# Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids
## 1 Introduction  
Stratified squamous epithelium of the esophagus comprises the basal and the suprabasal compartments. The basal compartment contains proliferative basal/parabasal epithelial cells (keratinocytes) that undergo post-mitotic terminal differentiation within the suprabasal cell layers. Differentiating keratinocytes establish cell junctions to provide epithelial barrier function while undergoing desquamation into the lumen at the superficial cell layers, allowing continuous esophageal epithelial renewal [1; 2]. The homeostatic proliferation-differentiation gradient of the esophageal epithelium is subjected to regulation by various factors and conditions including growth factors, hormones, and cell-cell contact. Among these factors are epidermal growth factor (EGF), transforming growth factor (TGF)-\(\beta\), bone morphogenetic proteins (BMP), Notch, and retinoids/vitamins (see Tables 1 and 2 for abbreviations). EGF and other ligands for epidermal growth factor receptor (EGFR) stimulate keratinocyte proliferation [3; 4; 5; 6; 7; 8; 9; 10]. All-trans retinoic acid, a key metabolite of vitamin A, stimulates basal cell proliferation to increase progenitor cells committed to terminal differentiation [11; 12; 13]. During terminal differentiation, Notch signaling activates transcription of differentiation-related genes such as Involuric (IVL) [3; 14; 15]. Belonging to the TGF\(\beta\)s super family, BMP promotes squamous cell differentiation through activation of the antioxidant response pathway [16]. These pathways are perturbed in a variety of esophageal disease conditions such as eosinophilic esophagigitis (EoE), whose pathogenesis involves disrupted mucosal integrity and barrier functions as manifested by basal cell hyperplasia (BCH), a histopathologic feature characterized by expansion of basal cells and delayed terminal differentiation [17; 18; 19]. The 3D organoid system is a robust cell culture system where a small number of tissue-dissociated cells (1000-10,000) are embedded in the Matrigel\({}^{\text{\textregistered}}\) matrix, the basement-membrane extract, to form single cell-derived spherical structures that arise within 2 weeks [20; 21]. 3D organoids are amenable to genetic and pharmacological modifications and are utilized \begin{table}
\begin{tabular}{l l} \hline \hline
**Abbreviation** & **Name** \\ \hline AI & Artificial Intelligence \\ BCH & basal cell hyperplasia \\ BMP & bone morphogenetic proteins \\ DAPI & 4\({}^{\prime}\),6-diamidino-2-phenylindole \\ DMSO & Dimethyl sulfoxide \\ EoE & eosinophilic esophagigitis \\ H\&E & Hematoxylin and Eosin \\ IF & immunofluorescence \\ IHC & immunohistochemistry \\ IL & interleukin \\ IVL & Involuric \\ OFR & Organoid formation rate \\ PBS & phosphate-buffered saline \\ PDL & population doubling level \\ PDO & Patient-derived organoids \\ qRT-PCR & quantitative reverse-transcription polymerase chain reaction \\ RAR & retinoic acid receptor \\ KXR & retinoic-X receptor \\
3D & three-dimensional \\ TGF-\(\beta\) & transforming growth factor-beta \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Abbreviations of general terms. to explore how pathogenic agents such as alcohol and pro-inflammatory cytokines may influence esophageal epithelial homeostasis [22]. Patient-derived organoids (PDO) are translatable in personalized medicine to predict therapeutic response and explore novel therapeutics, albeit limited to oncology to date [23; 24; 25; 26; 27; 28; 29]. To date, ADF is the most widely utilized base medium to generate organoids representing various types of normal and abnormal human epithelial conditions, including esophageal intestinal metaplasia, preneoplasia, and cancers [21; 23; 30]. We have previously described that ADF is suboptimal to grow normal human esophageal organoids and proposed an alternative KSFMC medium with a final concentration of 0.6 mM Ca2+ added to keratinocyte serum-free medium (KSFM) that allows recapitulation of not only the normal squamous epithelial differentiation gradient but also pathologic changes such as BCH [22].