A-83-01

Alveolar regeneration requires coordinated alveolar type 1 (AT1)/alveolar type 2 (AT2) responses and an intact epithelial barrier. In vitro systems, however, rarely couple lineage control with high resistance. Here we establish a concise workflow in which human AT2-like progenitors are expanded in a medium containing a small-molecule cocktail (Y-27632/A-83-01/CHIR99021; YAC), followed by air-liquid interface (ALI) culture in medium without the YAC cocktail (hereafter referred to as the YAC(-) condition). Compared with submerged culture, ALI increased AT1(AQP5, AGER, and PDPN) and AT2 (SFTPB, SFTPC, and SFTPD) markers. Within ALI, β-catenin inhibition by XAV939 elevated AT1 features while retaining AT2 protein expression, but was accompanied by reduced AT2 marker expression at the transcriptional level compared with the YAC(-) condition. Transepithelial electrical resistance entered the kΩ range by ALI day 10 and remained high across ALI conditions, with hematoxylin and eosin staining confirming a continuous single-layer epithelium. Passage reduced absolute marker levels and resistance but preserved the rank order across conditions. In scratch assays, ALI monolayers closed wounds over 24-48 h, and spatial immunofluorescence staining showed surfactant protein-C enrichment at the leading edge and podoplanin predominantly behind the front. Collectively, these findings demonstrate that β-catenin modulation within ALI biases the alveolar epithelial profile toward AT1-like features, whereas the YAC(-) condition sustains a balanced AT1/AT2 state with superior epithelial barrier performance and repair capacity. This platform provides a single-monolayer system suitable for dissecting alveolar epithelial differentiation and functional maintenance.