类型特异性分子缺失 HSC维持至关重要的导致血管内皮细胞（EC）和 CXCL12丰富的网状（CAR）细胞是HSC小众的两种主要细胞类型。但是，删除此类因素会影响整个BM中存在的所有EC和汽车单元，因此并不具体他们在HSC利基市场中的本地影响条款。直接成像有可能发现哪些细胞类型在与HSC密切接触，前提是所有涉及的细胞类型都可以使用特定标记。作为 HSC的标记现在才刚刚开始出现，次要细胞类型的可视化仍然是挑战，直接的成像方法没有解决HSC是否处于接近度之外到EC，汽车电池或骨衬骨细胞。成像本身也不提供分子了解如何将利基市场信号传达给HSC的信息至关重要。我们建议该领域需要两件事才能前进。首先，开发HSC特定的记者小鼠将使内源性干细胞在其天然微环境中识别没有移植。其次，开发一种方法，可以选择性地分离出细胞的近端 HSC将实现细胞类型及其分子特征的公正分析（例如，单细胞RNA测序）参与HSC维护。我们现在已经采取了措施来解决这两个这些需求。首先，我们开发了（Camargo Lab）在限制记者的小鼠中的双重遗传策略仅在HSC室（LT-HSC）的最静止的长期子集附近标记。这记者线完全与钙化bm中当前的弹刻成像方法完全兼容，并且可以实现基于绿色荧光蛋白（GFP）的表达的天然HSC（LIN LAB）的动物跟踪一个人，无需其他标记，而无需移植。此外，我们已经开发了直接从BM下的单细胞和细胞簇的微目抽吸技术在两光子下直接从BM 图像引导，实现具有高空间定义的单细胞分析。在这里，我们建议带上两个团队共同努力制定一种集成方法，用于标记，隔离和分析本地HSCS 以及他们相邻的“小众细胞”，他们的细胞类型将被追溯地从转录组轮廓。