CRISPR-engineered human lung organoids with a biomolecular condensate reporter enable mechanistic toxicity monitoring

Understanding how chemical stress perturbs human lung physiology requires models that capture dynamic molecular responses in real time. Here, we established a CRISPR/Cas9-engineered human induced pluripotent stem cell (hiPSC)-derived lung organoid expressing endogenous G3BP1–mCherry, enabling live, non-destructive visualization of stress granule (SG) formation under toxicant exposure. The organoids recapitulated airway and alveolar epithelial diversity and displayed lamellar body-like ultrastructures, indicating advanced maturation. Time-lapse imaging revealed rapid and reversible SG dynamics across chemically distinct stressors, while cytotoxicity assays showed that these organoids are significantly more sensitive than conventional 2D or cancer-derived lung models. Importantly, SG dynamics were linked to exposure duration–dependent changes in epithelial barrier integrity, indicating that SG formation precedes overt epithelial injury and serves as an early indicator of toxicant-induced cellular stress. Integration with high-content screening enabled quantitative, image-based analysis of cellular stress phenotypes, greatly enhancing throughput and mechanistic insight, thereby provided next-generation New Approach Methodologies for lung toxicity assessment. Together, this hiPSC-derived lung organoid SG reporter platform links early molecular stress adaptation to tissue-level responses, offering a predictive and mechanistically informative framework for human-relevant lung toxicity evaluation. ● CRISPR-engineered human lung organoids express endogenous G3BP1–mCherry reporter ● Live imaging enables rapid and reversible visualization of stress granules ● Stress granule dynamics act as an early and sensitive biomarkers of chemical stress ● Lung organoids show higher chemical sensitivity than immortalized and cancer cell models ● Reporter enables non-destructive, real-time monitoring of lung toxicant responses