The Esophageal Organoid System Reveals Functional Interplay Between Notch and Cytokines in Reactive Epithelial Changes

Abstract

Background & AimsAberrations in the esophageal proliferation-differentiation gradient are histologic hallmarks in eosinophilic esophagitis (EoE) and gastroesophageal reflux disease. A reliable protocol to grow 3-dimensional (3D) esophageal organoids is needed to study esophageal epithelial homeostasis under physiological and pathologic conditions.MethodsWe modified keratinocyte-serum free medium to grow 3D organoids from endoscopic esophageal biopsies, immortalized human esophageal epithelial cells, and murine esophagi. Morphologic and functional characterization of 3D organoids was performed following genetic and pharmacologic modifications or exposure to EoE-relevant cytokines. The Notch pathway was evaluated by transfection assays and by gene expression analyses in vitro and in biopsies.ResultsBoth murine and human esophageal 3D organoids displayed an explicit proliferation-differentiation gradient. Notch inhibition accumulated undifferentiated basal keratinocytes with deregulated squamous cell differentiation in organoids. EoE patient-derived 3D organoids displayed normal epithelial structure ex vivo in the absence of the EoE inflammatory milieu. Stimulation of esophageal 3D organoids with EoE-relevant cytokines resulted in a phenocopy of Notch inhibition in organoid 3D structures with recapitulation of reactive epithelial changes in EoE biopsies, where Notch3 expression was significantly decreased in EoE compared with control subjects.ConclusionsEsophageal 3D organoids serve as a novel platform to investigate regulatory mechanisms in squamous epithelial homeostasis in the context of EoE and other diseases. Notch-mediated squamous cell differentiation is suppressed by cytokines known to be involved in EoE, suggesting that this may contribute to epithelial phenotypes associated with disease. Genetic and pharmacologic manipulations establish proof of concept for the utility of organoids for future studies and personalized medicine in EoE and other esophageal diseases.

Modeling Epithelial Homeostasis and Reactive Epithelial Changes in Human and Murine Three‐Dimensional Esophageal Organoids

“…We have found that the well described immortalized non‐transformed EPC2‐hTERT organoids passage indefinitely (Kasagi et al., 2018). Organoids from biopsies do not grow beyond passage four to five.…”

“…Our recent publication demonstrates the use of this technique to recapitulate the unique epithelial changes associated with EoE by stimulation with EoE‐relevant cytokines (IL‐13, IL‐4; Kasagi et al., 2018). We evaluated the effect of the EoE milieu on Notch signaling and utilized genetic and pharmacologic inhibition of Notch signaling of organoids to simulate the reactive epithelial changes that occur in vivo.…”

“…Methodologies to evaluate differentiation may vary and complementary methods may be utilized. In our recently published work (Kasagi et al., 2018), we utilized an expert pathologist to evaluate the organoids for basaloid cell content and complimented this with immunohistochemistry and flow cytometry analysis for involucrin and CD29, respectively.…”

“…Their methods involved utilizing advanced Dulbecco's modified Eagle's medium with multiple growth factors including: Glutamax, B27, p38 kinase inhibitor, EGF, TGFβ inhibitor, R-spondin, Noggin, and Wnt3A. We found that human esophageal organoids did not grow or stratify in this enriched medium (Kasagi et al, 2018). Instead, we utilized a simplified medium of KSFM with additional calcium that produced a stratified squamous epithelium from both murine and human tissue with almost 100% reliability.…”

“…We have established the single‐cell‐derived esophageal 3D organoid culture system to mimic in vivo tissue architecture to model epithelial homeostasis and reactive changes associated with esophageal disease conditions (Kasagi et al., 2018). We describe protocols to (1) prepare single‐cell suspensions from endoscopic biopsies from human subjects or epithelial sheets isolated from murine esophagi as starting materials; (2) grow, passage, and cryopreserve 3D organoids; (3) determine growth kinetic of 3D organoids; and (4) perform morphological and functional evaluation of 3D organoids coupled with or without pharmacologic or genetic manipulations.…”