CFC assays were performed on 7-d cultures of 30 EpCAM++ fetal cells (green), 60 EpCAM+CD49f+ adult basal cells (blue), 100 EpCAM++CD49flow/−CD61+ adult luminal cells (red), and 300 adult EpCAM+ cells (purple) using various additives to determine their effects on CFC outputs. (A) Comparison of the effect of 80% 3T3 cell CM, 160 ng/ml Wnt3a±400 ng/ml R-Spondin 1 (R-Spo), or 16 ng/ml bFGF relative to added 3T3 cells (set = 100%). The concentrations shown are the final concentrations in the 250 µl cultures. Results are pooled from 3–6 experiments. The difference in CFC output between CM and no added 3T3 cells was significant for cultures initiated with all types of cells (p<0.05, one-way ANOVA with Bonferroni's multiple comparison test). (B) Effect of Wnt pathway inhibitors: XAV939 (0.8 µM for adult, 4 µM for fetal) or mDKK1 (160 ng/ml) in cultures with irradiated 3T3 cells (set = 100%). Results are pooled from 3–9 experiments. Only the effect of added XAV939 was significant, and only for basal cells (p = 0.04, one-way ANOVA with Bonferroni's multiple comparison test). (C) Effect of 40 ng/ml HGF and 16 ng/ml CSF-1 on adult EpCAM+ cells. The difference in CFC output between added HGF or CSF-1 and no added 3T3 cells is not significant (p>0.99, one-way ANOVA with Bonferroni's multiple comparison test).