Single‐cell transcriptomics of human organoid‐derived enteroendocrine cell populations from the small intestine

Gut hormones control intestinal function, metabolism and appetite, and have been harnessed therapeutically to treat type 2 diabetes and obesity. Our understanding of the enteroendocrine axis arises largely from animal studies, but intestinal organoid models make it possible to identify, genetically modify and purify human enteroendocrine cells (EECs). This study aimed to map human EECs using single-cell RNA sequencing. Organoids derived from human duodenum and ileum were genetically modified using CRISPR-Cas9 to express the fluorescent protein Venus driven by the chromogranin-A promoter. Fluorescent cells from CHGA-Venus organoids were purified by flow cytometry and analysed by 10X single-cell RNA sequencing. Cluster analysis separated EEC populations, allowing an examination of differentially expressed hormones, nutrient-sensing machinery, transcription factors and exocytotic machinery. Bile acid receptor GPBAR1 was most highly expressed in L-cells (producing glucagon-like peptide 1 and peptide YY), long-chain fatty acid receptor FFAR1 was highest in I-cells (cholecystokinin), K-cells (glucose-dependent insulinotropic polypeptide) and L-cells, short-chain fatty acid receptor FFAR2 was highest in ileal L-cells and enterochromaffin cells, olfactory receptor OR51E1 was notably expressed in ileal enterochromaffin cells, and the glucose-sensing sodium glucose cotransporter SLC5A1 was highly and differentially expressed in K- and L-cells, reflecting their known responsiveness to ingested glucose. The organoid EEC atlas was merged with published data from human intestine and organoids, with good overlap between enteroendocrine datasets. Understanding the similarities and differences between human EEC types will facilitate the development of drugs targeting the enteroendocrine axis for the treatment of conditions such as diabetes, obesity and intestinal disorders. KEY POINTS: Gut hormones regulate intestinal function, nutrient homeostasis and metabolism and form the basis of the new classes of drugs for obesity and diabetes. As enteroendocrine cells (EECs) comprise only ∼1% of the intestinal epithelium, they are under-represented in current single-cell atlases. To identify, compare and characterise human EECs we generated chromogranin-A labelled organoids from duodenal and ileal biopsies by CRISPR-Cas9. Fluorescent chromogranin-A positive EECs were purified and analysed by single-cell RNA sequencing, revealing predominant cell clusters producing different gut hormones. Cell clusters exhibited differential expression of nutrient-sensing machinery including bile acid receptors, long- and short-chain fatty acid receptors and glucose transporters. Organoid-derived EECs mapped well onto data from native intestinal cell populations, extending coverage of EECs.