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: 9906877
• 负责人: Keisuke Ito
• 金额: $ 37.58万
• 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/9906877
• 关键词: Ablation; Adipose tissue; Autophagocytosis; Behavior; Biological Assay; Biological Markers; Biosensor; Bone Marrow; Carnitine Palmitoyltransferase I; Cell Maintenance; Cell division; Cells; Cellular Assay; Clinical; Cues; Data; Embryo; Environment; Enzymes; Equilibrium; Event; Fatty Acids; Gene Expression; Genes; Genetic Models; Goals; Hematological Disease; Hematopoietic; Hematopoietic Stem Cell Research; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Heterogeneity; Homeostasis; Image; In Vitro; Individual; Knockout Mice; Knowledge; Lipase; Lipids; Maintenance; Mass Spectrum Analysis; Measurement; Measures; Metabolic; Metabolic Pathway; Metabolism; Microscope; Mitochondria; Molecular; Monitor; Mus; NADP; Natural graphite; Non-Malignant; Nonesterified Fatty Acids; Outcome; Pathway interactions; Pattern; Play; Population; Process; Production; Regimen; Research; Role; Solid; Source; System; Techniques; Testing; Time; Transplantation; Work; base; bioinformatics tool; cell behavior; clinical practice; conditional knockout; daughter cell; fatty acid metabolism; fatty acid oxidation; hematopoietic stem cell expansion; hematopoietic stem cell fate; hematopoietic stem cell self-renewal; image guided; improved; in vivo; innovation; insight; lipid metabolism; metabolome; metabolomics; mitochondrial metabolism; multiphoton microscopy; novel therapeutics; prevent; prospective; self-renewal; stem cell biology; stem cell division; stem cells; transcriptome; transcriptomics

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，也是一个前沿问题。 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。我们假设脂质代谢通过精确控制分裂有助于 HSC 的维持 模式。单细胞方法已发现脂肪酸可增强受损线粒体的清除能力 氧化是HSC自我更新扩张的重要机制。然而，我们的理解 HSC 自我更新和脂质代谢之间的关系是有限的，因为对个体 HSC 分裂的分析 可用的 HSC 富集组分的异质性和技术水平都阻碍了模式的发展 体内 HSC 命运成像的挑战。此外，完整代谢组学分析所需的细胞数量 事实证明，稀有造血干细胞群体的数量令人望而却步。检查脂肪酸氧化上游的活性 HSC，我们已经培育出了影响脂肪酸关键基因的造血特异性条件敲除小鼠 氧化途径和/或脂肪酸流。一种用于评估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物质最相关的代谢模式。 HSC 的平衡，以及基因表达导向的生物信息学工具石墨已被用来识别 特定的代谢依赖性途径。为了阐明体内单个 HSC 的行为，我们有 建立了新技术方案，其中包括基于 Tie2 前瞻性分离高纯度 HSC positivity，一种局部移植技术，可在多光子显微镜引导下输送单个 HSC 注入活体小鼠骨髓，直接微量移液器吸取分裂后的单细胞 来自骨髓用于功能或转录组测定。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。这反过来又将促进新的治疗方法 通过代谢操纵将 HSC 的分裂平衡转向自我更新的策略，以及 可能有助于改善 HSC 移植治疗非恶性血液疾病后的临床结果。 因此，该提案的目标有三个：（1）在目标1中，我们将研究线粒体脂肪的功能 HSC分裂对称性中的酸氧化并探索满足要求的潜在脂肪酸来源 HSC 数量； (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目标 HSC 命运的代谢并探索 HSC 中细胞代谢组的测量； （3）最后，我们 在目标 3 中建议直接检查体内 HSC 分裂对称性，以及由此产生的脂肪细胞分裂平衡 酸氧化缺陷的 HSC 将明确显示脂肪酸代谢与 HSC 命运的体内相关性。如果 如果成功的话，拟议的研究将通过提供更深入的了解来对 HSC 领域产生积极影响 控制 HSC 命运决定的代谢线索。