Skip to Content
Merck
  • Labeling and Characterization of Human GLP-1-Secreting L-cells in Primary Ileal Organoid Culture.

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

Cell reports (2020-07-02)
Deborah A Goldspink, Van B Lu, Emily L Miedzybrodzka, Christopher A Smith, Rachel E Foreman, Lawrence J Billing, Richard G Kay, Frank Reimann, Fiona M Gribble
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
DAPT, ≥98% (HPLC), solid
Sigma-Aldrich
PD 0325901, ≥98% (HPLC)
Sigma-Aldrich
Angiotensin II human, ≥93% (HPLC), powder
Sigma-Aldrich
Amyloid Protein Non-Aβ Component, ≥80% (HPLC)
Sigma-Aldrich
G 418 disulfate salt, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
L-(−)-Glucose, ≥99%
Sigma-Aldrich
Forskolin, from Coleus forskohlii, ≥98% (HPLC), powder
Sigma-Aldrich
AR231453, ≥98% (HPLC)
Sigma-Aldrich
GPBAR-A, ≥98% (HPLC)
Sigma-Aldrich
Amphotericin B from Streptomyces sp., ~80% (HPLC), powder