Background: The Hedgehog (HH) signaling pathway is essential for lung branching morphogenesis, yet its role in human alveologenesis remains unclear. Bronchopulmonary dysplasia (BPD), a chronic lung disease of preterm infants, is characterized by arrested alveolarization and elevated expression of HH pathway components. We hypothesized that HH pathway regulation is crucial for alveolar type II (AT2) cell differentiation during late-stage lung development. To further investigate mechanisms contributing to BPD we modeled hyperoxic injury in prenatal human lung explants. Methods Fresh human prenatal lung explants (18-22 weeks gestation) were cultured at air-liquid interface in CK+DCI medium to promote alveolar-like differentiation. Explants were treated with either a HH pathway activator (SAG) or inhibitor (5E1) and/or exposed to hyperoxic (40% O 2 ) or normoxic conditions for 7 days. RT-qPCR, immunofluorescence (IF), and fluorescent in situ hybridization (FISH) were used to assess target gene expression, cell fate, primary cilia (PC) dynamics, and tissue morphology. Bronchopulmonary dysplasia (BPD) lung tissues were collected, and the HH pathway, proliferation, PC, and progenitor markers were further evaluated by RT-qPCR and combined FISH-IF. Publicly available BPD RNA sequencing datasets were used to compare and correlate with in vitro results. Results: Under normoxia, SAG treated explants exhibited large distal cysts, increased proliferation, and higher PC frequency in mesenchymal and epithelial compartments (p< 0.05). We also observed decreased expression of AT2/AT1 markers as compared to non-treated explants (ABCA3, LAMP3, AGER and HOPX) (p< 0.05). Results were confirmed by FISH and IF analyses with a reduction in SFTPC alongside SOX9 (p< 0.05). Although SAG-treated explants contained fewer intermediate cells (KRT8 + /SFTPC + /CLDN4 + ), this population exhibited a 1.5-fold increase in PC compared with untreated controls. HH activation induced senescence signatures (CDKN1A, CDKN2A, TP53, P21, γH2AX; p< 0.01). Similarly, hyperoxia lead to HH activation, cystic morphology, enhanced proliferation, expansion of KRT8 + /SFTPC + /CLDN4 + intermediate cells with higher PC, and reduced distal/AT2/AT1 programs, including SOX9 (p< 0.05). Under hyperoxia, 5E1 partially rescued hyperoxia-induced defects such as reduced cysts and senescence, restored AT2/AT1 markers and intermediate-state balance, decreased proliferation (p< 0.05), and significantly lowered PC prevalence (p< 0.01). Public BPD datasets confirm HH, senescence, and PC-related gene upregulation with reduced alveolar markers. Conclusions: In our CK+DCI hyperoxia model of late human alveologenesis, we demonstrated that pharmacologic HH activation recapitulates hyperoxia by blocking AT2 maturation and differentiation, and inducing cystic remodeling, PC accumulation, proliferation, and senescence. 5E1 partially rescues these defects, and BPD tissues/public datasets corroborate the signature, nominating the HH-PC axis as a key focus to deepen mechanistic understanding of BPD. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Belgacemi et al. (Fri,) studied this question.