# Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids
## 4 Discussion  
In optimizing medium conditions to generate normal human esophageal organoids utilizing ADF medium, we found that the removal of exogenous EGF and the addition of A83-01, a TGF\(\beta\) receptor I inhibitor, is most helpful to improve organoid formation efficiency. Our optimized HOME0 medium contains a limited number of supplements compared to other ADF-based media such as ADF3+ (Table 3), described previously [11; 40]. The HOME0-grown organoids displayed a well-organized proliferation-differentiation gradient that recapitulates the histologic architecture of the stratified squamous epithelium of the normal human esophagus. Furthermore, HOME0 medium appeared to be permissive for modeling BCH that represents reactive epithelial changes common in EoE, gastroesophageal reflux diseases, and other esophageal disease conditions. One remarkable premise is that exogenous EGF is dispensable for HOME0 and that addition of EGF at supraphysiological concentration (50 ng/mL) is detrimental for optimal organoid growth of human normal esophageal epithelial cells. This may explain why previously reported ADF-based media including ADF3+ performed poorly compared to other medium formulations such as KSFMC containing 1 ng/mL recombinant human EGF that is closer to a human serum level [38]. Our data do not preclude the importance of EGFR signaling for normal human esophageal organoid formation in HOME0, as EGFR tyrosine phosphorylation was detectable in the absence of exogenous EGF (Figure S4) and EGFR tyrosine kinase inhibitor AG-1478 decreased organoid formation and cell viability (Figures 3 and S1), reinforcing the role of endogenous EGFR activity in organoid formation. However, 0.1 ng/mL EGF added into HOME0 did not induce EGFR phosphorylation significantly compared to HOME0 (control) (Figure S4B,C), suggesting that a minimal and a physiologically-relevant level of EGFR ligands (e.g., \(\sim\)0.25 ng/mL EGF in the serum [38]) is sufficient for human normal esophageal organoid formation in HOME0. Interestingly, murine normal esophageal organoids grow well in either KSFMC or ADF-based media, including our own mouse organoid medium (MOM) containing 50 ng/mL EGF (Table 3) [11; 20], suggesting species differences in EGFR signaling and EGF sensitivity in esophageal epithelial cells. Given the oncogenic role of EGFR in esophageal carcinogenesis, normal human esophageal cells may negate malignant transformation via degenerative changes in response to excessive mitogenic stimuli as observed in HOME50 (Figure 1). In agreement, EGFR overexpression alone induces TGF\(\beta\)-mediated senescence in EPC1 and EPC2 cells while concurrent inactivation of cell-cycle checkpoint functions is permissive for transformation with epithelial-mesenchymal transition [39]. Since we did not compare ADF3+ with or without EGF in this study, we cannot exclude the possibility that the benefit we observe by HOME0 may be accounted for by the lack of other factors and agents present in ADF3+ (e.g., FGFs and CHIR9021). However, they are not required for murine organoid culture in MOM [20] (Table 3). Additionally, the lack of ADF3+ components in HOME50 does not account for the degenerative morphological changes (Figures 2 and 4), reduced OFR and shorter PDL (Figures 1 and S2) which were observed in both ADF3+ and HOME50 compared to HOME0 and KSFMC. Unlike ADF3+, HOME media contain the N2 supplement [51] consisting of insulin, transferrin, progesterone, selenium, and putrescine, which are all redundant factors present in B27 [52] and the ADF-base medium. We are currently testing HOME0 and HOME50 devoid of the N2 supplement without substantial differences (unpublished observations). Therefore, we may establish a simplified ADF3+ to identify a minimal standard media formulation except for the increased A83-01 concentration.