The impact of inflammation on hematopoietic stem cell specification

造血干细胞(HSCs)是人类骨髓中罕见的细胞，它既负责 所有血细胞系的终生补充和骨髓移植的疗效。 人类诱导多能干细胞的产生为细胞再生疗法带来了巨大的希望，但 目前还不可能指示这些细胞在体外产生HSCs。此应用程序的目标是 确定通过核因子-kB的促炎信号如何指导脊椎动物胚胎中的HSC命运，以及这是如何实现的 过程受原颗粒a(PgRNA)调节，最终目标是在体外复制HSC的生成。 临床应用。我的工作假设是，早期的促炎输入会聚在一起激活核因子-kB，这在 TURN激活HSC命运规范中的关键信号事件。这些促炎信号需要 在HSC规范后不久下调；我的初步结果表明pgRNA以这种方式发挥作用。 为了验证这些假说，这项建议包括两个目标：(1)表征核因子-kB在HSC中的作用 规范；以及(2)确定pgRNA在HSC出现中的作用。这项研究将在斑马鱼身上进行， 它们是直接可视化造血干细胞的理想系统，并已成为研究人类的模式生物 疾病。为了实现这一应用的目标，将产生新的转基因和突变株系，并通过qPCR， 将利用流式细胞仪分选、RNA-SEQ和共聚焦显微镜技术。 埃斯平·帕拉森博士是加州大学洛杉矶分校大卫·特拉弗实验室的博士后研究员，他的终极职业生涯 目标是在一家主要研究机构领导一个专注于干细胞的独立研究小组。她的短裤- 学期目标是(1)确定血液内皮中核因子-kB的时空需求，以及 下游基因被调控以指定HSC；以及(2)阐明pgRNA如何调控HSC的出现。 她被招募加入特拉弗博士的团队是因为她在免疫学和斑马鱼方面有很强的背景 动物模型。该提案中概述的项目将允许ESpin Palazon博士从一名 从科学职位到独立的研究生涯，帮助她获得了FACS分类、RNA-seq、 基因组编辑技术和HSC生物学，所有这些都是建立她独立实验室的基础。Dr。 ESpin Palazon将在年内会见并向发育、免疫学和血液学专家展示她的工作 除了将她的数据提交给由资深专家组成的正式指导委员会外，该委员会将帮助她 过渡到独立研究人员。加州大学圣迭戈分校提供了许多埃斯平·帕拉松博士将参加的课程，如 以及关于职业发展和实验室管理的研讨会。充满活力、协作的科学 加州大学圣迭戈分校的氛围是Espin Palazon博士在指导阶段发展的理想环境 她的获奖，将有助于为这些研究的未来成功奠定基础。她很好 准备好执行拟议的工作，实现她的职业发展和培训目标，并做出贡献 对科学界的研究产生影响。

Hematopoietic stem cells (HSCs) are rare cells within human bone marrow that are responsible both for the life-long replenishment of all blood cell lineages and for the curative effects of bone marrow transplantation. The creation of human induced pluripotent stem cells holds great promise for cellular regeneration therapies, but it is not currently possible to instruct these cells to generate HSCs in vitro. The goal of this application is to determine how pro-inflammatory signaling via NF-kB instructs HSC fate in the vertebrate embryo, and how this process is regulated by Progranulin a (Pgrna), with the ultimate goal of replicating HSC generation in vitro for clinical utility. My working hypothesis is that early pro-inflammatory inputs converge to activate NF-kB, which in turn activates key signaling events in the specification of HSC fate. These pro-inflammatory signals need to be downregulated soon after HSC specification; my preliminary results suggest that Pgrna functions in this manner. To test these hypotheses, this proposal consists of 2 aims: (1) Characterize the role of NF-kB in HSC specification; and (2) identify the role of Pgrna in HSC emergence. This study will be conducted in zebrafish, which are an ideal system for direct visualization of HSCs and have served as a model organism to study human disease. To achieve this application’s goals, novel transgenic and mutant lines will be generated, and qPCR, FACS-sorting, RNA-seq and confocal microscopy techniques will be utilized. Dr. Espin Palazon is a postdoctoral fellow in David Traver’s laboratory at UCSD whose ultimate career goal is to lead an independent research group focused on stem cells at a major research institution. Her short- term goals are (1) to determine the spatiotemporal requirements of NF-kB within hemogenic endothelium, and the downstream genes regulated to specify HSCs; and (2) to elucidate how Pgrna governs HSC emergence. She was recruited to join Dr. Traver’s group because of her strong background in immunology and the zebrafish animal model. The project outlined in this proposal will allow Dr. Espin Palazon to transition from a mentored scientific position to an independent research career, helping her gain expertise in FACS sorting, RNA-seq, genome editing techniques, and HSC biology, all of which are fundamental to establish her independent lab. Dr. Espin Palazon will meet and present her work to experts in development, immunology and hematology, in addition to presenting her data to a formal mentorship committee comprised of senior experts that will aid her transition to an independent researcher. UCSD offers numerous courses that Dr. Espin Palazon will attend, as well as seminars on career development and laboratory management. The vibrant, collaborative scientific atmosphere at UCSD is an ideal environment for Dr. Espin Palazon to develop during the mentored phase of her award, and will be instrumental in forming the foundation for the future success of these studies. She is well poised to execute the proposed work, achieve her career development and training goals and to contribute high impact research to the scientific community.