Fig. 2. IPF-ABCs generate more bronchospheres and increase fibroblast proliferation and collagen production compared to non-IPF ABCs in a 3D organoid model.

a Airway basal cells w/wo human lung fibroblasts were cultured in matrigel applying a transwell system. b IPF-ABCs form large spheres which become hollow tube-like structures after 21 days of 3D culture. In the co-culture system of ABCs with lung fibroblasts, fibroblasts surround bronchospheres and form a mesh-like structure. Upper panel: Masson trichrome staining, scale bars 100 µm or 200 µm as indicated. Lower panel: Confocal immunohistochemistry demonstrates tube formation by IPF-ABCs and close interaction with lung fibroblasts (red = vimentin, yellow = KRT5/6, blue = TO-PRO-3 = nuclei, n = 10, scale bar 10 µm and 20 µm). c Immuno-histochemistry of evolving bronchospheres stained for KRT5/6 in red, p40 in turquoise and beta6 integrin in brown (n = 9, scale bars 200 µm). d IPF-ABCs (n = 23) generated significantly more and larger spheres than HV-ABCs (n = 7; P < 0.0001) and NU-ABCs (n = 15, P < 0.0001); mean ± SD. e Cell proliferation was also significantly increased in IPF-ABCs (n = 23) compared to HV-ABCs (n = 7, P < 0.0001) and NU-ABCs (n = 6, P < 0.0001) as measured by MTT assay at d21 (mean ± SD). f Amphiregulin levels were increased in conditioned medium of bronchospheres from IPF-ABCs (n = 23) compared to HV-ABCs (n = 7) and NU-ABCs (n = 15) and correlated closely with bronchosphere counts. g CTGF levels (mean ± SD) were increased in conditioned medium of bronchospheres from IPF-ABCs (n = 42) compared to HV-ABCs (n = 7) and NU-ABCs (n = 12). h Bright field images of raster microscopy of an original experiment (10 independent experiments in triplicate). Lung fibroblasts do not form spheres. Sphere formation by IPF-ABCs is easily detectable. i, In the presence of lung fibroblasts (n = 5 IPF-Fib, n = 5 HV-Fib), IPF-ABCs and HV-ABCs generate increased numbers of bronchospheres (mean ± SD). j Fibroblast cell lines were transduced with lentiviral vectors encoding GFP. Fibroblast proliferation was highly increased in the presence of IPF-ABCs. k Mean fluorescence intensity was significantly increased in fibroblast cell lines co-cultured with IPF-ABCs (n = 5; mean ± SD). l Collagen levels were detected in conditioned medium and matrigel by sircol assay at day 63. Collagen production by lung fibroblasts cultured with IPF-ABCs was significantly increased (n = 5, mean ± SD). m–o in addition, normal lung fibroblasts cultured for 48 h in conditioned medium of IPF-ABC-derived bronchospheres (high BS) showed an increase in fibronectin and collagen 1A expression compared to conditioned medium of bronchosphere cultures derived from NU-ABCs (low BS). n Normalized expression levels of fibronectin from n = 7 fibroblast lines (mean ± SD). o Normalized expression levels of collagen-1 from n = 11 fibroblast lines (mean ± SD). p Normal lung fibroblasts were treated for 20 min with conditioned media of bronchospheres which were harvested at day 14. Pooled conditioned media of bronchospheres derived from IPF-ABCs (High BS) resulted in EGFR phosphorylation compared to conditioned media of bronchospheres derived from NU-ABCs (Low BS). For statistical comparison (d, e, g) one-way ANOVA with Tukey correction for multiple testing, f Pearson correlation, (h, j, k) repeated measures one-way ANOVA with Tukey correction for multiple testing, (n, o) two-tailed Mann–Whitney test was used.