Figure 7.

Phenotypic characterization of mucus and goblet cells in human colonoids after removal of Wnt3a, Noggin, and R-spondin from cultures. Human colonoids derived from the Donor 1 biopsy were cultured for one day in WNR CM, and either kept in this medium or switched to rENR and rE as differentiation culture condition. After three additional days, phenotypic characterization of mucus and goblet cells was performed by histology and qPCR. (A) Immunohistochemistry for Ki67 (proliferation), GPA33 (all epithelial cells) and Alcian blue staining for goblet cells and mucus and (B) quantification of goblet cells; (C) Immunohistochemistry for Mucin 2, GPA33 (all epithelial cells) and Hematoxylin (nuclei); and (D) quantification of Mucin 2 positive Goblet cells; (E) Immunohistochemistry for Mucin 1 with Hematoxylin (nuclei) and (F) quantification of Mucin 1 positive cell area. For automated analysis of Alcian blue and Mucin 2 data is the average number for three slides and is representative of two independent studies. For each slide, the average number was determined by collecting and analyzing 15 random 40x images (containing an average of 25 colonoids each) to determine % positive cells per colonoid or per image. The Mucin 1 staining was defined as the area of Mucin 1 immunopositive pixels per colonoid area pixels after luminal white space was subtracted. Mucin 1 analysis was applied to the entire slide image, each of which contained an average of 570 colonoids. Scale bars indicate 50 microns. Statistical significance was determined by one-way ANOVA.