Abstract
SummaryGlucagon-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.
Highlights
Enteroendocrine cells (EECs) produce gut hormones that regulate energy metabolism and appetite as well as local processes such as nutrient digestion and intestinal motility (Gribble and Reimann, 2019)
Growth of hGLUVenus organoids in IF medium (Fujii et al, 2018; STAR Methods), or modified medium (IF*) containing inhibitors of mitogen activated protein kinase kinases (MEK1/2) and Notch (Basak et al, 2017; Beumer et al, 2018) with reduced Wnt3A to enhance EEC development, resulted in the appearance of scattered yellow-fluorescent cells that morphologically resembled EECs, with processes extending across the epithelial layer (Figure 1B)
The intensities of Venus and proglucagon staining were positively correlated, as predicted since Venus expression was driven by the proglucagon promoter in this line (Figure 1F)
Summary
Enteroendocrine cells (EECs) produce gut hormones that regulate energy metabolism and appetite as well as local processes such as nutrient digestion and intestinal motility (Gribble and Reimann, 2019). An alternative therapeutic approach under investigation is to stimulate release of the body’s endogenous supplies of gut hormones, thereby mimicking the effects of Roux-en-Y gastric bypass (RYGB) surgery, which results in high post-prandial plasma levels of hormones including GLP-1, peptide YY (PYY), and neurotensin (NTS) as a consequence of rapid nutrient delivery to the distal small intestine and enhanced stimulation of ileal EECs (Gribble and Reimann, 2019). Understanding the physiology and stimulus responsiveness of human GLP-1-secreting L-cells is key to the development of these new classes of therapeutics. Murine L-cells are electrically active and utilize a variety of sensory proteins for the detection of food ingestion, including sodium-coupled glucose transporters (SGLT1s) for sensing ingested glucose and G-protein-coupled receptors (GPCRs) for detection of many other nutrients (e.g., fatty acids, amino acids) and bile acids (Gribble and Reimann, 2016)
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