Figure 6.

Combination of Fibroblast Ligands and Signaling Pathway Inhibitors Induce the Formation of Fibroblast-Free Alveolospheres

(A) Experimental scheme of the fibroblast-free alveolosphere culture.

(B–D) Representative wells (entire Transwell stored in 24-well plates is shown) from fibroblast-free alveolospheres formation assays on day 8 (B) or 14 (D) and representative fibroblast-free alveolosphere (epithelial cells; EGFP) on day 12 (C). Confocal micrographs (B, left and D, left) and bright-field images (B, right and D, right) are shown.

(E–G) Representative sections of fibroblast-free alveolospheres on day 8 (E and F) and day 14 (G) labeled with antibodies against Aqp5/Sftpc (E and G), Scgb1a1/acetylated tubulin (F left), or Krt5 (F right), showing Sftpc⁺ Aqp5⁺ cells on the outside and no cells inside (E) and Sftpc⁺ AEC2 on the outside and Aqp5⁺ AEC1 on the inside (G).

(H–J) qPCR analysis of AEC1 (H), AEC2 (I), and non-alveolar epithelial (J) markers using cells obtained from epithelial-fibroblast co-culture (day 14) or fibroblast-free alveolosphere formation assays (day 14). “Fibroblasts” refer to sorted fibroblasts before co-culture and “Epi; before culture” refers to sorted epithelial cells before co-culture. mRNA levels were normalized to that of Actb. Data represent mean ± SEM (n = 3 wells for cultured cells and n = 3 animals for sorted cells) and are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Tukey's post hoc test).

Scale bars: 1 mm in B and D; 100 μm in C and E–G. See also Figures S5 and S6.