Cytochrome P450 expression, induction and activity in human induced pluripotent stem cell-derived intestinal organoids and comparison with primary human intestinal epithelial cells and Caco-2 cells

Abstract

Human intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by β-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine.

Reversing Epithelial Polarity in Pluripotent Stem Cell-Derived Intestinal Organoids

“…These results indicate that organoids on day 15 already express intestine-specific markers but are fairly immature, whereas by day 30 the in vitro maturation culminates. This is in accordance with findings from Spence et al (2011) and Janssen et al (2020) . After 50 days in culture, we observed a general decrease in the expression of most markers, although still in detectable levels, showing that organoids’ functionality in culture gradually deteriorates from day 30 days.…”

“…These stainings were performed at 30 days post Matrigel embedment of hindgut organoids or their transfer to suspension culture. This time-point was selected according to Spence et al (2011) and Janssen et al (2020) , who demonstrated that intestinal organoids embedded in Matrigel can reach sufficient maturation after approximately 28 days. Organoids from both culture conditions showed similar expression patterns of the mentioned markers.…”

Differential gene expression in iPSC-derived human intestinal epithelial cell layers following exposure to two concentrations of butyrate, propionate and acetate

“…iPSC derived HIOs consist of enterocytes, goblet cells, mesenchymal and enteroendocrine cells [25][26][27][28] . Furthermore, iPSC derived HIOs have shown improved cytochrome P450 and transporter expression in comparison with Caco-2 cells 29,30 . More recently, iPSCs have been used to create 2D intestinal epithelial cell (IEC) layers that emulate the full cellular complexity of the 3D HIOs, but have an easily accessible apical and basolateral side in comparison with their 3D HIO variants which are closed spheres.…”

Farm and Companion Animal Organoid Models in Translational Research: A Powerful Tool to Bridge the Gap Between Mice and Humans

“…Successful recovery of cryopreserved organoids ( 44 , 145 ) can provide a banked resource for future use. Furthermore, organoids are useful for standard biological analyses such as live cell imaging using inverted phase-contrast and confocal microscopies ( 101 ), gene expression analysis ( 14 ), genetic modification ( 146 ), and enzyme activity assay ( 14 ).…”

“…Although many studies have been conducted on organoids in humans and mice ( 5 , 14 , 15 ), studies focusing on organoids in farm and companion animal species have been gaining attention in various fields including veterinary and translational medicine as well as biomedical research. This is because companion animals that develop spontaneous chronic disorders similar to those of humans have been suggested to serve as better models of human patients than traditional murine models where the disorders are usually genetically or chemically induced ( 16 , 17 ).…”