Highly functional intestinal monolayer through organoids derived from human iPS cells for drug discovery research

Human induced pluripotent stem (iPS) cell-derived enterocyte-like cells (ELCs) are expected to evaluate intestinal absorption and metabolism of orally administered drugs. However, it was difficult to generate large amounts of ELCs with high quality because they cannot proliferate and be passaged. In this study, we established the organoids from ELCs (ELC-org), which could be passaged and maintained for more than a year. When ELC-org were dissociated into single cells and seeded on cell culture inserts (ELC-org-mono), they formed a tight monolayer in 3 days. Both ELC-org and ELC-org-mono were composed exclusively of epithelial cells, being nearly 100% of EpCAM-positive. Gene expressions of many drug-metabolizing enzymes and drug transporters were enhanced in ELC-org-mono compared to those in ELCs and ELC-org. Their levels were similar to those in human adult intestine. The CYP3A4 activity level in ELC-org-mono was comparable or higher than that in primary cryopreserved human small intestinal cells and their expression level was up-regulated by inducers such as rifampicin. ELC-org-mono had the efflux activities of P-gp and BCRP. Importantly, ELC-org-mono cultured for more than a year maintained high intestinal functions. Even when the cryopreserved organoid-derived cells were directly seeded and cultured on cell culture inserts, they maintained high intestinal functions similar to the cells without cryopreservation. RNA-seq analysis showed that ELC-org-mono were more mature as intestinal epithelial cells than ELC and ELC-org. Therefore, we have successfully improved the function and convenience of ELCs by utilizing organoid technology. Overall design: Human iPS cell-derived enterocyte-like cells (ELCs), ELC-derived intestinal organoids (ELC-org) and ELC-org-derived monolayers