The impact of inflammation on hematopoietic stem cell specification

炎症对造血干细胞规范的影响

• 批准号: 9432324
• 负责人: Raquel Espín Palazón
• 金额: $ 14.65万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-08 至 2022-09-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9432324
• 关键词: Address; Anemia; Animal Model; Aorta; Award; Blood; Blood Cells; Bone Marrow; Bone Marrow Transplantation; Cell Lineage; Cell Therapy; Cells; Cellular biology; Clinical; Communities; Confocal Microscopy; Cytokine Receptors; Data; Development; Discipline; Dorsal; Embryonic Development; Endothelium; Environment; Event; Focus Groups; Foundations; Future; Generations; Genes; Genetic; Goals; Hematological Disease; Hematology; Hematopoietic; Hematopoietic Stem Cell Research; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hemoglobinopathies; Human; Imagery; Immunologics; Immunology; In Vitro; Individual; Inflammation; Inflammation Mediators; Inflammatory; Institution; Joints; Knowledge; Laboratories; Lead; Life; Mentors; Mentorship; Modeling; Molecular Biology; Multipotent Stem Cells; NF-kappa B; Natural regeneration; Organism; Outcome; PGRN gene; Patients; Phase; Play; Positioning Attribute; Postdoctoral Fellow; Process; Property; Protocols documentation; Recruitment Activity; Regulator Genes; Reporting; Research; Research Personnel; Role; Signal Transduction; Sorting - Cell Movement; Source; Specific qualifier value; Stem cells; System; TNF gene; Techniques; Testing; Therapeutic; To specify; Training; Transgenic Organisms; Work; Zebrafish; base; career; career development; cytokine; experimental study; genetic manipulation; genome editing; hematopoietic stem cell fate; human disease; improved; in vivo; induced pluripotent stem cell; leukemia; mutant; notch protein; novel; pgRNA; reconstitution; scientific atmosphere; self renewing cell; skills; spatiotemporal; success; tool; transcriptome sequencing; vertebrate embryos

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.

造血干细胞 (HSC) 是人类骨髓中的稀有细胞，负责造血干细胞和造血干细胞 所有血细胞谱系的终生补充以及骨髓移植的疗效。 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，但是 目前还不可能指导这些细胞在体外生成 HSC。该应用程序的目标是 确定 NF-kB 促炎症信号如何指导脊椎动物胚胎中 HSC 的命运，以及这如何 该过程由颗粒体蛋白前体 a (Pgrna) 调节，最终目标是在体外复制 HSC 生成 临床实用性。我的工作假设是，早期促炎症输入会聚合激活 NF-kB，这在 转激活 HSC 命运规范中的关键信号事件。这些促炎信号需要 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。 为了检验这些假设，该提案包含 2 个目标：(1) 表征 NF-kB 在 HSC 中的作用 规格; (2) 确定 Pgrna 在 HSC 出现中的作用。这项研究将在斑马鱼中进行， 这是直接可视化 HSC 的理想系统，并已作为研究人类的模型生物 疾病。为了实现该应用的目标，将产生新的转基因和突变株系，并进行 qPCR、 将利用 FACS 分选、RNA 测序和共焦显微镜技术。 Espin Palazon 博士是加州大学圣地亚哥分校 David Traver 实验室的博士后研究员，他的最终职业生涯 目标是在一个主要研究机构领导一个专注于干细胞的独立研究小组。她的短篇—— 术语目标是 (1) 确定造血内皮细胞内 NF-kB 的时空需求，以及 下游基因被调节以指定HSC； (2) 阐明 Pgrna 如何控制 HSC 的出现。 由于她在免疫学和斑马鱼方面的深厚背景，她被招募加入 Traver 博士的团队 动物模型。该提案中概述的项目将使 Espin Palazon 博士能够从受指导的人转变为 科学地位转向独立研究职业，帮助她获得 FACS 分选、RNA-seq、 基因组编辑技术和 HSC 生物学，所有这些都是建立她的独立实验室的基础。博士。 Espin Palazon 将在以下地点与发育、免疫学和血液学专家见面并向他们展示她的工作 除了向由高级专家组成的正式指导委员会提供她的数据之外，该委员会将帮助她 转型为独立研究员。加州大学圣地亚哥分校提供 Espin Palazon 博士将参加的众多课程，例如 以及职业发展和实验室管理研讨会。充满活力、协作的科学 UCSD 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未来的成功奠定基础。她很好 准备好执行拟议的工作，实现职业发展和培训目标，并做出高贡献 对科学界的影响研究。