# Project-3: Modeling EA/TEF in human organoids

> **NIH NIH P01** · CINCINNATI CHILDRENS HOSP MED CTR · 2022 · $397,500

## Abstract

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.

## Key facts

- **NIH application ID:** 10458162
- **Project number:** 2P01HD093363-06
- **Recipient organization:** CINCINNATI CHILDRENS HOSP MED CTR
- **Principal Investigator:** James M Wells
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $397,500
- **Award type:** 2
- **Project period:** 2017-08-15 → 2027-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10458162

## Citation

> US National Institutes of Health, RePORTER application 10458162, Project-3: Modeling EA/TEF in human organoids (2P01HD093363-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10458162. Licensed CC0.

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