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）：在历史上，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在几种生理功能中的作用的证明，认识到甚至比以前认识到的更多的器官系统可能受到AHR配体的影响。我们对原代人细胞的计算分析表明，在造血过程中Ahr上调，并且Ahr与对干细胞、红系细胞和巨核细胞发育至关重要的几个基因共调节。为了进一步研究AHR在人类造血中的作用，我们开发了一种独特的平台，用于在化学定义的无血清和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AHR激动剂有效产生几乎无限数量的生血内皮细胞（HEC）、双能造血祖细胞（HPC）、产生血红蛋白的红系（Ery）细胞和多倍体巨核细胞的能力。使用这个系统，我们产生了令人信服的数据，支持中心假设，即AHR在几个关键决策点起着关键作用 在正常的造血细胞发育过程中。作为一个推论，我们提出，环境AHR配体有可能改变这一严格监管的过程。我们提出了三个具体的目标来测试这些假设：具体目标1：地图AHR调节的命运决定点在人类造血细胞的发展。使用经基因工程改造以表达AHR驱动的报告基因或诱导型AHR特异性shRNA的人iPSC，我们将构建标称人造血细胞分化期间AHR表达、活化和功能的时间图，为AHR调节的转录组的全局分析（Aim 2）和验证人源化小鼠模型（Aim 3）建立基础。具体目标2：读取AHR依赖性细胞命运决定的基因组特征。这一目标补充了目标1的功能分析，提供了一个公正的，高分辨率的地图AHR依赖的转录程序在HEC，HPC和成红细胞。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。将大量的标记有β-淀粉酶和表达shAHR的人iPSC衍生的HPC移植到免疫功能低下的小鼠中，并充分表征它们的分化和功能。在这三个目标中，将比较假定的内源性和环境AHR配体对人红系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：1）比较不同AHR配体对造血的影响，2）揭示AHR控制血细胞发育背后的基本生物学，3）将技术推向产生临床级，可转移的RBC和血小板的目标。他们还利用了AHR信号传导（Sherr博士）和干细胞生物学（Murphy博士）的共同PI以及计算生物学的共同研究者Monti博士的综合专业知识。