Figure 3.

Switching culture conditions from Wnt3a-conditioned medium to Wnt3a free medium induces most efficient differentiation of mouse colonoids. Mouse colonoids derived from C57Bl/6 mice crypts were cultured for two days in WNR CM, Wnt3a CM + rENR or rWENR medium before medium change to rENR or maintenance in original media. After four additional days of culture, colonoids were assayed for markers of epithelial cell types by ISH/IHC and qPCR. (A, B) Colonoids were stained with in situ hybridization for LGR5 for the quantification of stem cells (brown dots), immunohistochemistry for EpCam (epithelial cells) and hematoxylin (nuclei); (C) Quantification of Ki67 histochemistry for measurement of proliferation; (D) immunohistochemistry for Chromogranin A (enteroendocrine cells) and Alcian Blue counterstain (mucus and Goblet cells); (E) quantification of goblet cells; (F) quantification of Mucin 2 mRNA levels; (G) immunohistochemistry of Ki67 as a proliferation marker, with an Alcian Blue counterstain (mucus and Goblet cells); (H) quantification of chromogranin A immunohistochemistry; (I) quantification of enterocytes using mRNA transcript levels of alkaline phosphatase (ALPI). qPCR analysis was performed on biological triplicates for each treatment group at each timepoint. Graphs in (B, C) are the average expression by IHC/ISH of the indicated markers in the stem cell conditions from three independent studies. Graphs in (E, F, H, I) are the average fold increase in markers by IHC (E) or qPCR (F, H, I) after differentiation compared to levels in the stem cell conditions from three independent experiments. Scale bars in A indicate 20 microns and in C and E indicate 50 microns. IHC/ISH quantification was performed on an average of 30 colonoids per slide. Graphs and images are representative of the average of biological duplicates from three independent experiments. Statistical significance was determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.005.