Porcine Esophageal Submucosal Gland Culture Model Shows Capacity for Proliferation and Differentiation

Abstract

Background & AimsAlthough cells comprising esophageal submucosal glands (ESMGs) represent a potential progenitor cell niche, new models are needed to understand their capacity to proliferate and differentiate. By histologic appearance, ESMGs have been associated with both overlying normal squamous epithelium and columnar epithelium. Our aim was to assess ESMG proliferation and differentiation in a 3-dimensional culture model.MethodsWe evaluated proliferation in human ESMGs from normal and diseased tissue by proliferating cell nuclear antigen immunohistochemistry. Next, we compared 5-ethynyl-2′-deoxyuridine labeling in porcine ESMGs in vivo before and after esophageal injury with a novel in vitro porcine organoid ESMG model. Microarray analysis of ESMGs in culture was compared with squamous epithelium and fresh ESMGs.ResultsMarked proliferation was observed in human ESMGs of diseased tissue. This activated ESMG state was recapitulated after esophageal injury in an in vivo porcine model, ESMGs assumed a ductal appearance with increased proliferation compared with control. Isolated and cultured porcine ESMGs produced buds with actively cycling cells and passaged to form epidermal growth factor–dependent spheroids. These spheroids were highly proliferative and were passaged multiple times. Two phenotypes of spheroids were identified: solid squamous (P63+) and hollow/ductal (cytokeratin 7+). Microarray analysis showed spheroids to be distinct from parent ESMGs and enriched for columnar transcripts.ConclusionsOur results suggest that the activated ESMG state, seen in both human disease and our porcine model, may provide a source of cells to repopulate damaged epithelium in a normal manner (squamous) or abnormally (columnar epithelium). This culture model will allow the evaluation of factors that drive ESMGs in the regeneration of injured epithelium. The raw microarray data have been uploaded to the National Center for Biotechnology Information Gene Expression Omnibus (accession number: GSE100543).

Single cell RNA-seq reveals profound transcriptional similarity between Barrett’s oesophagus and oesophageal submucosal glands

“…in gene or protein expression and cellular appearance) between BO and selected normal tissues - including the intestine, gastric pylorus, gastric corpus and gastric cardia – have found some shared attributes 11 , 12 . There is also evidence suggesting BO may originate directly from native oesophageal squamous 13 or submucosal gland cells 14 – 17 , from recruitment of circulating stem cells 18 , or from reactivation of dormant p63 − /KRT7 + residual embryonic cells (RECs) in situ 19 . In contrast to p63 − /KRT7 + RECs, a recent study identified p63 + /KRT5 + /KRT7 + cells derived from the squamocolumnar junction as the cells of origin of BO in a transgenic mouse model with ectopic expression of CDX2 in KRT5 + epithelium 20 .…”

Pathogenesis and Cells of Origin of Barrett's Esophagus

“…64 Porcine ESMGs placed in 3-dimensional culture also proliferated and single cells derived from these ESMGs formed 2 types of spheroids, which recapitulated the squamous and columnar phenotypes associated with ESMGs in the human and dog studies. 65 One spheroid type had a solid, squamous appearance and expressed squamous markers, such as p63, whereas the other was hollow and expressed columnar cell markers found in Barrett's metaplasia, including AGR2, MUC13, KRT18, MUC1, KRT8, and SOX9. Research is underway in pigs to elucidate factors that affect expression of squamous vs columnar markers.…”

Esophageal 3D Culture Systems as Modeling Tools in Esophageal Epithelial Pathobiology and Personalized Medicine

“…Jiang et al 142 identified unique p63-positive transitional basal cells expressing cytokeratins K5 and K7 as a putative BE cell of origin along with functional validation in 3D organoid assays. In addition, von Furstenberg et al 143 used a porcine model of epithelial injury and human tissues to identify 2 distinct esophageal submucosal gland–derived cells expressing p63 or K7 that give rise to squamous and ductal 3D spheroid structures, respectively.…”