# Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids
## 4 Discussion
As A83-01 significantly improved organoid formation, this study also highlights that TGF\(\beta\) may act as a potential safeguard mechanism against aberrant proliferation of normal human esophageal epithelial cells exposed to excessive mitogenic stimuli. A83-01 is not required in KSFMC medium to form normal human esophageal organoids [22]. UnlikeKSFMC, however, ADF and its common supplements such as N2 and B27 contain certain agents and factors redundantly. Amongst them, insulin (>10-15 ug/mL altogether) stimulates IGF-I receptor at such supraphysiologic concentrations [3; 53] as to provide a mitogenic stimulus as well as a cytoprotection [54], the latter via the PI3K-AKT pathway. In the absence of exogenous EGF, IGF-I stimulates esophageal basal cell proliferation [53]. It is plausible that A83-01 limits TGF\(\beta\)-mediated cell cycle arrest in normal human esophagael keratinocytes under a variety of stressors [31; 39]. In line with previous attempts [22], all biopsy-derived normal human esophagael PDOs ceased proliferation within 40-60 PDLs regardless of the medium (Figure 5), suggesting that pharmacological inhibition of TGF\(\beta\) receptor I-mediated signaling does not prevent telomere attrition and replicative senescence [33]. Y-27632, a selective inhibitor of Rho-associated, coiled-coil containing protein kinase and a common ingredient of ADF-based organoid media, may immortalize keratinocytes [54]. Y-27632 improved growth and OFR of hTERT-immortalized EPC1 and EPC2 cells (Figure 3) but did not extend the replicative lifespan of non-immortalized primary esophageal keratinocytes grown in PDOs EN1-EN3 (Figure 5). In fact, organoids made with immortalized EPC1 and EPC2 cells grew indefinitely in HOME0. Interestingly, murine normal esophageal organoids grow indefinitely (i.e., over 20 passages, exceeding 250 PDLs) in ADF-based medium without A83-01 [20], highlighting another species difference. Our optimized organoid culture condition in HOME0 recaptured a proper proliferation-differentiation gradient in the normal stratified squamous epithelium of the esophagus as well as perturbation relevant to esophageal epithelial pathologies. Normal human esophageal organoids grown in HOME0 display a modest, if any, cornification compared to those grown in KSFMC (Figures 2 and 5). HOME0 may excel KSFMC in this regard, as the human oral/esophageal squamous epithelium does not normally undergo cornification, unlike the epidermis or the oral/esophageal squamous epithelia in mice. Keratinocyte differentiation depends on calcium [55; 56]. Although calcium is a key ingredient that permits esophageal organoid formation and squamous cell differentiation in KSFMC [22], calcium does not account for limited cornification in HOME0 because ADF contains a higher calcium concentration than KSFMC (Table 3). As terminal differentiation occurs in post-mitotic cells, the differential quantity of mitogenic factors and hormones such as transferrin, insulin, and vitamin A present in these media may account for the differences in degree of observed differentiation. Although the content of KSFM is not fully disclosed by the manufacturer, it is possible that retinoic acid signaling by vitamin A in the B27 supplement added to HOME0 may delay terminal differentiation, as was enhanced by UAB30 (Figure 6). Both UAB30 and IL-13 had similar and profound impacts upon the differentiation gradient through basaloid cell expansion and delayed terminal differentiation (Figures 5 and 6). Interestingly, UAB30 did not induce degenerative changes observed in the presence of 1 ng/mL EGF (Figure 2). Nevertheless, the extent of the UAB30-induced EGFR phosphorylation was comparable to the effect of 1 ng/mL EGF (Figure S4B,C), suggesting that UAB30 and EGF have differential biological effects. EGFR limits terminal differentiation by suppressing Notch signaling [14]. Our data indicate that intrinsic EGFR ligands such as HB-EGF may contribute to BCH. Interestingly, IL-13 and other EoE-related cytokines suppress Notch activation [22].