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

摘要 造血干细胞（HSC）自我更新和分化之间的平衡直接影响造血干细胞的功能。 造血稳态我们假设来自骨髓微环境（或"生态位"）的信号， 与来自细胞内在网络的线索一起，有助于微调这种平衡。然而，我们的理解是， 目前的做法依赖于顺序删除个别监管机构， 在可用的小鼠模型中的候选细胞的因子，以及分析单个HSC及其体内 与生态位的相互作用也受到可用HSC富集级分的异质性的阻碍， 在体内成像HSC命运的技术挑战。为了阐明单个HSC在体内的行为，我们 已经建立了一种新的技术方案，包括基于高纯度的HSC的前瞻性分离， Tie2阳性，一种在多光子显微镜下递送单个HSC的局部移植技术 引导至活小鼠的骨髓中，并在分裂后用微量移液管抽吸以提取单细胞 用于转录组学测定。我们的项目将利用这些进展来描述 HSC命运选择的分子基础。这反过来将促进新的治疗策略，为细胞- 命运操纵可以加速移植后的造血恢复，并可能有助于 涉及提高非恶性血液病的移植效率。因此，本提案的目标是 三重：（1）鉴定增强HSC对称自我更新分裂的分子机制，（2） 了解支配HSC分裂平衡的生态位因素，以及（3）评估非- 遗传毒性条件作用如果成功，拟议的研究将通过确定 这些分子靶点将改善移植后的造血恢复，并能够改善移植后的造血功能。 小生境模型的离体工程。

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.