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

说明(申请人提供)：造血干细胞是所有造血细胞的来源，并在整个生物体生命周期内根据需要补充造血室。由于这种平衡的改变极大地影响了干细胞的维持，调节干细胞命运的分子机制在临床应用中具有很大的前景。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，到目前为止，关于脂代谢在干细胞动态平衡中的作用还知之甚少。为了更好地了解参与干细胞命运和维持的关键代谢途径，我们提出了以下具体目标：1.研究干细胞中PPAR-脂肪酸氧化失活的影响；我们先前已经证明，干细胞比承诺的祖细胞表现出更高的Ppard表达和脂肪酸氧化，并假设脂代谢在其再繁殖能力中起作用。根据这一前提，我们还发现，在体外抑制脂肪酸氧化会导致长期培养启动细胞能力的降低。此外，Ppard消融可减少造血干细胞室中的脂肪酸氧化。目前的建议旨在阐明体内Ppard基因缺失对移植环境中干细胞重建能力的影响。使用Ppard基因敲除模型进行干细胞分裂分析将使我们能够测试Ppard基因敲除是否会导致干细胞在分裂过程中承诺增加。2.通过激活PPAR信号通路，以最少的供体细胞实现长期植入；我们将在体内使用不同的低剂量PPAR激活剂，观察它们对Ppard野生型和基因敲除小鼠干细胞长期维持的影响。这将以PPAR依赖的方式提供明确的证据，证明PPAR激活剂在干细胞室中的潜在好处。然后，我们将通过使用与人骨髓细胞移植的异种小鼠模型来确定，PPAR信号的药理学激活是否诱导移植的最低数量的人造血干细胞，以最大限度地提高其在体内的长期再繁殖能力。3.确定通过分裂平衡控制维持干细胞特性的细胞命运决定因素；与已知的无脊椎动物正常细胞的对称和不对称分裂形成鲜明对比的是，很难想象脊椎动物中大多数纯化的干细胞室的分裂模式。因此，我们建议建立敲入鼠系，用于干细胞分裂的实时成像，并研究调节干细胞决定的内在和外在信号。结合我们在纯化干细胞室进行的RNA-seq整个转录组分析的数据，这些小鼠模型的结果将有助于更深入地理解干细胞的细胞命运决定因素。这些拟议的研究将为干细胞的细胞命运决定确定一个新的代谢开关，并反过来为操纵造血干细胞功能开辟新的治疗途径，可能还包括白血病干细胞的功能。这项工作将在下列专家的支持下进行：Michael A.Brownlee博士(新陈代谢)、李志浩博士(新陈代谢)、David E.Avigan(血液学/肿瘤学)、Julie Teruya-Feldstein(血液病理学)、Toshio sua(干细胞)、Jan Vijg(遗传学)和温弗里德·埃德尔曼(基因打靶)。重要的是，Paul S.Frenette博士(干细胞利基)与Arthur Skoultchi博士(血液学)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).