Aim: Organoid cultures are a powerful model system for the study of cell biology and human disease. Manipulation of the composition of the culture medium has been used to promote cellular differentiation in gastric organoids to allow more accurate modelling of the mature epithelial cells present in the stomach. However, methods to promote the differentiation of neuroendocrine cells and specifically enterochromaffin-like (ECL)-cells within a gastric organoid system have not yet been established.
Methods: Gastric organoids were produced from adult C57BL/6 mice and following ethical approval and informed consent from adult H. pylori negative patients who had a normal stomach at the time of diagnostic gastroscopy. We investigated whether altering the composition of the culture medium and the addition of a small molecule that is known to induce neural differentiation in other culture systems promoted neuroendocrine cell differentiation in mouse and human gastric organoids. The abundance of Chromogranin A and other markers of differentiated cells were assessed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence.
Results: Mouse and human gastric corpus organoids that were cultured in conventional culture media showed high expression of the gastrointestinal epithelial stem cell marker Lgr5 and low abundances of the markers of differentiated stomach cell types. Reduction of the amount of Wnt in the culture medium directed organoid differentiation towards the pit cell lineage, with a reduced abundance of Lgr5 and increased expression of the mucus neck cell marker MUC5AC. Incubation of mouse and human gastric organoids with a small molecule that is known to induce neural differentiation resulted in increased abundance of Chromogranin A as well as other markers associated with ECL-cell differentiation such as histidine decarboxylase and the gastrin/CCK-2 receptor.
Conclusions: Mature differentiated parietal and enterochromaffin-like cells were rare in tissue derived mouse and human gastric organoids that were cultured using standard conditions. Cellular differentiation could however be promoted by manipulating the media composition and we additionally established a novel method for promoting neuroendocrine cell differentiation in gastric organoid cultures. This methodology will hopefully provide a platform to investigate gastric ECL-cell physiology as well as the development of gastric neuroendocrine tumours.