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，也是一个重要的研究方向。 我们研究的目标是确定调节造血干细胞(Hsc)的关键代谢途径。 命运。我们假设脂代谢通过对分裂的精确控制来促进HSC的维持。 模式。单细胞方法已经证实了脂肪酸对受损线粒体的增强清除作用 氧化是HSCs自我更新扩增的重要机制。然而，我们对此的理解 HSC的自我更新和脂代谢之间的关系是有限的，正如对HSC个体分裂的分析一样 模式受到可用HSC富集组分的异质性和技术上的阻碍 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 事实证明，罕见的造血干细胞种群的数量令人望而却步。为了检测脂肪酸氧化上游的活性 HSCs，我们已经产生了影响脂肪酸的关键基因的造血特异性条件性基因敲除小鼠 氧化途径和/或脂肪酸流动。一种用于评价活体脂肪酸氧化活性的新型生物传感器 同样，细胞也被建立来确定与受控者最相关的代谢模式 HSCs的平衡，以及面向基因表达的生物信息学工具，石墨已被用于识别 特定的代谢物依赖途径。为了阐明单个造血干细胞在体内的行为，我们有 建立了新的技术方案，包括基于Tie2的高纯度HSCs的前瞻性分离 阳性，一种在多光子显微镜引导下移植单个HSC的局部移植技术 进入活体小鼠的骨髓，微吸管抽吸直接提取分裂后的单个细胞 从骨髓中提取，用于功能或转录分析。我们的项目将利用这些进步来测试我们的 关于脂代谢在HSC命运选择中作用的假说。这反过来将促进新的治疗方法 通过代谢调控将造血干细胞的分裂平衡转向自我更新的策略，以及 可能有助于改善非恶性血液病的HSC移植后的临床结果。 因此，这项提议的目标有三个：(1)在目标1中，我们将研究线粒体脂肪的功能 酸氧化在HSC分裂对称性和潜在脂肪酸来源方面的满足要求 (2)在目标2中，我们将使用生物传感器来识别脂肪酸的关键下游代谢靶点 代谢对HSC命运的影响，并探索HSCs细胞代谢物的测定；(3)最后，我们 在目标3中建议直接检查体内HSC的分裂对称性，并由此得到脂肪的分裂平衡 酸性氧化缺陷的HSC将在体内明确地显示脂肪酸代谢与HSC命运的相关性。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的了解，对HSC领域产生积极影响 主宰HSC命运决定的代谢线索。

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.