Fig. 3: HTII-280⁺-derived organoids maintain a stable AT2 phenotype in CHIR medium.

a HE staining and phase-contrast images showing the morphology of HTII-280⁺-derived organoids in CHIR medium. Scale bar: 100 µm. b Representative confocal images showing expression of the pneumocyte marker SFTPC (I, red) and maintained apical polarity (II, HTII-280, green,) in the long-term organoid culture (P3, ~3 months). Scale bar: 50 µm. c Representative transmission electron microscopy (TEM) images revealing fully formed junctions (arrow) and prominent microvilli (asterisk) of AT2 cells (yellow square). Abundantly secreted vesicles with lamellar membrane structures are present in the organoid lumen (violet square). Scale bars: 2.5 µm, 1 µm, 100 nm. d Protein levels of AT2 markers (SFTPC, NAPSIN A, HTII-280, and HOPX) in HTII-280⁺-derived alveolar organoids (AvO) compared to airway organoids (AO) of the same donor reveal sustained levels in all four lines, while no signal was detected in controls. e scRNA-seq analysis of alveolar organoids identifies clusters of AT2 cells, but also intermediate and airway cell types (secretory and basal cells) (n = 3 donors, 7784 cells). f Expression of HOPX, SFTPC, KRT5, and SCGB1A1 in the clusters derived from scRNA-seq of alveolar organoids. g Fractions of all identified cell types within the HTII-280⁺-derived organoids. h Relative expression level of SFTPC in HTII-280⁺-derived alveolar organoids treated with CHIR or CHIR+EGF relative to pool organoids grown in AOM for two different donors.