Maturation of pluripotent stem cell-derived alveolar type II cells at air-liquid interface and response to environmental stimuli

Human pulmonary alveolar type II cells (AT2s) are facultative progenitors of the distal lung epithelium and secrete surfactant, a protein-lipid mixture that lowers alveolar surface tension and prevents alveolar collapse during expiration. AT2s exist at a physiological interface between inhaled air and pulmonary vasculature, and are among the pulmonary cell types that are directly exposed to inhaled environmental stimuli such as respiratory viruses and cigarette smoke. Primary human AT2s can be cultured in vitro in three-dimensional organoids known as alveolospheres, but are difficult to culture in the physiologically relevant air-liquid interface (ALI) format due to their tendency to lose their AT2 phenotype and senesce in culture. Human induced pluripotent stem cells (iPSCs) can be directed to differentiate to iPSC-derived AT2s (iAT2s) in three-dimensional spheres, where they transcriptomically resemble primary human fetal lung. Here we report the successful adaptation of iAT2s to ALI culture, which promotes their maturation and permits exposure to inhaled environmental stimuli. We transcriptomically profile iAT2s cultured at ALI and find that they upregulate key markers of AT2 maturation as they downregulate cell cycle-associated transcripts. We then evaluate the extent of iAT2 maturation at ALI within the developmental context by transcriptomic comparison to cultured and freshly isolated primary AT2s. We find that iAT2s cultured at ALI are more similar to primary AT2s than iAT2s cultured as spheres, and that the main differences between iAT2s at ALI and primary AT2s are due to primary AT2s’ response to immune stimuli. We then test the capacity of iAT2s to respond to immune stimuli and serve as useful in vitro model system for human respiratory viral infections by infecting iAT2s at ALI with SARS-CoV-2, the virus that causes Coronavirus Disease 2019 (COVID-19). We find that iAT2s are permissive to SARS-CoV-2 infection, mount an epithelial-intrinsic interferon and inflammatory response to infection, and can serve as an in vitro platform for testing antiviral therapeutics. Finally, we demonstrate that iAT2s at ALI also have utility as a system for modeling the response to cigarette smoke and electronic cigarette vapor, enabling the direct comparison of these two common inhaled noxious stimuli. Overall, we describe a novel disease modeling platform that enables future exploration of gene-environment interactions unique to inhaled exposures of the alveolar epithelium

CRISPR interference interrogation of COPD GWAS genes reveals the functional significance of desmoplakin in iPSC-derived alveolar epithelial cells

Genome-wide association studies (GWAS) have identified dozens of loci associated with chronic obstructive pulmonary disease (COPD) susceptibility; however, the function of associated genes in the cell type(s) affected in disease remains poorly understood, partly due to a lack of cell models that recapitulate human alveolar biology. Here, we apply CRISPR interference to interrogate the function of nine genes implicated in COPD by GWAS in induced pluripotent stem cell–derived type 2 alveolar epithelial cells (iAT2s). We find that multiple genes implicated by GWAS affect iAT2 function, including differentiation potential, maturation, and/or proliferation. Detailed characterization of the GWAS gene DSP demonstrates that it regulates iAT2 cell-cell junctions, proliferation, mitochondrial function, and response to cigarette smoke–induced injury. Our approach thus elucidates the biological function, as well as disease-relevant consequences of dysfunction, of genes implicated in COPD by GWAS in type 2 alveolar epithelial cells.This work was supported by a CJ Martin Early Career Fellowship from the Australian National Health and Medical Research Council awarded to R.B.W.; NIH grant F30HL147426 awarded to K.M.A.; NIH grants U01TR001810, R01DK101501, and R01DK117940 awarded to A.A.W.; NIH grants R01HL135142, R01HL137927, and R01HL147148 awarded to M.H.C.; and NIH grants R01HL127200 and R01HL148667 awarded to X.Z

Air-liquid interface culture promotes maturation and allows environmental exposure of pluripotent stem cell–derived alveolar epithelium

Type 2 alveolar epithelial cells (AT2s), facultative progenitor cells of the lung alveolus, play a vital role in the biology of the distal lung. In vitro model systems that incorporate human cells, recapitulate the biology of primary AT2s, and interface with the outside environment could serve as useful tools to elucidate functional characteristics of AT2s in homeostasis and disease. We and others recently adapted human induced pluripotent stem cell-derived AT2s (iAT2s) for air-liquid interface (ALI) culture. Here, we comprehensively characterize the effects of ALI culture on iAT2s and benchmark their transcriptional profile relative to both freshly sorted and cultured primary human fetal and adult AT2s. We find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. We then leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor

β2-Adrenoreceptor is a regulator of the α-synuclein gene driving risk of Parkinson’s disease

Copy number mutations implicate excess production of α-synuclein as a possibly causative factor in Parkinson's disease (PD). Using an unbiased screen targeting endogenous gene expression, we discovered that the β2-adrenoreceptor (β2AR) is a regulator of the α-synuclein gene (SNCA). β2AR ligands modulate SNCA transcription through histone 3 lysine 27 acetylation of its promoter and enhancers. Over 11 years of follow-up in 4 million Norwegians, the β2AR agonist salbutamol, a brain-penetrant asthma medication, was associated with reduced risk of developing PD (rate ratio, 0.66; 95% confidence interval, 0.58 to 0.76). Conversely, a β2AR antagonist correlated with increased risk. β2AR activation protected model mice and patient-derived cells. Thus, β2AR is linked to transcription of α-synuclein and risk of PD in a ligand-specific fashion and constitutes a potential target for therapies

Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system. [Display omitted] •SARS-CoV-2 infection in induced lung cells is characterized by phosphoproteomics•Analysis of response reveals host cell signaling and protein expression profile•Comparison to studies in undifferentiated cell lines shows unique pathology in iAT2s•Systems-level predictions find druggable pathways that can impede viral life cycle Hekman et al. describe how a layer of primary stem cells (iAT2s) recapitulating lung biology responds to infection with SARS-CoV-2. They compare their work to previous studies with immortalized cell lines. Their data predict what effect the virus has on a lung cell and which drugs may slow infection