Role of slug in hematopoietic stem cell regeneration

描述（由申请人提供）：造血干细胞（HSC）是造血区室中罕见的自我更新细胞，可促进数百万种不同类型的分化血细胞的产生。造血干细胞移植已成为白血病、再生障碍性贫血、镰状细胞病和其他造血系统疾病患者的常用临床治疗方法。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用的HSC数量，因此HSC在体内和体外的扩增已被认为是高度优先的。不幸的是，在这一研究领域的进展一直受到阻碍的有限的理解的决定HSC自我更新或分化的分子机制。在拟议的项目中，我们的目标是确定鼻涕虫，进化上保守的锌指转录抑制因子，在调节HSC自我更新的作用。 我们最近的研究表明，Slug对正常的造血功能是有害的，但对骨髓移植或5-氟尿嘧啶（5- FU）治疗后抑制血细胞再生至关重要。其他研究表明，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Id 3）的有效负调节因子，而它在HSC中受到SCF/c-Kit信号的正调节。这些发现在逻辑上导致本申请的中心假设：Slug在SCF/c-kit信号传导途径中的新型负反馈环中起作用，并且其缺失将在造血系统的再增殖期间促进HSC的对称分裂。这些预测将通过检查Slug缺陷对骨髓毒性损伤后HSC完整性的影响（Aim 1）;通过确定Slug是否在造血再生期间调节HSC的对称细胞分裂（Aim 2）;以及通过阐明Slug在SCF/c-kit信号通路中的作用（Aim 3）进行测试。这些目标的成功完成将通过专门为此项目开发的创新方法和策略来促进，包括基于PKH 2 -26染料和Notch-GFP报告基因的单细胞分裂测定，以区分对称和不对称分裂，以及新的HSC自我更新竞争测定。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.