Regulation of Hematopoietic Stem Cell Self Renewal

描述(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创造的。因此，干细胞在分裂过程中的命运决定是组织动态平衡的基础。像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代细胞。由于HSC的不受控制的扩张和丧失对生物体都是致命的，因此必须严格控制自我更新还是分化的决定。因此，了解决定HSC命运的分子机制(S)具有相当重要的临床意义。这项应用着重于了解p190-B RhoGAP在HSC自我更新和命运决定中的作用。P190-B是Rho GTP酶活性的负调控因子。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不可少的。我们发现，在连续移植过程中，p190-B的丢失会导致长期植入的增强。与初次受者相比，二次受者骨髓中的WT-HSCs数量减少，而p190-B-空的受者数量在二次受者和初次受者之间保持不变。同时，p190-B-空的造血干细胞仍具有多向分化潜能。因此，在连续移植过程中，p190-B的丢失可防止HSC耗尽以维持造血。在机制水平上，p190-B缺乏并不改变HSC的细胞周期动力学或在体内和体外的存活-因此，提示p190-B在细胞分裂时调节HSC的自我更新活性。这些数据表明，p190-B是HSC自我更新的关键调节因子，通过调节HSC在分裂过程中的命运决定。我们建议确定p190-B在HSC自我更新中调控的信号通路，并确定p190-B及其相关信号通路调控HSC命运决定的机制(S)。我们计划利用HSC功能分析、视频显微镜、免疫荧光成像来研究(1)IGF-1信号和p16INK4a在p190-B介导的HSC自我更新中的作用；(2)确定p190-B及其下游信号通路在影响细胞命运决定因素分离的细胞极性机制中的作用，以控制不对称/对称自我更新分裂的平衡。我们认为，对p190-B-空的HSC的分析提供了一个难得的机会来确定对HSC命运决定重要的干细胞特异性基因。如果p190-B通过IGF-1/p16INK4a调控HSC命运的假设被证实，这将显著改变对IGF-1信号和p16INK4a在HSC自我更新中作用的看法。造血干细胞重建造血系统的巨大潜力使临床移植治疗多种疾病成为可能。这些拟议的研究有望为设计新的临床造血干细胞移植方案提供理论依据。 公共卫生相关性：造血干细胞自我更新是维持终生血细胞持续生产的基本过程。取消对HSC自我更新的调控会导致癌症或组织退化。HSC重建造血系统的巨大潜力使临床HSC移植得以发展，用于治疗多种疾病，包括骨髓衰竭或白血病。然而，由于我们无法控制HSC的功能，HSC移植的成功是有限的。本申请中提出的研究将为p190-B RhoGAP在调节HSC自我更新中的生理作用提供重要信息。因此，我们的研究将指导设计通过抑制p190-B Rho间隙来调节HSC功能的策略，未来可以用于设计临床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.