AbstractObjectiveHuman gastric epithelial stem/progenitor cells are important for stomach homeostasis; however, the in vitro culture system of these cells remains immature. Although three‐dimensional (3D) organoid culture has fundamentally changed the in vitro study of gastrointestinal tract, its use is limited by inaccessible luminal compartment, and difficulties of imaging and manipulation. To overcome these limitations of 3D organoid culture system, we established adult human gastric epithelial progenitor‐like (hGEPL) cell lines using a novel robust monolayer cell culture system.Materials and MethodsWe established an in vitro gel‐based monolayer culture system for normal human adult gastric epithelium, and compared it with traditional two‐dimensional (2D) and 3D organoid culture systems using transcriptomics, immunofluorescence and cell viability experiments. At the same time, we used single‐cell transcriptomics to compare the differences of the hGEPL cells in conditioned medium (Cond.) and in chemically defined medium (Chem.), the two most common media for organoid culture, in maintaining the stemness and proliferative activity of hGEPL cells. Finally, we explored the role of key niche factors in inducing hGEPL cell differentiation.ResultsThe hGEPL cells were similar to the in vivo gastric epithelial stem/progenitor cells, which could stably proliferate in culture for a long time. Based on the established culture system, we explored signalling pathways that were important for the homeostasis of hGEPL cells. We found that after blocking the WNT signalling pathway or activating the BMP signalling pathway, hGEPL cells could differentiate into mucous surface cells.ConclusionOur culture system of hGEPL cells from adults is robust and easy to operate, and has the transformative potential of personalized and precision medicine, laying a solid foundation for studying the self‐renewal and differentiation potentials of gastric epithelial stem/progenitor cells as well as modelling of related gastric diseases.