Addition of NOG restores viability and stimulates expression of esophageal markers. (A) hEOUs grown for 10 days from 5 different patients. NOG inhibitor was added to the culture for 48 hours, washed, and cultures were allowed to grow for 10 days. IHC analysis for expression of esophageal and tracheal markers. (B) Fluorescence intensity of CK13, TFF3, CC10, CK4, NOG, and BMP was evaluated using Image J (n = 3–5), ***P < 0.0009. (C) hEOUs isolated from EA/TEF patients at the time of surgical repair and cultured for 10 days with or without NOG. hEOUs were fixed and paraffin sections were used to evaluate their morphology and protein expression. (D) Fluorescence intensity of CK13, TFF3, CC10, CK4, NOG, and BMP was evaluated using Image J (n = 3–8), *P < 0.05, **P < 0.005. Scale bars: 100 μm.