An hiPSC-derived multi-lineage lung model exhibiting proximal-distal epithelial features for modeling pulmonary fibrosis

Background Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible interstitial lung disease with limited therapeutic options. Existing hiPSC-derived lung organoid models are largely restricted to single epithelial lineages and cannot endogenously integrate multiple pulmonary cell types, limiting mechanistic studies and drug screening. Methods We established a staged directed differentiation protocol to sequentially differentiate hiPSCs into definitive endoderm, anterior foregut endoderm, and lung progenitor cells, ultimately generating a multi-lineage lung model on Transwell inserts. The model was characterized by bright-field microscopy, hematoxylin-eosin (H&E) staining, scanning and transmission electron microscopy, immunofluorescence, quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and single-cell RNA sequencing. Fibrosis-like phenotypes were induced by transforming growth factor-beta 1 (TGF-β1) stimulation. Transcriptomic similarity to human IPF was assessed by RNA sequencing (RNA-seq) combined with bidirectional gene set enrichment analysis (GSEA), and drug responsiveness was validated with pirfenidone. Results Without genetic editing or exogenous cell supplementation, the model endogenously generated proximal duct-like epithelium, distal alveolar epithelium, fibroblasts, endothelial cells, and CD68 + macrophage-like cells confirmed by immunofluorescence. Single-cell sequencing identified 13 transcriptional subclusters and revealed epithelial-mesenchymal co-development. TGF-β1 stimulation induced epithelial barrier disruption, ferroptosis-like mitochondrial ultrastructural alterations, extracellular matrix (ECM) remodeling, and aberrant secretion of multiple IPF-associated biomarkers. Transcriptomic analysis showed that upregulated genes in the fibrosis model group were significantly enriched in ECM organization, cell adhesion, and the PI3K-Akt pathway. Protein-protein interaction (PPI) network analysis identified COL1A1 , FN1 , and integrin family members as central hubs. Bidirectional GSEA validation confirmed that the transcriptomic signature of the model was highly similar to human IPF, with differentially expressed genes significantly enriched in three independent IPF cohorts. Pirfenidone reversed TGF-β1-induced ECM deposition and myofibroblast activation, and partially restored type II alveolar epithelial (AT2) cell marker expression. Conclusion We successfully established a humanized multi-lineage lung model that captures epithelial-mesenchymal co-development in vitro . Its responsiveness to TGF-β1 stimulation and sensitivity to pirfenidone highlight its potential for antifibrotic drug screening. This platform offers a humanized tool with the potential to be standardized for investigating cell fate determination during early lung development and the pathogenesis of IPF.