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， 国际吧COPD包括气道疾病以及肺泡上皮破坏，但是 肺泡上皮细胞对香烟烟雾的反应比气道的反应研究得少 上皮这是由于与气道细胞相比，肺泡上皮细胞的可及性差，后者是 其可以通过支气管镜检查和刷拭获得，并且缺乏可以支持其的体外培养系统。 研究香烟烟雾暴露的人肺泡上皮细胞。原代人肺泡上皮2型 细胞（AEC 2s）可以在三维类器官中培养，但难以在气-液界面上培养 (ALI)这是因为它们在培养中倾向于转分化和衰老。本项目建议设立 一个新的模型系统，以询问肺泡上皮细胞损伤响应香烟烟雾的基础上，人类 诱导多能干细胞（iPSC）分化为肺泡上皮2型细胞（iAEC 2），并在 阿里我们假设iAEC 2对香烟烟雾损伤表现出不同的分子反应， 同源气道上皮细胞和AATD肺泡上皮细胞本质上更易受 香烟烟雾伤害比野生型对应物。在目标1中，我们将定义iAEC 2转录组学 对香烟烟雾伤害的反应。我们将产生iAEC 2，并将其暴露于气相香烟烟雾中， 阿里将通过iAEC 2 ALI培养物的单细胞RNA测序评估缓解， 香烟烟雾暴露，与来自同一患者的气道上皮细胞ALI培养物的反应进行比较。 这将导致阐明肺泡上皮细胞特异性烟雾反应转录签名， 与来自相同遗传背景的气道上皮细胞相比。在aim 2中，我们将把我们的模型扩展到 仅单基因型COPD，α-1抗胰蛋白酶缺乏症（AATD）。我们将利用AATD特异性iPSC， 对于AAT的聚合形式是纯合的，这是一种被称为"PiZZ"的基因型， 野生型同基因对应物（"PiMM"）。我们假设α-1抗胰蛋白酶（AAT）在胰腺癌中上调， 肺泡上皮细胞对香烟烟雾的敏感性增加，而PiZZ iAEC 2表现出对 香烟烟雾与同基因PiMM iAEC 2相比，由于AAT的Z形式的功能获得性毒性， 表现为未折叠蛋白反应和ER应激途径的激活。在目标3中，我们将建立一个 使用患者远端肺组织的PiZZ特异性转录组疾病特征。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，所有患者 研究有吸烟史。我们假设疾病之间的转录组差异 状态将包括PiZZ中未折叠蛋白反应和ER应激途径的更大途径富集 远端肺这些目标的完成将导致对肺上皮细胞类型特异性 这将增加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.