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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE240322 | GEO | 2024/03/06

REPOSITORIES: GEO

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Project description:We performed transcriptomic profiling of freshly isolated primary adult human alveolar epithelial type 2 cells (AEC2s), their isogenic cultured progeny, and human induced pluripotent stem cell-derived AEC2s (iAEC2s). Distal lung preparations from adult donor lung explants from two individuals were cryopreserved using methods we have previously described. Primary AEC2s were purified using fluorescence activated cell sorting (FACS) to isolate cells co-expressing EPCAM and HTII-280. To establish cultures of primary AEC2s, these cells were combined with MRC5 fibroblasts at an 1:10 ratio on cell culture inserts in CK+DCI medium. iAEC2s were derived using our previously published lung directed differentiation protocol. We used the SPC2 human iPSC line and specifically the SPC2-ST-B2 (SFTPC tdT/WT ) clone containing a SFTPC tdTomato knock-in reporter. SFTPC tdTomato+ cells were sorted on day 41 of differentiation. iAEC2s were single-cell passaged in self-renewing feeder-free 3D cultures approximately every 2 weeks. We used the 10X Chromium System to generate single-cell transcriptomic data from 5 samples prepared in parallel and sequenced on the same day. We profiled (a) preculture primary AEC2s, (b) cultured primary AEC2s, (c) 3D feeder-free iAEC2s, (d) feeder-free iAEC2 cultured on cell culture inserts, and (e) iAEC2s cultured on cell inserts with MRC5 fibroblasts. The cultured primary AEC2 samples and the iAEC2 sample co-cultured with MRC5s were sorted for live EPCAM+ cells prior to encapsulation. All other samples were sorted for live cells. We find that although each population (fresh, primary cultured, and iPSC-derived AEC2s) occupies a distinct transcriptomic space, both cultured primary AEC2s and cultured feeder-free iAEC2s closely resemble their primary pre-cultured counterparts. We also find a continuum of progressively more proliferative states (from fresh primary to cultured primary to cultured iAEC2s) inversely associated with progressively less mature AEC2 states.

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

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