Pluripotent stem cell-based modeling of cigarette smoke injury

摘要 慢性阻塞性肺疾病(COPD)是美国发病率和死亡率的重要原因 全世界。COPD包括呼吸道疾病和肺泡上皮破坏，但 肺泡上皮细胞对香烟烟雾的反应比对呼吸道的反应要少得多。 上皮组织。这是由于与呼吸道细胞相比，肺泡上皮细胞的可及性较差，后者 可通过支气管镜检查和刷检获得，以及缺乏能够支持 香烟烟雾暴露人肺泡上皮细胞的研究。人肺泡上皮2型原代培养 细胞(AEC2)可以在三维有机物中培养，但很难在气液界面培养。 (ALI)由于他们在文化中有跨分化和衰老的趋势。本项目拟设立 基于人体的香烟烟雾致肺泡上皮损伤模型系统的建立 诱导多能干细胞(IPSCs)分化为2型肺泡上皮细胞(IAEC2s) 阿里。我们假设iAEC2对香烟烟雾损伤表现出明显的分子反应。 同基因的呼吸道上皮细胞和AATD肺泡上皮细胞在本质上更易患上 吸烟对人体的伤害要比野生型同龄人大。在目标1中，我们将定义iAEC2转录 对香烟烟雾伤害的反应。我们将产生iAEC2，并将它们暴露在气相香烟烟雾中 阿里。反应将通过有和没有iAEC2 ALI培养的单细胞RNA测序来评估 香烟烟雾暴露，与同一患者的呼吸道上皮细胞ALI培养的反应进行比较。 这将导致阐明肺泡上皮细胞特异的烟雾反应转录特征 与来自相同遗传背景的呼吸道上皮细胞进行比较。在目标2中，我们将把我们的模型扩展到 只有单基因形式的COPD，α-1抗胰蛋白酶缺乏症(AATD)。我们将利用特定于AATD的IPSC 是AAT聚合型的纯合子，一种被称为“PIZZ”的基因型，以及他们的CRISPR编辑的 野生型等基因等位基因(“PIMM”)。我们假设α-1抗胰蛋白酶(AAT)在 肺泡上皮细胞对香烟烟雾的反应，以及Pizz iAEC2表现出对 香烟烟雾与同基因的PIMM iAEC2相比，是由于AAT的Z型功能获得毒性所致 表现为激活未折叠的蛋白反应和内质网应激途径。在目标3中，我们将建立一个 使用患者远端肺组织的PIZZ特异性转录型疾病特征。我们将利用现有的 来自PIZZ和PIMM COPD患者肺实质组织的微阵列数据集。重要的是，这里所有的病人 研究有吸烟暴露的历史。我们假设这种疾病在转录水平上的差异 各州将包括PIZZ中未折叠蛋白反应和内质网应激途径的更大途径丰富 远端肺。完成这些目标将增加对肺上皮细胞类型特异性的理解 对香烟烟雾的反应，并将增加AATD疾病发病机制的范例。

Abstract Chronic obstructive pulmonary disease (COPD) is an important cause of morbidity and mortality in the US and worldwide. COPD encompasses disease of the airways as well as alveolar epithelial destruction, but the response of the alveolar epithelium to cigarette smoke has been much less well-studied than that of the airway epithelium. This is a result of poor accessibility of alveolar epithelial cells compared to airway cells, the latter of which can be obtained by bronchoscopy and brushing, and a lack of in vitro culture systems that can support the study of cigarette smoke exposure of the human alveolar epithelium. Primary human alveolar epithelial type 2 cells (AEC2s) can be cultured in three-dimensional organoids, but are difficult to culture at air-liquid interface (ALI) due to their tendency to transdifferentiate and senesce in culture. This project proposes the establishment of a novel model system to interrogate alveolar epithelial injury in response to cigarette smoke based on human induced pluripotent stem cells (iPSCs) differentiated to alveolar epithelial type 2 cells (iAEC2s) and cultured at ALI. We hypothesize that iAEC2s exhibit a distinct molecular response to cigarette smoke injury relative to syngeneic airway epithelial cells and that AATD alveolar epithelial cells are intrinsically more susceptible to cigarette smoke injury than their wild type counterparts. In aim 1, we will define the iAEC2 transcriptomic response to cigarette smoke injury. We will generate iAEC2s and expose them to gas-phase cigarette smoke at ALI. The response will be assessed by single-cell RNA sequencing of iAEC2 ALI cultures with and without cigarette smoke exposure, in comparison to the response of airway epithelial ALI cultures from the same patient. This will result in the elucidation of an alveolar epithelial-specific smoke response transcriptional signature when compared to airway epithelial cells from the same genetic background. In aim 2, we will extend our model to the only monogenic form of COPD, alpha-1 antitrypsin deficiency (AATD). We will utilize AATD-specific iPSCs that are homozygous for the polymerizing form of AAT, a genotype known as “PiZZ”, alongside their CRISPR-edited wild type isogenic counterparts (“PiMM”). We hypothesize that alpha-1 antitrypsin (AAT) is upregulated in the alveolar epithelium in response to cigarette smoke, and that PiZZ iAEC2s exhibit increased susceptibility to cigarette smoke compared to isogenic PiMM iAEC2s due to gain-of-function toxicity of the Z form of AAT that manifests as activation of the unfolded protein response and ER stress pathways. In aim 3, we will establish a PiZZ-specific transcriptomic disease signature using patient distal lung tissue. We will leverage an existing microarray dataset from PiZZ and PiMM COPD patient lung parenchymal tissue. Importantly, all patients in this study have a history of smoke exposure. We hypothesize that transcriptomic differences between the disease states will include greater pathway enrichment of the unfolded protein response and ER stress pathways in PiZZ distal lung. Completion of these aims will result in an increased understanding of lung epithelial cell type-specific responses to cigarette smoke and will add to the paradigm of disease pathogenesis in AATD.