Image guided profiling of the native HSC niche

干细胞生态位的概念对于从根本上理解干细胞是如何 由他们的微环境调节，并针对微环境的临床翻译 改善治疗结果。造血干细胞(HSCs)所在的骨髓(BM)是一种 拥挤的空间，挤满了来自造血系和非造血系的各种细胞类型 先驱物。研究HSC生态位的一个主要挑战是很难识别罕见的HSC 以及它们在天然BM微环境中的邻近细胞。优雅的细胞类型特异性分子缺失 对于HSC的维持至关重要的是导致了血管内皮细胞(ECs)的鉴定和 CXCL12-丰富的网状(CAR)细胞是HSC生态位的两种主要细胞类型。然而，删除这类 影响存在于整个BM中的所有EC和CAR细胞的因素，因此在 关于它们在HSC利基市场的本地影响。直接成像有可能揭示哪些细胞类型在 与造血干细胞密切接触，前提是所有涉及的细胞类型都有特定的标记。AS 造血干细胞的标志物现在才刚刚开始出现，而对次要细胞类型的可视化仍然是一项 挑战，直接成像方法除了解决造血干细胞是否在附近之外，还没有取得进展 内皮细胞、CAR细胞或骨衬成骨细胞。成像本身也不能提供分子 对于理解来自生态位的信号如何传递给HSC来说至关重要的信息。我们 提出这一领域需要两件事才能向前发展。首先，开发专门针对HSC的 报告小鼠将能够在其天然微环境中鉴定内源性干细胞 不需要移植。第二，开发了一种有选择地分离邻近细胞的方法 HSC将能够无偏见地分析细胞类型及其分子特征(例如，通过 单细胞RNA测序)参与了HSC的维持。我们现在已经采取措施解决这两个问题 这些需求。首先，我们(Camargo Lab)在小鼠身上开发了一种双重遗传策略，限制了记者 仅在HSC隔室中最静止的长期子集(LT-HSC)附近进行标记。这 REPORTER LINE与当前颅骨BM活体成像方法完全兼容，并支持实时 基于绿色荧光蛋白(GFP)表达的天然造血干细胞(LIN Lab)的动物示踪 单独，不需要额外的标记，也不需要移植。此外，我们还开发了一种 双光子直接从骨髓中吸单细胞和细胞团的微吸管技术 图像引导，支持高空间清晰度的单细胞分析。在这里，我们建议将这两个 团队合作，共同开发用于标记、隔离和分析本地HSC的集成方法 以及它们邻近的“利基细胞”，它们的细胞类型将从 转录组特征。

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.