Defining the Role of the AHR in Blood Cell Specifications

 描述(由申请人提供)：进化上保守的AHR因其在环境化学诱导的毒性中的作用而被历史地研究。随着AHR在几个生理功能中的作用的展示，人们意识到AHR配体可能比以前认识到的更多的器官系统受到影响。我们对原代人类细胞的计算分析表明，在造血过程中，Ahr上调，并与几个对干细胞、红系细胞和巨核细胞发育至关重要的基因共同调节。为了进一步研究AHR在人类造血中的作用，我们开发了一个独特的平台，用于在化学定义的、无血清和无饲养层细胞的培养条件下定向分化多能干细胞。这一平台依赖于无毒的AHR激动剂有效地产生几乎无限数量的造血内皮细胞(HECs)、双潜能造血祖细胞(HPC)、产生血红蛋白的红系(Ery)细胞和多倍体巨核细胞。使用这个系统，我们产生了令人信服的数据，支持AHR在几个关键决策点发挥关键作用的中心假设 在正常的造血细胞发育过程中。因此，我们认为环境中的AHR配体有可能改变这一严格调控的过程。我们提出了三个特定的目标来验证这些假说：特定的目标1：在人类造血细胞发育中映射AHR调节的命运决定点。使用基因工程的人IPSCs表达AHR驱动的报告或可诱导的AHR特异性shRNA，我们将构建AHR在名义上的人类造血细胞分化过程中表达、激活和功能的时间图，为AHR调节的转录组的全球分析(AIM 2)和验证人源化的小鼠模型(AIM 3)奠定基础。具体目标2：阅读AHR依赖的细胞命运决定的基因组签名。这一目的是对AIM 1功能分析的补充，它提供了HECS、HPC和红细胞中AHR依赖的转录程序的无偏见、高分辨率图谱。具体目标3：在过继移植动物模型中确定AHR在人血细胞扩增和分化中的作用。大量荧光素酶标记和Shahr表达的人iPSC来源的HPC将被移植到免疫低下的小鼠体内，并对其分化和功能进行充分表征。在所有这三个目标中，假定的内源性和环境AHR配体对人类红系和巨核细胞谱系发育的影响将被比较。这些研究具有非常重要的意义，因为它们使用了独特的策略：1)比较不同的AHR配体对造血的影响；2)揭示AHR控制血细胞发育背后的基本生物学；3)将这项技术推向产生临床分级、可转移的红细胞和血小板的目标。他们还利用了共同PI在AHR信号(Sherr博士)和干细胞生物学(Murphy博士)以及蒙蒂博士(共同研究员)在计算生物学方面的综合专业知识。

 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.