Role of slug in hematopoietic stem cell regeneration

描述(申请人提供)：造血干细胞(HSCs)是造血室中罕见的自我更新细胞，为数百万不同类型的分化血细胞的产生提供燃料。造血干细胞移植已成为治疗白血病、再生障碍性贫血、镰状细胞病等造血系统疾病的常用方法。由于移植的成功在很大程度上取决于外周血和脐带血资源中可利用的HSCs的数量，因此在体内和体外扩增HSCs已成为当务之急。不幸的是，由于对造血干细胞决定自我更新或分化的分子机制的了解有限，这一研究领域的进展一直受到阻碍。在拟议的项目中，我们的目标是确定Slug在HSC自我更新调控中的作用，Slug是一种进化上保守的锌指转录抑制因子。我们最新的工作表明，鼻涕对于正常的造血是必不可少的，但对于抑制骨髓移植或5-氟尿嘧啶(5-FU)治疗后的血细胞再生成是至关重要的。更多的研究表明，Slug是c-kit和其他细胞周期调节因子(包括细胞周期蛋白D1、c-Myc和ID3)的强有力的负调控因子，而在HSC中它受到SCF/c-Kit信号的正调控。这些发现合乎逻辑地引出了当前应用的中心假设：在SCF/c-kit信号通路中，Slug在一个新的负反馈环中发挥作用，并且它的缺失将促进造血系统重新繁殖过程中HSC的对称分裂。这些预测将通过以下方法得到验证：检测鼻涕蛋白缺乏对骨髓毒性损伤后HSCs完整性的影响(目标1)；通过确定在造血再生过程中是否调节HSCs的对称细胞分裂(目标2)；以及通过阐明鼻涕蛋白在SCF/c-kit信号通路中的作用(目标3)。这些目标的成功实现将通过专门为该项目开发的创新方法和战略来促进，包括基于PKH2-26染料和Notch-GFP报告的单细胞分裂分析，以区分对称和不对称分裂，以及一种新的HSC自我更新竞争分析。我们期望产生的信息将有可能改善HSC在体外和体内的扩增。

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) are rare self-renewing cells in the hematopoietic compartment that fuel the production of millions of differentiated blood cells of various types. HSC transplantation has become a common clinical treatment for patients suffering from leukemia, aplastic anemia, sickle cell disease, and other disorders of hematopoietic systems. Because transplantation success depends heavily on the number of available HSCs in peripheral and cord blood resources, expansion of HSCs both in vivo and in vitro has assumed a high priority. Unfortunately, progress in this research area has been impeded by the limited understanding of the molecular mechanisms governing the decision of HSCs to self-renew or differentiate. In the proposed project, we aim to define the role of Slug, an evolutionarily conserved zinc-finger transcriptional repressor, in the regulation of HSC self-renewal. Our most recent work shows that Slug is dispensable for normal hematopoiesis, but is critical for suppression of blood cell repopulation after bone marrow transplantation or treatment with 5-fluorouracil (5- FU). Additional studies suggest that Slug is a potent negative regulator of c-kit and other cell cycle regulators (including cyclin D1, c-Myc, and Id3), while it is positively regulated by the SCF/c-Kit signaling in HSCs. These findings led logically to the central hypothesis of the current application: that Slug functions in a novel negative- feedback loop in SCF/c-kit signaling pathway, and that its deletion will promote symmetric division of HSCs during repopulation of the hematopoietic systems. These predictions will be tested by examining the effects of Slug deficiency on the integrity of HSCs following myelotoxic injury (Aim 1); by determining if Slug regulates symmetric cell division of HSCs during hematopoietic regeneration (Aim 2); and by elucidating Slug's role in the SCF/c-kit signaling pathway (Aim 3). Successful completion of these aims will be facilitated by innovative methods and strategies that were developed specifically for this project, including single cell division assays based on PKH2-26 dye and Notch-GFP reporters to discriminate between symmetric and asymmetric division, and a new HSC self-renewal competitive assay. The information we expect to generate will have the potential to improve HSC expansion both in vitro and in vivo.