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Signaling basis of gastric epithelial cell differentiation revealed by human and mouse organoids [RNA-Seq]

ABSTRACT: Purpose:One goal of this bulk RNA-seq is to compare genome-wide transcriptome profiles in gastric organoids cultured in CM and differentiation media including Nog WD, BMP4, EB, Isx-9, BI, NRC and NRCA in mouse and EB, NRCSI and BIMDP in human. The other aim is to identify the differential biological process underpinning gastric epithelial cell differentiation and the conservative or divergent process between human and mouse. Methods: For human and mouse gastric organids, we isolated and digested with Tryple and then extracted total RNA. Bulk RNA-seq was performed using the Illumina Hiseq X. Sequence reads that passed quality filters were analysed at the transcript isoform level using two methods: Burrows-Wheeler Aligner (BWA) followed by ANOVA and TopHat followed by Cufflinks. Results: By comparing RNA-seq data, we identified the signature genes involved in gastric epithelial cell lineage and enrichment of biological processes during differentiation. Conclusion: EB is optimal medium for pit cell differentiation in both human and mouse, BI and BIMDP media are optimal media for parietal cell differentiation in human and mouse respectively, NRCS and NRCSI media are optimal media for chief cell differentiation in human and mouse respectively.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE226195 | GEO | 2025/05/29

REPOSITORIES: GEO

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Project description:Objective: Enteroendocrine cells (EECs) survey the gut luminal environment and co-ordinate hormonal, immune and neuronal responses to it. They exhibit well characterized physiological roles ranging from the control of local gut function to whole body metabolism, but little is known regarding the regulatory networks controlling their differentiation, especially in human gut. The small molecule Isoxazole-9 (ISX-9) has been shown to stimulate neuronal and pancreatic beta-cell differentiation, both closely related to EEC differentiation. Our aim was to use ISX-9 as a tool to explore EEC differentiation. Methods: We investigated the effects of ISX-9 on EEC differentiation in mouse and human intestinal organoids, using real time quantitative PCR, fluorescent activated cell sorting, immunostaining and single cell RNA sequencing. Results: ISX-9 increased the number of neurogenin3 (Ngn3) positive endocrine progenitor cells and upregulated NeuroD1 and Pax4, transcription factors which play roles in mouse EEC specification. Single cell analysis revealed induction of Pax4 expression in a developmentally late Ngn3+ population of cells and potentiation of genes associated with progenitors biased towards serotonin-producing enterochromaffin (EC) cells. This coincided with enrichment of organoids with functional EC cells which was partly dependent on stimulation of calcium signalling in a population of cells residing outside the crypt base. Inducible Pax4 overexpression, in ileal organoids, uncovered its importance as a component of early human endocrine specification and highlighted the potential existence of two major endocrine lineages, the early appearing enterochromaffin lineage and the later developing peptidergic lineage which contains classical gut hormone cell types. Conclusion: Our data provide proof-of-concept for the controlled manipulation of specific endocrine lineages with small molecules, whilst also shedding new light on human EEC differentiation and its similarity to mouse. Given their diverse roles, understanding endocrine lineage plasticity and its control could have multiple therapeutic implications.

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Signaling basis of gastric epithelial cell differentiation revealed by human and mouse organoids [RNA-Seq]

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