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Single-cell transcriptome analysis identifies distinct cell types and niche signaling in a primary gastric organoid model.

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.

SUBMITTER: Chen J

PROVIDER: S-EPMC6418230 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Single-cell transcriptome analysis identifies distinct cell types and niche signaling in a primary gastric organoid model.

Chen Jiamin J Lau Billy T BT Andor Noemi N Grimes Susan M SM Handy Christine C Wood-Bouwens Christina C Ji Hanlee P HP

Scientific reports 20190314 1

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-Se ...[more]

PMID: 30872643

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Project description:BACKGROUND:Single-cell genomic methods now provide unprecedented resolution for characterizing the component cell types and states of tissues such as the epithelial subsets of the gastrointestinal tract. Nevertheless, functional studies of these subsets at scale require faithful in vitro models of identified in vivo biology. While intestinal organoids have been invaluable in providing mechanistic insights in vitro, the extent to which organoid-derived cell types recapitulate their in vivo counterparts remains formally untested, with no systematic approach for improving model fidelity. RESULTS:Here, we present a generally applicable framework that utilizes massively parallel single-cell RNA-seq to compare cell types and states found in vivo to those of in vitro models such as organoids. Furthermore, we leverage identified discrepancies to improve model fidelity. Using the Paneth cell (PC), which supports the stem cell niche and produces the largest diversity of antimicrobials in the small intestine, as an exemplar, we uncover fundamental gene expression differences in lineage-defining genes between in vivo PCs and those of the current in vitro organoid model. With this information, we nominate a molecular intervention to rationally improve the physiological fidelity of our in vitro PCs. We then perform transcriptomic, cytometric, morphologic and proteomic characterization, and demonstrate functional (antimicrobial activity, niche support) improvements in PC physiology. CONCLUSIONS:Our systematic approach provides a simple workflow for identifying the limitations of in vitro models and enhancing their physiological fidelity. Using adult stem cell-derived PCs within intestinal organoids as a model system, we successfully benchmark organoid representation, relative to that in vivo, of a specialized cell type and use this comparison to generate a functionally improved in vitro PC population. We predict that the generation of rationally improved cellular models will facilitate mechanistic exploration of specific disease-associated genes in their respective cell types.

Dataset Information

Single-cell transcriptome analysis identifies distinct cell types and niche signaling in a primary gastric organoid model.

Publications

Single-cell transcriptome analysis identifies distinct cell types and niche signaling in a primary gastric organoid model.

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