PROJECT 3 | SUMMARY: MODELING EA IN HUMAN PSC-DERIVED EMBRYONIC TISSUES The esophagus is composed of a layer of stratified epithelium that is surrounded by layers of innervated muscle that regulates the unidirectional movement of food from the mouth to the stomach. Esophageal atresia (EA) with or without associated tracheoesophageal fistula (TEF) is the most common Tracheoesophageal birth defect (TED), yet most mutations causing EA are unidentified. This proposal aims to use human pluripotent stem cell (PSC)-derived esophageal tissues both to understand EA and as a future source for reconstruction. We developed human esophageal organoids (HEOs) and esophageal organotypic rafts (HEORs) with stratified esophageal epithelium that is highly similar to human esophagus. We used these to investigate how SOX2, an HMG-box transcription factor that is essential for esophageal development in mice, might cause EA in humans. Transcriptional profiling of organoids at different stages of differentiation that are analogous to critical stages of esophageal development identified that SOX2 regulates expression of Wnt antagonists like SFRP2 in the dorsal foregut endoderm that gives rise to the esophagus. Loss of SOX2 expression resulted in loss of esophageal fate and impaired morphogenesis, resulting in EA. In humans, patients with EA can have persistent motility defects suggesting that EA-associated genes may affect development of the smooth muscles or the enteric nervous system (ENS) of the esophagus. Project 1 has identified new TED-associated mutations that we predict may act in specific germ layers to cause EA. Published and preliminary data indicate that the transcription factors RFX6 and SOX2 act in the epithelium whereas FOXF1 and SMAD6 act in the mesenchyme. Since esophageal organoids only contained epithelium, they were not useful to study mutations impacting mesenchyme or ENS. We therefore engineered several new organoid systems containing mesenchymal and ENS cell types all derived from PSCs. We propose to use these novel human esophageal systems to test the hypothesis that EA-associated genes impact different germ layer components and can cause later developmental and functional deficits in the esophagus. Here we propose using PSC-derived HEOs and HEORs that contain all three germ layers to identify how EA-causing mutations impact epithelial, mesenchymal and ENS development. We will then determine how EA- causing mutations impact the function of human engineered esophageal tissue. Lastly we will use EA-causing mutations to identify epithelial- and mesenchyme-specific transcriptional programs in the developing human esophagus.