RT Journal Article
SR Electronic
T1 Human intestinal enteroids with inducible neurogenin-3 expression as a novel model of gut hormone secretion
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 579698
DO 10.1101/579698
A1 Alexandra L. Chang-Graham
A1 Heather A. Danhof
A1 Melinda A. Engevik
A1 Catherine Tomaro-Duchesneau
A1 Umesh C. Karandikar
A1 Mary K. Estes
A1 James Versalovic
A1 Robert A. Britton
A1 Joseph M. Hyser
YR 2019
UL http://biorxiv.org/content/early/2019/03/16/579698.abstract
AB Background Enteroendocrine cells (EECs) are specialized epithelial cells that produce molecules vital for intestinal homeostasis, but due to their limited numbers, in-depth functional studies have remained challenging. Human intestinal enteroids (HIEs) that are derived from intestinal crypt stem cells are a biologically relevant in vitro model of the intestinal epithelium. HIEs contain all intestinal epithelial cell types; however, like the intestine, HIEs spontaneously produce few EECs, which limits their study.Methods To increase the number of EECs in HIEs, we used lentivirus transduction to stably engineer jejunal HIEs with doxycycline-inducible expression of neurogenin-3 (NGN3), a transcription factor that drives EEC differentiation (tetNGN3-HIEs). We examined the impact of NGN3 induction on EECs by quantifying the increase in the enterochromaffin cells and other EEC subtypes. We functionally assessed secretion of serotonin and EEC hormones in response to norepinephrine and rotavirus infection.Results Treating tetNGN3-HIEs with doxycycline induced a dose-dependent increase of chromogranin A (ChgA)-positive and serotonin-positive cells, demonstrating increased enterochromaffin cell differentiation. Despite increased ChgA-positive cells, other differentiated cell types of the epithelium remained largely unchanged by gene expression and immunostaining. RNA sequencing of doxycycline-induced tetNGN3- HIEs identified increased expression of key hormones and enzymes associated with several other EEC subtypes. Doxycycline-induced tetNGN3-HIEs secreted serotonin, monocyte chemoattractant protein-1, glucose-dependent insulinotropic peptide, peptide YY, and ghrelin in response to norepinephrine and rotavirus infection, further supporting the presence of multiple EEC types.Conclusions We have combined HIEs and inducible-NGN3 expression to establish a flexible in vitro model system for functional studies of EECs in enteroids and advance the molecular and physiological investigation of EECs.Synopsis Enteroendocrine cells have low abundance but exert widespread effects on gastrointestinal physiology. We engineered human intestinal enteroids with inducible expression of neurogenin-3, resulting in increased enteroendocrine cells and facilitating investigations of host responses to the dynamic intestinal environment.(NGN3)neurogenin-3(tet)tetracycline(dox)doxycycline(GI)gastrointestinal(HIEs)human intestinal enteroids(HIOs)human intestinal organoids(SCFAs)short chain fatty acids(ChgA)chromogranin A(PP)pancreatic polypeptide(GIP)glucose-dependent insulinotropic peptide(GLP-1)glucagon-like peptide-1(PYY)peptide YY(MCP-1)monocyte chemoattractant protein-1(Tph1)tryptophan hydroxylase(VIL1)villin(SI)sucrase isomaltase(RV)rotavirus(hr)hour(hpi)hours post infection(RT-qPCR)reverse transcriptase – quantitative polymerase chain reaction(IF)immunofluorescence(H&E)hematoxylin and eosin(2D)two-dimensional(3D)three-dimensional(CMGF-)complete medium without growth factors(CMGF+)complete media with growth factors(hW-CMGF+)high Wnt complete media with growth factors