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 wi...