Regulation of Hematopoietic Stem Cell Self Renewal

描述（由申请人提供）：组织多样性是通过干细胞不对称自我更新分裂产生的。因此，在分裂过程中干细胞的命运决定是组织稳态的基础。与其他类型的成体干细胞一样，造血干细胞（HSC）可以产生具有不同命运的子细胞。由于不受控制的HSC扩增以及HSC的丧失对于生物体都是致命的，因此必须严格控制自我更新与分化的决定。因此，对HSC命运决定的分子机制的理解具有相当大的临床重要性。本申请着重于了解p190-B RhoGAP（Rho GT3活性的负调节因子）在HSC自我更新和命运决定中的作用。我们对p190-B缺失的HSC的分析表明，p190-B对于移植后HSC自我更新的调节是必需的。我们发现p190-B的缺失导致连续移植过程中长期植入增强。虽然与初次接受者相比，二次接受者骨髓中WT HSC的数量减少，但p190-B-无效HSC的数量在二次和初次接受者之间保持不变。与此同时，p190-B-null HSC保持多潜能谱系分化。因此，p190-B的缺失阻止了HSC耗竭以维持连续移植期间的造血。在机制水平上，p190-B-缺陷并没有改变HSC细胞周期动力学或存活在体内和体外-因此，表明p190-B调节HSC自我更新活性作为细胞分裂。这些数据表明，假设p190-B是一个关键的调节HSC自我更新，通过调节HSC的命运决定在分裂。本研究拟探讨p190-B调控HSC自我更新的信号通路，以及p190-B及其相关信号通路调控HSC命运决定的机制。我们计划利用HSC功能检测、视频显微镜、免疫荧光成像等方法来研究（目的1）IGF-1信号通路和p16 Ink 4a在p190-B介导的HSC自我更新中的作用;（目的2）确定p190-B和aim 1中鉴定的下游信号通路在影响细胞命运决定子分离的细胞极性机制中的作用，以控制不对称/非对称性细胞周期的平衡。对称自我更新分裂我们认为，p190-B-null HSC的分析提供了一个难得的机会，以确定干细胞特异性基因，是重要的HSC命运的决定。如果我们的假说p190-B通过IGF-1/p16 Ink 4a调节HSC命运决定被证实，这将显著改变对IGF-1信号和p16 Ink 4a在HSC自我更新中功能的看法。HSC重建造血系统的巨大潜力使得临床HSC移植治疗多种疾病成为可能。这些研究将为临床HSC移植提供新的治疗方法。 造血干细胞自我更新是维持整个生命中持续血细胞生产的基本过程。HSC自我更新的失调导致癌症或组织变性。HSC重建造血系统的巨大潜力使得临床HSC移植的发展能够治疗多种疾病，包括骨髓衰竭或白血病。然而，由于我们无法控制HSC功能，HSC移植的成功是有限的。本申请中提出的研究将为p190-B RhoGAP在HSC自我更新调节中的生理作用提供重要信息。因此，我们的研究将指导设计策略，通过抑制p190-B Rho GAP来调节HSC功能，可以在未来用于设计临床HSC移植方案的新治疗方法。

DESCRIPTION (provided by applicant): Tissue diversity is created through stem cell asymmetric self renewal division. Stem cell fate decision during division is thus fundamental for tissue homeostasis. Like other types of adult stem cells, hematopoietic stem cells (HSC) can produce daughter cells with distinct fates. Since both uncontrolled HSC expansion as well as loss of HSC is fatal for the organism, the decision of self renewal versus differentiation must be tightly controlled. An understanding of the molecular mechanism(s) of HSC fate decision is thus of considerable clinical importance. This application focuses on understanding the role of p190-B RhoGAP, a negative regulator of Rho GTPase activity, in HSC self renewal and fate decision. Our analysis of p190-B-null HSC shows that p190-B is essential for the regulation of HSC self renewal following transplantation. We show that loss of p190-B results in enhanced long term engraftment during serial transplantation. While the number of WT HSCs in the bone marrow of secondary recipients decreases in comparison with primary recipients, the numbers of p190-B-null HSCs is maintained between the secondary and primary recipients. At the same time, p190-B-null HSCs retain multipotential lineage differentiation. Thus, loss of p190-B prevents HSC exhaustion to maintain hematopoiesis during serial transplantation. At a mechanistic level, p190-B-deficiency did not alter HSC cell cycle kinetics or survival both in vivo and in vitro - thus, suggesting that p190-B modulates HSC self renewal activity as the cells divide. These data suggest the hypothesis that p190-B is a critical regulator of HSC self renewal by modulating HSC fate decision during divisions. We propose to identify the signaling pathway modulated by p190-B in HSC self renewal; and to determine the mechanism(s) by which p190-B and the associated signaling pathway regulates HSC fate decision. We plan to use functional assays of HSC functions, video microcopy, immunofluorescence imaging to examine (Aim 1) the role of IGF-1 signaling and p16Ink4a in p190-B-mediated HSC self renewal; (Aim 2) determine the role of p190-B and downstream signaling pathway identified in aim1 in mechanism of cell polarity that influences the segregation of cell fate determinant to control the balance of asymmetric/symmetric self renewal divisions. We believe that analysis of p190-B-null HSC offers a rare opportunity to identify stem cell-specific genes that are important for HSC fate decision. If our hypothesis that p190-B regulates HSC fate decision via IGF-1/p16Ink4a is validated, this will significantly change the view of IGF-1 signaling and p16Ink4a functions in HSC self renewal. The tremendous potential of HSC to reconstitute the hematopoietic system has allowed the development of clinical HSC transplantation to treat a wide variety of diseases. The proposed studies are expected to provide rationales to devise new therapeutic approaches of clinical HSC transplantation protocols. PUBLIC HEALTH RELEVANCE: Hematopoietic stem cell self renewal is a fundamental process to maintain continuous blood cell production throughout life. Deregulation of HSC self renewal leads to cancer or tissue degeneration. The tremendous potential of HSC to reconstitute the hematopoietic system has allowed the development of clinical HSC transplantation to treat a wide variety of diseases, including bone marrow failure or leukemias. The success of HSC transplantation is however limited due to our inability to control HSC functions. The studies proposed in this application will provide important information of the physiologic role of p190-B RhoGAP in the regulation of HSC self renewal. Therefore, our study will guide designing strategies to modulate HSC functions via inhibition of p190-B Rho GAP that could be used in the future to device new therapeutic approaches of clinical HSC transplantation protocols.