An in vitro model of bleomycin-induced epithelial injury using stem cell-derived alveolar type 2 epithelial cells

Human alveoli constitute most of the lung parenchyma and play a pivotal role in respiratory diseases. Traditional alveolar type 2 epithelial cells, such as A549 and H441, have been widely used to study pulmonary toxicity mechanisms. However, these cells have inherent limitations, including insufficient barrier function, lack of functional surfactant protein expression, and failure to recapitulate the physiological features of human alveolar epithelial cells. Here, we established differentiated AEC2s (diff-AEC2s) from human embryonic stem cells and validated their stage-specific differentiation. We confirmed key AEC2-like features in vitro, including lamellar body-like organelles, higher TEER values, and increased AEC2 marker expression relative to A549 and H-6053 cells under ALI culture. The diff-AEC2s were treated with bleomycin to evaluate their potential for modeling disease-related epithelial injury. Transcriptomic analysis with benchmark dose showed epithelial stress related to DNA damage and cell cycle arrest quantitatively. Bleomycin also induced the dose-dependent alterations of alveolar epithelial and damage-related markers, including LGALS3, KRT8, CTGF, and senescent phenotypes. This study presents a human stem cell-derived alveolar epithelial platform to quantify early epithelial stress and injury-associated responses in vitro.