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，并在生物体生命周期内根据需要补充造血室。由于该区室平衡的改变极大地影响了干细胞的维持，因此调节干细胞命运决定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，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的关键代谢途径，我们提出以下具体目标：探讨ppar脂肪酸氧化失活对干细胞的影响；我们之前的研究表明，干细胞比稳定的祖细胞表现出更高的Ppard表达和脂肪酸氧化，并假设脂质代谢在它们的再生能力中起作用。根据这一前提，我们还发现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消融导致造血干细胞室中脂肪酸氧化的减少。目前的建议旨在阐明体内Ppard基因缺失对移植环境下干细胞重建能力的影响。使用Ppard敲除模型进行干细胞分裂试验将允许我们测试Ppard消融是否会导致干细胞在分裂过程中增加承诺。2. 通过激活PPAR信号通路，以最少的供体细胞实现长期移植；我们将在体内使用不同的低剂量PPAR¿激活剂，观察它们对Ppard野生型和敲除小鼠干细胞长期维持的影响。这将以PPAR依赖的方式，为干细胞室中PPAR激活剂的潜在益处提供明确的证据。然后，我们将通过使用移植了人骨髓细胞的异种移植小鼠模型来确定，PPAR信号的药理激活是否会诱导移植最少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生能力。3. 鉴定通过分裂平衡控制维持干细胞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，在脊椎动物中对大多数纯化干细胞区室的分裂模式进行成像是极其困难的。因此，我们建议产生敲入小鼠系，用于干细胞分裂的实时成像，并研究调节干细胞决策的内在和外在信号。结合我们在纯化干细胞室中通过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细胞命运决定因素的更深入了解。这些拟议的研究将确定干细胞细胞命运决定的一种新的代谢开关，并反过来为操纵造血干细胞功能以及可能的白血病干细胞功能开辟新的治疗途径。这项工作将在以下专家的支持下进行：Drs。Michael A. Brownlee（代谢），Chih-Hao Lee（代谢），David E. Avigan（血液学/肿瘤学），Julie Teruya-Feldstein（血液学-病理学），Toshio Suda（干细胞），Jan Vijg（遗传学）和Winfried Edelman（基因靶向）。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项目。

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).