Background: Esophagus tissue engineering using the hybrid-construct approach involves assembly of the esophageal components to engineer the organ. This study investigated the dedifferentiation and loss in expression of esophageal smooth muscle after explant expansion in culture.Methods: Ovine esophagus smooth muscle cells (OESMC) were sourced from adult biopsies and expanded using the explant culture technique. The explants were maintained under static in vitro tissue culture. Flow cytometry analysis was performed for a-smooth muscle actin (a-SMA) expression at intervals of 4 weeks for up to 8 weeks.Results: After 4 weeks 58.5% OESMC exhibited a-SMA which decreased to 28.5% after 6 weeks in culture. After 8 weeks in culture a mere 1.3% OESMC demonstrated a-SMA expression.Conclusions: OESMC proliferated using the explant technique exhibit a total loss of a-SMA expression after 8 weeks of static in vitro culture. This information is crucial for tissue engineering of the esophagus using the hybridconstruct approach.