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

摘要 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， 我们的研究目标是确定调控造血干细胞（HSC）的关键代谢途径 命运我们推测脂质代谢通过精确控制分裂来维持HSC 模式.单细胞方法已经确定了脂肪酸对受损线粒体的增强清除作用， 氧化作为HSC自我更新扩增的重要机制。然而，我们对 HSC自我更新与脂质代谢之间的关系是有限的， 模式受到可用HSC富集级分的异质性和技术缺陷的阻碍。 在体内成像HSC命运的挑战。此外，完整代谢组学分析所需的细胞数量 罕见的造血干细胞群体已经被证明是禁止的。为了检查在脂肪酸氧化上游的活性， HSC，我们已经产生了造血特异性条件敲除小鼠的关键基因影响脂肪酸 氧化途径和/或脂肪酸流动。一种新的脂肪酸氧化活性生物传感器 同样，已经建立了细胞来确定与受控代谢最相关的代谢模式。 HSC的平衡，和基因表达导向的生物信息学工具，石墨，已经被用来识别 特定的代谢依赖途径。为了阐明单个HSC在体内的行为，我们 建立了新的技术方案，包括基于Tie 2的高纯度HSC的前瞻性分离 阳性，一种在多光子显微镜引导下提供单个HSC的局部移植技术 活鼠骨髓中，直接用微量吸液管吸取分裂后的单个细胞， 用于功能或转录组学测定。我们的项目将利用这些进展来测试我们的 关于脂代谢在HSC命运选择中的作用的假说。这反过来将有助于新的治疗方法 通过代谢操纵将HSC的分裂平衡向自我更新转移的策略，以及 可能有助于改善HSC移植治疗非恶性血液病后的临床结局。 因此，本研究的目的有三：（1）目的1：研究线粒体脂肪酸的功能， 酸氧化HSC分裂对称性和探索一种潜在的脂肪酸来源，以满足要求 目的2：利用该生物传感器研究脂肪酸下游代谢的关键靶点 代谢的HSC的命运，并探讨在HSC的细胞代谢组的测量;（3）最后，我们 在目的3中提出直接检查体内HSC分裂对称性，以及由此产生的脂肪细胞分裂平衡。 酸氧化缺陷型HSC将明确显示脂肪酸代谢与HSC命运的体内相关性。如果 如果成功，拟议的研究将通过提供对以下方面的更深入了解，对HSC领域产生积极影响： 决定HSC命运的代谢线索。

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.