Using Induced Pluripotent Stem Cells to Understand Esophageal-Tracheal Separation and Esophageal Atresia.

Raad Suleen¹, David Anu¹, Faure Christophe^1,2

1. Esophageal Development and Engineering Laboratory, Sainte-Justine Research Centre, Montreal, Quebec, Canada.
2. Esophageal Atresia Clinic and Division of Pediatric Gastroenterology Hepatology and Nutrition, CHU Sainte Justine, Université de Montréal, Montréal, Quebec, Canada

Esophageal atresia/tracheoesophageal fistula (EA/TEF) is a congenital anomaly occurring in 1 in 3000 births. The molecular and cellular mechanisms that regulate trachea-esophageal separation are poorly understood. Induced pluripotent stem cells (iPSCs) provide an opportunity to understand human embryonic development and could be used to elucidate mechanisms associated with EA/TEF.

To understand abnormal foregut specification, we used iPSCs derived from healthy individuals and EA/TEF patients. Directed differentiation of iPSCs towards different development stages, such as definitive endoderm, anterior foregut, esophageal and tracheal epithelium, were confirmed with immunofluorescence, qPCR, and RNA sequencing analysis. Furthermore, esophageal epithelial organoids were generated and matured to determine cellular organization and morphology in a 3D environment.

Expression of endodermal markers CXCR4, SOX17, GATA4, and FOXA2 were similar in both patient and healthy differentiated cells. Microscopic observation revealed differences in the structural characteristics between patient and healthy cells beginning at the anterior foregut stage, with decreased gene and protein expression of key transcriptional factor SOX2 in patient-derived cells. Additionally, a significant increase in the expression of NKX2.1, a tracheal marker, was observed in the patient-derived esophageal epithelium. Following histological analysis, we observed differences in the cellular organization, as well as a lower expression of KRT4, and an increased expression of NKX2.1 in patient-derived esophageal organoids. Furthermore, NKX2.1 expression was lower in patient-derived tracheal epithelium compared to its expression in healthy cells.

In conclusion, we hypothesize that a transient dysregulation of key transcription factors SOX2 and NKX2.1 in patient-derived cells could be responsible for abnormal esophageal and tracheal development.