Introduction
Esophageal submucosal glands (ESMGs) are thought to play a role in the protection and repair of the esophageal epithelium1. No human model of these cells exists, and, therefore, experimental data are lacking2. Barrett’s esophagus (BE), a premalignant condition linked to chronic gastro-esophageal reflux, has an unclear cellular origin. Literature shows patches of BE in a field of squamous mucosa associated with underlying ESMGs and the other way around3,4. A human ESMG model enables direct investigation of ESMG function, including their potential role in the origin of BE. This study aims to establish and characterize the first human organoid and 2D model of ESMG epithelial cells. ESMG pathophysiology is particularly relevant to gastro-esophageal reflux and esophageal metaplasia.
Aims & Methods
ESMGs were micro-dissected from esophagectomy specimens. Culture conditions were optimized and quantified using MTT proliferations assays with respect to EGF, fibroblast conditioned medium, and Wnt, Noggin, and R-spondin-conditioned medium concentrations. Cells were cultured either in Matrigel to achieve 3D cultures or on cell culture inserts with micropores to achieve multilayer 2D cultures. Marker expression (e.g., EpCAM, TP63, KRT5, KRT7, MUC1, OLFM4, and the HOXA family of genes) was analyzed by RT-qPCR. The potential of these cells to form a clone from a single cell was investigated through serial dilutions of the single cell suspension. Organoid morphology was assessed through bright-field microscopy, H&E staining, and immunohistochemistry.
Results
Human ESMGs could be identified and dissected from full-thickness esophageal wall specimens. These specimens were derived from patients treated with chemo(radio)therapy. ESMG cell cultures show increased proliferation when exposed to higher concentrations of EGF. The addition of medium conditioned by an immortalized esophageal fibroblast cell line also results in increased proliferation. ESMG cells can be maintained as 3D organoids and can form a 2D attaching multilayer. ESMG cells can be harvested from the glands and cultured under these conditions for several months. High expression of EpCAM and low expression of COL1A1 confirms the cells are epithelial in nature. ESMGs cell cultures show expression of both TP63, KRT5 and KRT13, being squamous epithelial markers and KRT7, MUC1, being columnar epithelial markers, and OLFM4, an intestinal stem cell marker. Additionally, cultures are characterized by expression of HOXA1 and 4-7, characteristic of the upper GI tract, but also by midgut genes HOXA9 and 10 and the colon specific HOXA13, also characteristic of columnar esophageal epithelia5. ESMG cells continue to proliferate, after large dilution of the original cell suspension, at extremely low seeding density. 3D cultured human ESMG organoids show heterogeneity in their morphology. Notably, organoids tend either take on a solid or hollow spheroid form.
Conclusion
This study reports on the first human esophageal submucosal gland epithelial cell model. Both a 3D organoid and 2D multilayered epithelium models have formed. This model enables the experimental interrogation of human ESMG epithelial cells for the first time. The cultures show molecular and morphological characteristics associated with both squamous and columnar esophageal epithelia.
References
1. Long, J.D. and R.C. Orlando, Esophageal submucosal glands: structure and function. Am J Gastroenterol, 1999. 94(10): p. 2818-24.
2. von Furstenberg RJ, Li J, Stolarchuk C, Feder R, Campbell A, Kruger L, Gonzalez LM, Blikslager AT, Cardona DM, McCall SJ, Henning SJ, Garman KS. Porcine Esophageal Submucosal Gland Culture Model Shows Capacity for Proliferation and Differentiation. Cell Mol Gastroenterol Hepatol. 2017 Aug 4;4(3):385-404.
3. Owen, R.P., White, M.J., Severson, D.T. et al. Single-cell RNA-seq reveals profound transcriptional similarity between Barrett’s oesophagus and oesophageal submucosal glands. Nat Commun 9, 4261 (2018).
4. Nowicki-Osuch, Karol et al. “Molecular phenotyping reveals the identity of Barrett's esophagus and its malignant transition.” Science (New York, N.Y.) vol. 373,6556 (2021): 760-767. doi:10.1126/science.abd1449
5. Janmaat, V.T., Nesteruk, K., Spaander, M.C.W. et al. HOXA13 in etiology and oncogenic potential of Barrett’s esophagus. Nat Commun 12, 3354 (2021).
