Abstract
The diverse cellular milieu of the gastric tissue microenvironment plays a critical role in normal tissue homeostasis and tumor development. However, few cell culture model can recapitulate the tissue microenvironment and intercellular signaling in vitro. We used a primary tissue culture system to generate a murine p53 null gastric tissue model containing both epithelium and mesenchymal stroma. To characterize the microenvironment and niche signaling, we used single cell RNA sequencing (scRNA-Seq) to determine the transcriptomes of 4,391 individual cells. Based on specific markers, we identified epithelial cells, fibroblasts and macrophages in initial tissue explants during organoid formation. The majority of macrophages were polarized towards wound healing and tumor promotion M2-type. During the course of time, the organoids maintained both epithelial and fibroblast lineages with the features of immature mouse gastric stomach. We detected a subset of cells in both lineages expressing Lgr5, one of the stem cell markers. We examined the lineage-specific Wnt signaling activation, and identified that Rspo3 was specifically expressed in the fibroblast lineage, providing an endogenous source of the R-spondin to activate Wnt signaling. Our studies demonstrate that this primary tissue culture system enables one to study gastric tissue niche signaling and immune response in vitro.
Highlights
Within the microenvironment of the stomach tissue, gastric epithelial cells have complex interactions with other cell lineages
Our analysis indicated that air-liquid interphase (ALI)-based primary gastric tissue explants underwent macrophage-based tissue remodeling
To study the epithelial-stromal interaction in vitro, we applied a high-throughput single cell RNA-Seq in a gastric organoid model that is composed of epithelial cell and stromal components
Summary
Within the microenvironment of the stomach tissue, gastric epithelial cells have complex interactions with other cell lineages. The ALI system can be applied to maintain gastrointestinal primary tissue cultures over long periods of time as well as develop engineered cancer models where driver mutations can be systematically introduced[8,9,10] This method of primary tissue culture has significant advantages compare to the conventional approaches. ALI-grown gastrointestinal organoids do not require supplementation of exogenous Wnt growth factors such as Wnt3A and Rspo[18,10] Rather, these primary tissue cultures are self-sustaining, suggesting that the medley of different cell lineages provides an endogenous source of www.nature.com/scientificreports/. For this study of the microenvironment in the ALI model, we identified different cellular lineages, determined the gene differential expression among the major cell types, and as a result identified growth factors that enable signaling crosstalk and maintain microenvironment cellular diversity. Our results point to the role of R-spondin, as provided by the cellular microenvironment, being important for maintaining gastric organoid epithelial cell populations in the ALI system
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