Image guided profiling of the native HSC niche

干细胞生态位的概念对于理解干细胞是如何 受其微环境的调节，以及靶向微环境的临床翻译， 改善治疗效果。造血干细胞（HSC）所在的骨髓（BM）是造血干细胞的主要来源。 一个拥挤的空间挤满了来自造血和非造血细胞类型的多样性 前体研究HSC生态位的一个主要挑战是难以识别罕见的HSC 以及它们在原生BM微环境中的邻近细胞。优雅细胞类型特异性分子缺失 对HSC维持至关重要的研究导致了对血管内皮细胞（EC）的鉴定， CXCL 12-丰富的网状（CAR）细胞作为HSC小生境的两种主要细胞类型。但是，删除这些 这些因子影响存在于整个BM中的所有EC和CAR细胞，因此在 他们在HSC利基的本地影响方面。直接成像有可能揭示哪些细胞类型在 与HSC密切接触，前提是所有涉及的细胞类型都有特异性标记。作为 HSC的标记物现在才刚刚开始出现，小细胞类型的可视化仍然是一个挑战。 然而，直接成像方法的进展还没有超出解决HSC是否在邻近区域的范围。 到内皮细胞、CAR细胞或骨衬成骨细胞。成像本身也不能提供分子 这些信息对于理解来自小生境的信号如何传递到HSC至关重要。我们 我认为，要使该领域向前发展，需要做两件事。首先，开发HSC特异性 报告小鼠将能够在其天然微环境中鉴定内源性干细胞 没有移植。第二，开发一种方法来选择性地分离细胞， HSC将能够无偏地分析细胞类型及其分子特征（例如，通过 单细胞RNA测序）。我们现在已经采取措施， 这些需求。首先，我们已经开发了（Camargo实验室）小鼠的双重遗传策略， 几乎专门标记HSC隔室的最静止的长期亚群（LT-HSC）。这 报告子线与颅骨BM中的当前活体成像方法完全兼容， 基于绿色荧光蛋白（GFP）表达的天然HSC的动物追踪（Lin Lab） 单独使用，不需要额外的标记物，也不需要移植。此外，我们还开发了一个 在双光子作用下直接从骨髓中用微管吸取单个细胞和细胞团的技术 图像引导，能够以高空间分辨率进行单细胞分析。在这里，我们建议将两个 团队共同致力于标记，分离和分析天然HSC的综合方法 以及它们邻近的“小生境细胞”，其细胞类型将从 转录组图谱。

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.