Advancing Esophageal Disease Modeling: Microfluidic Platforms for Adult Tissue-Resident Stem Cell Culture and Differentiation.

BACKGROUND AND AIMS: Classical organoid models of Barrett's esophagus (BE) lack accessible luminal surfaces, tissue depth and tractable stem cell and immune cell components. Herein, we aimed to seed adult tissue-resident stem cells (ASC) with advanced organ-on-a-chip (OOAC) microfluidics to provide a platform upon which to study the immunobiology of BE and associated high grade dysplasia (HGD). METHODS: ASCs from BE and HGD and gastroesophageal junction (GEJ) were obtained using methods analogous to ground-state stem cell culture and conditional reprogramming. ASC differentiation was achieved on Transwells and flexible OOAC with tissues examined by immunohistochemistry and single-cell RNA sequencing gene expression analysis. Monocytic cell line THP1 was utilized in transmigration experiments. RESULTS: Monolayered ASCs exhibited colony formation, self-renewal and air-liquid interface-mediated differentiation resulting in 3D furrow formation, mucus production and cell types reflective of tissue of origin-foveolar, goblet, enterocyte-like. Levels of intestinal marker Trefoil Factor 3 were abundant in BE tissue, significantly lower in HGD and absent in GEJ. The gastric marker Gastrokine1 was only expressed in HGD- and GEJ-differentiated ASCs. Comparatively, tissues derived from OOAC displayed significantly enhanced villus-like structure, height, and prolonged survival (<19 days) when compared to Transwell-differentiated equivalents. Single-cell RNAseq analysis detected populations representative of stem cells, transit amplifying stem cells, early and late enterocytes, goblet and enteroendocrine cells and CXCL8 positive immunomodulatory cells in both systems. However, significantly higher levels of proliferating cells, terminal enterocyte-like, Goblet cells, and enteroendocrine-like cells were observed in BE-OOAC systems. THP1 cells were tracked under flow conditions in BE-OOAC and their transmigration through the endothelial layer was evident only in the presence of organoid tissue above. CONCLUSION: These findings showed that OOACs offer more physiologically relevant environments, promote deeper cellular differentiation, and increased cellular tractability when seeded as ASCs rather than classical organoid aggregates.