mEOUs cultured for 4–12 days and either embedded in paraffin or taken as whole mount. IHC was used to detect expression of esophageal markers. Red scale bars: 100 μm; white scale bars: 1000 μm. (A) mEOUs 4 days in culture showing that most cells were Ki67+. (B) mEOUs 12 days in culture, larger in size, fewer cells were Ki67+. H&E staining demonstrates squamous epithelium surrounded by stromal components. Squamous epithelium shows maturation from basal layer to superficial layer. Differentiated cells expressed the squamous epithelium markers CK4 and involucrin, smooth muscle marker SMA, neuron marker TUJ, and other molecules important in foregut development: SHH, BMP4, NOG, SMAD1/5/9. (C) mEOUs 12 days in culture examined in whole-mount 3D images showing how different cells types come together to form EOUs.