Defining the Role of the AHR in Blood Cell Specifications

定义 AHR 在血细胞指标中的作用

• 批准号: 9193079
• 负责人: GEORGE J MURPHY
• 金额: $ 35.97万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9193079
• 关键词: AHR gene; Acute; Adoptive Transfer; Agonist; Animal Model; Aryl Hydrocarbon Receptor; Automobile Driving; Biological; Biology; Blood; Blood Cells; Blood Platelets; Bone Marrow; Cell Differentiation process; Cell Lineage; Cells; ChIP-seq; Chemically Induced Toxicity; Chemicals; Chronic; Clinical; Complement; Computational Biology; Computer Analysis; Data; Development; Developmental Biology; Endothelial Cells; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Etiology; Foundations; Genes; Genetic Engineering; Genetic Transcription; Goals; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hemoglobin; Human; Immunity; Immunocompromised Host; In Situ; Inflammation; Laboratories; Ligands; Luciferases; Maps; Mediating; Megakaryocytes; Methodology; Molecular; Mus; Physiological; Play; Pluripotent Stem Cells; Polyploidy; Process; Production; Receptor Signaling; Regulation; Reporter; Research Personnel; Resolution; Role; Serum; Signal Pathway; Signaling Protein; Spleen; Stem cells; System; Technology; Testing; Time; Toxicology; Transplantation; Up-Regulation; Western Blotting; Xenograft Model; aryl hydrocarbon receptor ligand; body system; clinically relevant; environmental chemical; erythroid differentiation; gene interaction; genomic signature; humanized mouse; in vivo; induced pluripotent stem cell; knock-down; mouse model; novel; programs; public health relevance; receptor expression; small hairpin RNA; stem cell biology; transcriptome; transcriptome sequencing; virtual

 DESCRIPTION (provided by applicant): The evolutionarily conserved AHR has been studied historically for its role in environmental chemical- induced toxicity. With the demonstration of th AHR's role in several physiological functions comes the realization that even more organ systems may be impacted by AHR ligands than previously appreciated. Our computational analyses of primary human cells indicate Ahr upregulation during hematopoiesis and Ahr co- regulation with several genes critical to stem cells, erythroid cells, and megakaryocyte development. To further study the role of the AHR in human hematopoiesis, we developed a unique platform for the directed differentiation of pluripotent stem cells in chemically-defined, serum- and feeder cell-free culture conditions. This platform relies on the ability of non-toxic AHR agonists to efficiently produce virtually unlimited numbers of hemogenic endothelial cells (HECs), bi-potential hematopoietic progenitor cells (HPCs), hemoglobin- producing erythroid (Ery) cells, and polyploid megakaryocytes. Using this system, we generated compelling data supporting the central hypothesis that the AHR plays a critical role at several key decision points throughout normal hematopoietic cell development. As a corollary, we propose that environmental AHR ligands have the potential to alter this tightly regulated process. We propose three specific aims to test these hypotheses: Specific Aim 1: Map AHR-regulated fate decision points in human hematopoietic cell development. Using human iPSCs genetically engineered to express an AHR-driven reporter or inducible AHR-specific shRNA, we will construct a temporal map of AHR expression, activation, and function during nominal human hematopoietic cell differentiation, establishing a foundation for global analysis of the AHR- regulated transcriptome (Aim 2) and for validating a humanized mouse model (Aim 3). Specific Aim 2: Read genomic signatures of AHR-dependent cell fate decisions. This aim complements the functional analyses of Aim 1 by providing an unbiased, high resolution map of AHR-dependent transcriptional programs in HECs, HPCs and erythroblasts. Specific Aim 3: Determine the role of the AHR in human blood cell expansion and differentiation in an adoptive transfer animal model. Large numbers of luciferase-tagged and shAHR-expressing human iPSC-derived HPCs will be transplanted into immunocompromised mice and their differentiation and function fully characterized. In all three aims, the effects of putative endogenous and environmental AHR ligands on human erythroid- and megakaryocyte-lineage development will be compared. These studies are highly significant in that they use unique strategies to: 1) compare the effects of disparate AHR ligands on hematopoiesis, 2) reveal the basic biology behind AHR control of blood cell development, and 3) advance the technology towards the goal of generating clinical grade, transferable RBCs and platelets. They also exploit the combined expertise of the co-PIs in AHR signaling (Dr. Sherr) and stem cell biology (Dr. Murphy) and of Dr. Monti, a co-investigator, in computational biology.

 描述（由申请人提供）：历史上已经研究了进化上保守的AHR在环境化学诱导毒性中的作用，随着AHR在多种生理功能中的作用的证明，人们认识到甚至更多的器官系统可能受到AHR配体的影响。我们对原代人类细胞的计算分析表明，Ahr 在造血过程中上调，并且与干细胞、红细胞和巨核细胞发育至关重要的几个基因共同调节。为了了解 AHR 在人类造血中的作用，我们开发了一个独特的平台，用于在化学成分确定的无血清和无饲养细胞的培养条件下定向分化多能干细胞，该平台依赖于无毒的 AHR 激动剂有效地定向分化。产生几乎无限数量的造血内皮细胞 (HEC)、双能造血祖细胞 (HPC)、产生血红蛋白的红细胞 (Ery) 细胞、使用该系统，我们生成了令人信服的数据，支持 AHR 在几个关键决策点发挥关键作用的中心假设。 作为推论，我们提出环境 AHR 配体有可能改变这一严格调控的过程。我们提出了三个具体目标来检验这些假设： 具体目标 1：绘制人类造血细胞中 AHR 调节的命运决策点。使用经过基因工程改造的人类 iPSC 来表达 AHR 驱动的报告基因或可诱导的 AHR 特异性 shRNA，我们将构建正常人类期间 AHR 表达、激活和功能的时间图。造血细胞分化，为 AHR 调节的转录组的全局分析（目标 2）和验证人源化小鼠模型（目标 3）奠定基础。 具体目标 2：读取 AHR 依赖性细胞命运决定的基因组特征。通过提供 HEC、HPC 和成红细胞中 AHR 依赖性转录程序的无偏、高分辨率图来对目标 1 进行功能分析。 具体目标 3：确定 AHR 在过继转移动物模型中人血细胞扩增和分化中的作用，将大量带有荧光素酶标记且表达 shAHR 的人 iPSC 衍生的 HPC 移植到免疫功能低下的小鼠中，并对其分化和功能进行充分表征。为了实现这三个目标，将比较假定的内源性和环境 AHR 配体对人类红细胞和巨核细胞谱系发育的影响。这些研究非常重要，因为它们使用独特的策略来： 1) 比较不同的 AHR 配体对造血的影响，2) 揭示 AHR 控制血细胞发育的基本生物学原理，3) 推进技术的发展，以实现产生临床级、可转移的红细胞和血小板的目标。联合首席研究员（Sherr 博士）和干细胞生物学（墨菲博士）以及联合研究员 Monti 博士在计算生物学方面的专业知识。