FIGURE 2.

FGFs promote formation, proliferation, and branching of lung organoids. (A–C) In 3D Matrigel, lung organoids are efficiently formed in response to different FGFs. (A) Primary lung organoid forming efficiency (OFE). The plot shows mean + SD; n = 3–5. ****P < 0.0001 (one-way ANOVA). (B) Branching efficiency (%) of lung organoids on day 21 of culture, shown as mean + SD; n = 3–4. *P < 0.05 (one-way ANOVA). (C) Representative photographs of primary lung organoids formed in 3D Matrigel in response to different FGFs (1 nM). Scale bar, 100 μm. (D) Increased concentration of FGFs increases organoid forming efficiency. The plot shows primary lung OFE as mean + SD; n = 3–5. ****P < 0.0001 (two-way ANOVA). (E–H) EGF is not essential for lung organoid formation in the presence of FGFs. (E) Representative photographs of primary lung organoids formed in 3D Matrigel in the presence of different FGFs (2 nM) and in the absence of EGF. Scale bar, 100 μm. (F,G) The plots show primary (F) and secondary (G) lung organoid forming efficiency as mean + SD; n = 3–4. **P < 0.01; ****P < 0.0001 (two-way ANOVA). (H) Analysis of organoid size. The sizes are relative to organoids formed in a medium with EGF and no FGF. The plots show mean + SD; n = 3, N = 15–20 organoids/treatment. The gray symbols indicate significance between the culture with and without EGF for the respective FGF. ****P < 0.0001 (two-way ANOVA). (I–K) Initial treatment with FGF2 for 7 days increases lung organoid formation and branching efficiency in response to FGF7, FGF9, and FGF10. (I) Representative photographs of primary lung organoids formed in 3D Matrigel after 7 days of culture with 1 nM FGF2, followed by culture with different FGFs (1 nM). The pictures are from day 20 of culture. Scale bar, 100 μm. (J,K) The plots show primary lung OFE (J) and branching efficiency of lung organoids (K) as mean + SD; n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-way ANOVA).