# Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids
## 3 Results
### RXR Stimulation Induces BCH-like Changes in Normal Esophageal Organoids Formed in HOME0  
EoE patient esophageal biopsies show upregulation of retinoic acid receptor (RAR)-\(\beta\) mRNA expression [43; 44]. In keratinocytes, biological effects of vitamin A are mediated by heterodimers of RARs and retinoic-X receptors (RXRs) that act as a transcription factor [45]. The effect of activation of retinoic acid signaling in the human esophageal epithelium remains elusive. We stimulated EPC2 organoids and a normal human PDO line EN1 with UAB30, a potent synthetic RXR agonist that enhances all-trans retinoic acid signaling in human epidermal keratinocytes in organotypic 3D culture [46]. In normal esophageal PDO EN1 and EPC2 organoids grown in HOME0, UAB30 induced BCH-like epithelial changes with increased Ki67-positive basaloid cells while decreasing IVL expression concurrently (Figure 6A-G). Since retinoic acid-induced epidermal hyperplasia involves heparin-binding EGF-like growth factor (HB-EGF), an EGFR ligand [47; 48; 49; 50], we next evaluated HB-EGF expression and the EGFR activity. UAB30 upregulated HB-EGF mRNA expression (Figure S4A) as well as the EGFR tyrosine phosphorylation to an extent that was comparable with the effect of 1 ng/mL EGF (Figure S4B,C), suggesting that HB-EGF may activate EGFR to mediate UAB30-stimulated BCH.  
Figure 5: IL-13 induces BCH-like epithelial changes in normal human esophageal organoids grown in HOME0. Established organoids were stimulated with or without recombinant human IL-13 and subjected to H&E staining, IHC for indicated markers, or IF for IVL (**A**). Halo AI and multiplex IHC modules were used for organoid segmentation, and Halo IHC was used for quantification of optical density (OD) (**B,C,E,F**). The OD average was calculated in each organoid. For IF, nuclei were counterstainedained with DAPI. IVL intensity was measured by ImageJ ([[https://imagej.net/](https://imagej.net/)]([https://imagej.net/](https://imagej.net/))) for each organoid (**D,G**). * Indicates \(p<0.05\), NS: non-significant. Student’s \(t\)-test was used for the comparison. Scale bars, \(100\) μm.  
Figure 6: UAB30 induces BCH-like epithelial changes in normal human esophageal PDO and EPC2 organoids grown in HOME0. Organoids were grown in the presence or absence of 5 μmU LAB30 and to H&E staining, IHC for indicated markers, or IF for IVL (**A**). Halo AI and multiplex IHC modules were used for organoid segmentation, and Halo IHC was used for quantification of optical density (OD) (**B,C,E,F**). The OD average was calculated in each organoid. For IF, nuclei were counterstainedained with DAPI. IVL intensity was measured by ImageJ ([[https://imagej.net/](https://imagej.net/)]([https://imagej.net/](https://imagej.net/))) for each organoid (**D,G**). * Indicates \(p<0.05\), NS: non-significant. Student’s \(t\)-test was used for the comparison. Scale bars, \(100\) μm.  
In aggregate, the above findings indicate that the normal proliferation-differentiation gradient and its perturbation are recapitulated in human esophageal organoids grown in the HOME0 medium conditions, thus establishing ADF-based organoid culture medium conditions.