Chimeric iPSC-Derived Lung Organoids Reveal the Minimum Wild-Type Cell Fraction Detectable by the Forskolin-Induced Swelling Assay in Cystic Fibrosis

Cystic fibrosis is a life-threatening inherited disease caused by pathogenic variants in the CFTR gene that impair chloride and bicarbonate transport across airway epithelia. Gene editing and cell replacement strategies hold promise for restoring CFTR function, yet they inevitably generate heterogeneous cell populations in which only a fraction of cells carry a corrected allele, raising the question of how large this fraction must be for functional rescue to be detectable and whether the forskolin-induced swelling (FIS) assay is sensitive enough to capture partial correction. To address this, we generated chimeric iPSC-derived lung organoids by co-culturing GFP-labeled wild-type airway basal cells with unlabeled F508del-homozygous cystic fibrosis basal cells at seven predefined ratios (0–100% wild-type), verifying the actual wild-type fraction by flow cytometry and quantifying 24-h forskolin-induced swelling as the area ratio. Chimeric organoids showed a dose-dependent increase in swelling with rising wild-type fractions, with a statistically significant FIS response first resolved at approximately 6% wild-type cells in the pooled analysis. Separately, ROC analysis of well-level data identified two area-ratio cutoffs for classifying organoid populations—1.16 (80% sensitivity, 80% specificity) and 1.21 (98% specificity, 70% sensitivity)—which the fitted mean response reaches at approximately 14% and 34% wild-type cells, respectively. The minimum statistically detectable fraction and the classification cutoffs are distinct quantities derived from different analyses and are not interchangeable; both describe the performance of the FIS assay in heterogeneous organoids rather than thresholds for therapeutic benefit and provide a practical reference framework for designing and interpreting gene editing and cell therapy studies in cystic fibrosis.