Endocrine Abstracts

Introduction: Enteroendocrine cells (EECs) are hormone-secreting cells within the intestinal epithelium that play an important role in regulating food absorption, insulin secretion and appetite. To understand the molecular mechanism governing the function of EECs, it is important to investigate cell-type-specific gene expression. The aim of this study was to identify the transcriptome of human EEC populations from organoid culture.

Methods: To label the full spectrum of human EECs in organoids from adult human proximal small intestine (duodenum) and distal small intestine (ileum), CRISPR-Cas9 followed by homology-directed repair was used to insert a P2A ribosomal stutter sequence, followed by the fluorescent protein Venus sequence at the 3’ end of the chromogranin-A (CHGA) coding sequence, a general marker of EECs, in chromosome 14. Venus-positive EECs were collected by flow cytometry. Single-cell RNA-seq was performed using the 10x Genomics 3, GEX V3.1 platform.

Results: The transcriptional profiles of 10,016 cells and 5,911 cells from the duodenal and ileal organoids respectively were analysed. Cluster analysis identified six major EECs populations in ileal organoids and ten in duodenal organoids. Gut hormones were amongst the top differentially expressed genes for each of the labelled clusters, including SST-expressing D cells, MLN/GHRL expressing M/X cells, and TPH1-expressing enterochromaffin cells in both regions, while GAST/GIP/CCK expressing G/K/l cells were detected in the duodenum, and GCG-expressing L cells in the ileum. Interestingly, clusters of mature EECs were detected in both duodenum and ileum that do not express known hormonal markers. The expression of G-protein coupled receptors differed between clusters, suggesting that EECs are regulated differentially.

Conclusion: In this study, we have generated high-resolution transcriptomic profiles of human EECs from proximal small intestine (duodenum) and distal small intestine (ileum) which provide an important foundation to guide future genomics-based interrogation of EECs functions and their sensory apparatus.