The roles of lipid metabolism in the maintenance of hematopoietic stem cells

脂质代谢在造血干细胞维持中的作用

• 批准号: 9857923
• 负责人: Keisuke Ito
• 金额: $ 14.23万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9857923
• 关键词: roles; lipid; metabolism; maintenance; hematopoietic

ABSTRACT The symmetry of stem cell division is one of the most fundamental questions in stem cell biology, and a leading goal of our research is identification of the key metabolic pathways that regulate hematopoietic stem cell (HSC) fate. We hypothesize that lipid metabolism contributes to HSC maintenance through precise control of division patterns. Single-cell approaches have identified the enhanced clearance of damaged mitochondria by fatty acid oxidation as an important mechanism of the self-renewing expansion of HSCs. However, our understanding of the relationship between HSC self-renewal and lipid metabolism is limited, as analyses of individual HSC division patterns have been hindered by both the heterogeneity of available HSC-enriched fractions and the technical challenges of imaging HSC fate in vivo. In addition, the number of cells required for full metabolomics analysis of rare populations of HSCs has proven prohibitive. To examine the activity upstream of fatty acid oxidation in HSCs, we have generated hematopoietic-specific conditional knockout mice for key genes impacting fatty acid oxidation pathway and/or fatty acid flow. A new biosensor for assessment of fatty acid oxidation activity in live cells has likewise been established to determine the metabolic modes which are most relevant to the controlled equilibrium of HSCs, and the gene-expression oriented bioinformatics tool, graphite, has been adapted to identify specific metabolite-dependent pathways. In order to illuminate the behavior of individual HSCs in vivo, we have established new technical regimens which include 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 bone marrow for functional or transcriptomic assay. Our project will utilize these advances to test our hypothesis regarding the roles of lipid metabolism in HSC fate choice. This in turn will facilitate novel therapeutic strategies for shifting the division balance of HSCs toward self-renewal through metabolic manipulation, and possibly contribute to improved clinical outcomes after HSC transplantation for non-malignant blood diseases. Thus, the goals of this proposal are three-fold: (1) In Aim 1, we will investigate the function of mitochondrial fatty acid oxidation in HSC division symmetry and explore a potential source of fatty acids to fulfill the requirements of HSCs; (2) In Aim 2, we will use the biosensor to identify key downstream metabolic targets of fatty acid metabolism for HSC fate and explore the measurement of the cellular metabolome in HSCs; and (3) finally, we propose in Aim 3 to directly examine in vivo HSC division symmetry, and the resulting division balance of fatty acid oxidation-defective HSCs will show definitively the in vivo relevance of fatty acid metabolism to HSC fate. If successful, the proposed research will positively impact the HSC field by providing a deeper understanding of the metabolic cues governing HSC fate decisions.

抽象的 干细胞分裂的对称性是干细胞生物学中最基本的问题之一，也是领先的 我们研究的目标是确定调节造血干细胞的主要代谢途径（HSC） 命运。我们假设脂质代谢通过精确控制分裂有助于HSC维持 模式。单细胞方法已经确定了通过脂肪酸损坏的线粒体的清除率 氧化是HSC自我更新扩张的重要机制。但是，我们对 HSC自我更新与脂质代谢之间的关系有限，因为单个HSC分裂的分析 可用的HSC增益分数和技术的异质性都阻碍了模式 体内成像HSC命运的挑战。此外，完整代谢组学分析所需的细胞数量 HSC的罕见人群已被证明是过分的。检查脂肪酸氧化上游的活性 HSC，我们为影响脂肪酸的关键基因产生了造血特异性条件敲除小鼠 氧化途径和/或脂肪酸流。用于评估脂肪酸氧化活性的新生物传感器 同样建立了细胞以确定与受控的最相关的代谢模式 HSC的平衡以及面向基因表达的生物信息学工具石墨已被调整以识别 特定代谢物依赖性途径。为了阐明体内单个HSC的行为，我们有 建立的新技术方案，包括基于TIE2的高纯度的前瞻性隔离HSC 阳性，一种局部移植技术，在多光子显微镜指导下提供单个HSC 进入活小鼠的骨髓，并在直接分裂后提取单个细胞 从骨髓进行功能或转录组测定。我们的项目将利用这些进步来测试我们的 关于脂质代谢在HSC命运选择中的作用的假设。反过来，这将有助于新颖的治疗 通过代谢操作将HSC平衡转移到自我更新的策略，并 HSC移植后，可能有助于改善非恶性血液疾病的临床结果。 因此，该提案的目标是三个折叠：（1）在AIM 1中，我们将研究线粒体脂肪的功能 HSC分裂对称中的酸氧化并探索脂肪酸的潜在来源以满足需求 HSC； （2）在AIM 2中，我们将使用生物传感器来识别脂肪酸下游代谢靶标 HSC命运的代谢并探索HSC中细胞代谢组的测量； （3）最后，我们 提议在AIM 3中直接检查体内HSC分裂对称性和最终的脂肪平衡 酸氧化缺陷的HSC将明确显示脂肪酸代谢与HSC命运的体内相关性。如果 成功的研究将通过对HSC领域产生积极影响，通过更深入地了解HSC领域 管理HSC命运决定的代谢提示。