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

描述（由申请人提供）：造血干细胞是所有造血细胞的来源，并在整个生物体寿命期间根据需要补充造血区室。由于该室平衡的改变极大地影响干细胞维持，因此调节干细胞的细胞命运决定的分子机制对于临床应用具有很大的希望。对基因工程小鼠模型的研究表明，代谢线索有助于控制这些细胞的自我更新能力。然而，迄今为止，关于脂质代谢在干细胞稳态中的作用知之甚少。为了更好地了解干细胞命运和维持中涉及的关键代谢途径，我们提出以下具体目标：1.为了研究在干细胞中灭活PPAR-脂肪酸氧化的影响，我们以前已经表明，干细胞表现出更高的Ppard表达和脂肪酸氧化比定向祖细胞，并假设脂质代谢在其再增殖能力中发挥作用。根据这一前提，我们还发现，体外脂肪酸氧化的抑制导致长期培养起始细胞能力的降低。此外，Ppard消融导致造血干细胞区室中脂肪酸氧化的减少。目前的建议旨在阐明体内Ppard基因丢失对移植环境中干细胞重建能力的影响。使用Ppard敲除模型的干细胞分裂测定将使我们能够测试Ppard消融是否导致干细胞在其分裂期间的承诺增加。2.为了通过激活PPAR信号传导实现最小供体细胞的长期植入，我们将在体内以低剂量使用不同的PPAR激活剂，以观察它们对来自Ppard野生型和敲除小鼠的小鼠干细胞的长期维持的影响。这将提供一个明确的证据，在一个依赖于过氧化物酶体增殖物激活物的方式，在干细胞隔室的过氧化物酶体增殖物激活物的潜在利益。然后，我们将通过使用移植有人骨髓细胞的异种移植小鼠模型来确定，PPAR信号传导的药理学激活是否诱导移植最小数量的人造血干细胞以最大化其体内长期再增殖能力。3.为了确定细胞命运的决定因素，通过分裂平衡控制维持干细胞的性;与已知的无脊椎动物中正常细胞的对称和不对称分裂形成鲜明对比，在脊椎动物中大多数纯化的干细胞隔室的分裂模式是非常困难的。因此，我们建议产生敲入小鼠系的干细胞分裂的实时成像和研究的内在和外在的信号调节干细胞的决定。结合我们在纯化的干细胞隔室中通过RNA-seq进行的全转录组分析的数据，这些小鼠模型的结果将导致对干细胞的细胞命运决定因素的更深入理解。这些拟议的研究将确定一种新的代谢开关，用于干细胞的细胞命运决定，并反过来为操纵造血干细胞功能开辟新的治疗途径，可能还有白血病干细胞的功能。这项工作将在以下专家的支持下进行：Brownlee（代谢），Chih-Hao Lee（代谢），大卫E. Avigan（血液学/肿瘤学）、Julie Teruya-Feldstein（血液病理学）、Toshio苏达（干细胞）、Jan Vijg（遗传学）和Winfried Edelman（基因靶向）。重要的是，保罗·S。Frenette（干细胞利基）与亚瑟Skoultchi博士（血液学）一起密切支持我们的研究计划。

DESCRIPTION (provided by applicant): Hematopoietic stem cells are the source of all hematopoietic cells, and replenish the hematopoietic compartment as required throughout organism lifespan. Since alterations in the equilibrium of this compartment greatly impact stem cell maintenance, the molecular mechanisms regulating the cell fate decisions of stem cells hold great promise for clinical applications. Studies of genetically-engineered mouse models suggest that metabolic cues contribute to the governance of these cells' self-renewal capacity. To date, however, little is known regarding the role of lipid metabolism in stem cell homeostasis. To better understand the key metabolic pathways involved in stem cell fate and maintenance, we propose the following Specific Aims: 1. To investigate the effects of inactivation of PPAR-fatty acid oxidation in stem cells; We have previously shown that stem cells exhibit higher Ppard expression and fatty acid oxidation than committed progenitor cells, and have hypothesized that lipid metabolism plays a role in their repopulation capacity. In accordance with this premise, we have also found that inhibition of fatty acid oxidation in vitro leads to a reduction of long-term culture-initiating cell capacity. Furthermore, Ppard-ablation leads to reduction of fatty acid oxidation in the hematopoietic stem cell compartment. The current proposal aims to elucidate the effect of genetic loss of Ppard in vivo on the reconstitution ability of stem cells in a transplantation setting. Stem cell division assays with Ppard knockout models will allow us to test whether Ppard-ablation leads to increased commitment of stem cells during their division. 2. To enable long-term engraftment with minimal donor cells by the activation of PPAR signaling; we will employ different activators of PPAR¿ at low doses in vivo to observe their effects on the long-term maintenance of murine stem cells from Ppard wild-type and knockout mice. This will provide a definitive proof, in a PPAR¿-dependent manner, of the potential benefit of PPAR¿ activators in the stem cell compartment. We will then determine, through the use of xenograft mouse models transplanted with human bone marrow cells, whether pharmacological activation of PPAR signaling induces a transplanted minimum number of human hematopoietic stem cells to maximize their long-term repopulation capacity in vivo. 3. To identify cell fate determinants that maintain stem cell-ness through division balance control; in stark contrast to what is known about symmetric and asymmetric division of normal cells in invertebrates, it has been extremely difficult to image the division pattern of most purified stem cell compartments in vertebrates. We therefore propose to generate knock-in mouse lines for real-time imaging of stem cell divisions and to study the intrinsic and extrinsic signals regulating stem cell decision. Combined with the data from our whole transcriptome analysis by RNA-seq in the purified stem cell compartment, the results from these mouse models will lead to a deeper understanding of the cell fate determinants of stem cells. These proposed studies will identify a novel metabolic switch for the cell fate decisions of stem cells, and in turn open new therapeutic avenues for the manipulation of hematopoietic stem cell function, and possibly the function of leukemia stem cells. This work will be conducted with the support of the following experts; Drs. Michael A. Brownlee (Metabolism), Chih-Hao Lee (Metabolism), David E. Avigan (Hematology/Oncology), Julie Teruya-Feldstein (Hemato- pathology), Toshio Suda (Stem Cells), Jan Vijg (Genetics), and Winfried Edelman (Gene Targeting). Importantly, Dr. Paul S. Frenette (Stem Cell niche) is closely supporting our research program along with Dr. Arthur Skoultchi (Hematology).