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

单细胞方法揭示体内调节 HSC 分裂对称性的因素

• 批准号: 9425824
• 负责人: Keisuke Ito
• 金额: $ 60.95万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9425824
• 关键词: Acceleration; Affect; Behavior; Biological Assay; Bone Marrow; Bone Marrow Cells; CD34 gene; Candidate Disease Gene; Cell division; Cells; Clinical; Cues; Data; Engineering; Engraftment; Environment; Equilibrium; Gene Expression Profiling; Gene Targeting; Goals; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Research; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Heterogeneity; High Dose Chemotherapy; Homeostasis; Image; Immunocompetent; Immunotoxins; Individual; Lasers; Marrow; Methods; Microscopy; Modeling; Molecular; Molecular Profiling; Molecular Target; Mus; Non-Malignant; Outcome; PTPRC gene; Pathway interactions; Pattern; Physiological; Proto-Oncogene Protein c-kit; Recovery; Regimen; Research; SLAM protein; Science; Severities; Signal Pathway; Signal Transduction; Site; Stem cells; Surface; System; Techniques; Testing; Time; Transplantation; base; bone; candidate validation; cell type; clinical practice; cohort; conditioning; cranium; cytopenia; daughter cell; differential expression; hematopoietic stem cell fate; improved; in vivo; insight; irradiation; mouse model; novel therapeutics; prospective; reconstitution; restoration; self-renewal; stem cell division; stem cell niche; transcriptome sequencing; transcriptomics

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.

抽象的 造血干细胞（HSC）自我更新和分化之间的平衡直接影响 造血稳态。我们假设来自骨髓微环境（或“利基”）的信号， 与细胞中心网络的提示一起，有助于微调这种平衡。但是，我们的理解 利基市场受到当前方法的限制，该方法依赖于单个调节的顺序删除 可用小鼠模型中候选细胞的因素，分析单个HSC及其体内的因素 可用的HSC富集分数的异质性和 成像体内HSC命运的技术挑战。为了阐明体内单个HSC的行为，我们 已经建立了一种新的技术方案，其中包括基于高纯度的HSC的预期隔离 在TIE2阳性上，局部移植技术在多光子显微镜下提供单个HSC 对活小鼠的骨髓进行引导，并在分裂后提取单个细胞的微量移液术。 直接从骨髓进行转录组测定。我们的项目将利用这些进步来描述 HSC命运选择的分子基础。反过来，这将有助于细胞的新型治疗策略 命运操纵可能会加速移植后造血恢复，并可能贡献 提高了非恶性血液疾病的移植效率。那是该提议的目标是 三倍：（1）确定增强HSC对称自我更新分裂的分子机制，（2） 了解有关HSC分区平衡的利基因素，（3）在非 - 遗传毒性调节。如果成功，拟议的研究将通过识别来对HSC领域产生积极影响 分子靶标可以改善移植后造血恢复，并能够改善 利基模型的体内工程。