Endocrine Abstracts

Backgrounds and aims: Enteroendocrine cells (EECs) are hormone-secreting cells within the intestinal epithelium that play an important role in regulating food absorption, insulin secretion and appetite. The somatostatin (SST)-producing D-cell is an EEC of particular interest due to the profound inhibition exerted by SST over other EECs, highlighting D-cells as critical regulator of the enteroendocrine axis. The aim of this study was to profile the transcriptome of human intestinal D-cells from organoids culture and to identify the key signalling pathways involved in the regulation of SST secretion.

Materials and methods: To label somatostatin secreting D-cells in human duodenal organoids CRISPR-Cas9 followed by homology-directed repair was used to insert Internal Ribosome Entry Site sequence, followed by the fluorescent protein tdTomato sequence and puromycin resistant cassette under control of the endogenous somatostatin promoter. Fluorescence-activated cell sorting (FACS) was used to purify organoids-derived D-cells. Bulk RNA sequencing of FACS-purified SST-tdTomato positive and negative cells was performed. 

Results: The transcriptional profiles of FACS purified D-cells and control populations were analysed. The principal component analysis exhibited a wide separation between these two populations on the first component (87% of variance), and narrow separation on the second component (8 % of variance). tdTomato-positive cells were strongly enriched for SST gene, which was found at ~1000-fold-higher levels in fluorescent compared to non-fluorescent cells (p < 0.001). RNA sequencing identified enriched expression of several G-protein coupled receptors in D-cells including short-chain fatty acids receptor (FFAR2), bile acids receptor (GPBAR1), amino acids receptor (GPR142), trace amines receptor (TAAR1) and the vasopressin receptor (AVPR1B).

Conclusion: This study provides the first in-depth transcriptomic analysis of human intestinal D-cells which provide an important foundation to guide future studies for functional characterisation of this cell type.