Labeling and Characterization of Human GLP-1-Secreting L-cells in Primary Ileal Organoid Culture

Glucagon-like peptide-1 (GLP-1) from intestinal L-cells stimulates insulin secretion and reduces appetite after food ingestion, and it is the basis for drugs against type-2 diabetes and obesity. Drugs targeting L- and other enteroendocrine cells are under development, with the aim to mimic endocrine effects of gastric bypass surgery, but they are difficult to develop without human L-cell models. Human ileal organoids, engineered by CRISPR-Cas9, express the fluorescent protein Venus in the proglucagon locus, enabling maintenance of live, identifiable human L-cells in culture. Fluorescence-activated cell sorting (FACS)-purified organoid-derived L-cells, analyzed by RNA sequencing (RNA-seq), express hormones, receptors, and ion channels, largely typical of their murine counterparts. L-cells are electrically active and exhibit membrane depolarization and calcium elevations in response to G-protein-coupled receptor ligands. Organoids secrete hormones in response to glucose and other stimuli. The ability to label and maintain human L-cells in organoid culture opens avenues to explore L-cell function and develop drugs targeting the human enteroendocrine system. [Display omitted] •Enteroendocrine differentiation and labeling in human small intestinal organoids•RNA-seq and peptide LC-MS profiling of purified human GLP-1-secreting L-cells•Human ileal L-cells are electrically active and respond to nutritional stimuli Development of anti-diabetic drugs targeting the hormone-secreting intestinal enteroendocrine system is limited by a lack of human cellular models. Goldspink et al. generate human ileal organoids with fluorescently-labeled GLP-1-secreting L-cells, which respond to nutritional stimuli and are used for transcriptomic, peptidomic, and single-cell functional analyses.