Hedgehog Pathway regulates Alveolar Type II Cells in Human Lung Development

Rationale: The Hedgehog (HH) pathway plays an important role in human lung branching morphogenesis, yet its role in human alveologenesis remains understudied. Bronchopulmonary Dysplasia (BPD) patients exhibit simplified alveolar structures, accompanied with increased expression of HH components. Therefore, we hypothesize that HH pathway regulation is crucial for alveologenesis, specifically for proximo-distal patterning and AT2 cell differentiation. Methods: Human fetal lung explants aged 18-22 weeks gestation (WG) were cultured in CKDCI media to induce alveolar marker expression, in the presence or absence of HH activator (SAG). AT2 progenitor cells (HTII280+/CD31-/CD45-) were isolated from fresh human lung tissues (18-22 WG) and plated in Matrigel for 14 days to form organoids. A subset of organoids was treated with SAG for 7 days while in matrigel, while others were lifted to air-liquid interface (ALI) culture and treated for 14 days. RT-qPCR, fluorescent in situ hybridization (FISH), and immunofluorescence staining (IF) were carried out at various steps to assess cell fate. Results: SAG treated human fetal lung explants displayed gross morphological differences compared to the control, e.g. enlarged distal cysting. HH pathway upregulation resulted in a significant decrease of AT2 markers expression ( ABCA3, LAMP3; p<0.05) and increased expression of proximal markers ( TP63, SCGB3A2; p<0.01) as compared to control. FISH for SFTPC (AT2 marker) showed a 3-fold decrease ( p<0.05) following HH pathway activation accompanied by a loss of SOX9+ cells (2.5-fold vs control, p<0.05). The loss of SFTPC+ cells was confirmed by IF ( p<0.05). A decrease in KRT8+, a marker of intermediate AT2 cells, was also observed (1.5-fold vs control, p<0.05). Similarly, in organoid cultures, we found decreased SFTPC+, SOX9+ and KRT8+ cells (1.5-; 2- and 1.5-fold vs control, p<0.05). Furthermore, SAG treatment resulted in the formation of fewer and smaller-sized organoids (1-fold and 1.5-fold vs control, p<0.05). Staining on ALI cultures revealed fewer SFTPC+ cells and more TP63+ cells (1.5-fold vs control, p<0.05). Tight junction proteins (ZO1 and Claudin18) were compromised on ALI membranes demonstrating that HH pathway activation results in larger and fewer tight junctions, as indicated by the TIJOR score (2-fold vs control, p<0.01). Conclusion: In our alveologenesis-like model, HH pathway activation reduces AT2 markers, favoring a proximal fate, ultimately suggesting that the HH pathway regulates late human lung development. Understanding the role of the HH pathway in regulating alveologenesis is essential in pediatric conditions like BPD and could facilitate translational discoveries. CIRM EDUC4-12837 (BR) NIH/NHLBI R01HL141856 (DA). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.