Single cell approach to uncovering factors regulating HSC division symmetry in vivo

摘要 造血干细胞(HSC)的自我更新和分化之间的平衡直接影响 造血动态平衡。我们假设来自骨髓微环境(或“生态位”)的信号， 与来自细胞固有网络的信号一起，有助于微调这种平衡。但是，我们的理解是 已经受到当前依赖于顺序删除个人监管机构的方法的限制 可利用的小鼠模型中候选细胞的因子以及单个HSCs及其体内分析 与生态位的相互作用也受到可利用的HSC富集组分和 活体成像HSC命运的技术挑战。为了阐明单个造血干细胞在体内的行为，我们 已经建立了一种新的技术方案，包括预期分离高纯度的HSCs 在多光子显微镜下移植单个HSC的局部移植技术--Tie2阳性 引导进入活体小鼠的骨髓，并通过微吸管抽吸分离后的单个细胞 直接从骨髓中提取进行转录分析。我们的项目将利用这些进步来描述 HSC生态位命运选择的分子基础。这反过来将促进新的细胞治疗策略- 命运操纵可以加速移植后的造血恢复，并可能有助于 以提高非恶性血液病的移植效率。因此，这项提议的目标是 三个方面：(1)确定促进造血干细胞对称性自我更新分裂的分子机制；(2) 了解控制HSC分工平衡的生态位因素，以及(3)评估非 基因毒性条件作用。如果成功，拟议的研究将通过确定 分子靶点，将改善移植后的造血恢复，并使 利基模型的体外工程学。

ABSTRACT The balance between hematopoietic stem cell (HSC) self-renewal and differentiation directly impacts hematopoietic homeostasis. We hypothesize that signals from the bone marrow microenvironment (or “niche”), together with cues from cell-intrinsic networks, contribute to fine-tuning this balance. However, our understanding of the niche has been limited by the current approach relying on sequential deletion of individual regulatory factors from candidate cells in available mouse models, and analysis of individual HSCs and their in vivo interactions with the niche has also been hindered by the heterogeneity of available HSC-enriched fractions and the technical challenges of imaging HSC fate in vivo. To illuminate the behavior of individual HSCs in vivo, we have established a new technical regimen which includes prospective isolation of HSCs with high purity based on Tie2 positivity, a local transplantation technique which delivers a single HSC under multiphoton microscopy guidance into the bone marrow of a live mouse, and micropipette aspiration to extract single cells after division directly from the marrow for transcriptomic assay. Our project will utilize these advances to describe the molecular basis of HSC fate choice in the niche. This in turn will facilitate novel therapeutic strategies for cell- fate manipulation which could accelerate hematopoietic recovery after transplantation, and possibly contribute to improved transplantation efficiency for non-malignant blood diseases. Thus, the goals of this proposal are three-fold: (1) to identify molecular mechanisms which enhance symmetric self-renewing division of HSCs, (2) to understand the niche factors governing HSC division balance, and (3) to assess the HSC niche under non- genotoxic conditioning. If successful, the proposed research will positively impact the HSC field by identifying molecular targets that will improve hematopoietic recovery after transplantation, and enable improvements in the ex vivo engineering of niche models.