Functional intestinal monolayers from organoids derived from human iPS cells for drug discovery research.

BACKGROUND: Human induced pluripotent stem (iPS) cell-derived enterocyte-like cells (ELCs) are expected to be useful for evaluating the intestinal absorption and metabolism of orally administered drugs. However, it is difficult to generate large amounts of ELCs with high quality because they cannot proliferate and be passaged. METHODS: To solve the issue above, we have established intestinal organoids from ELCs generated using our protocol. Furthermore, monolayers were produced from the organoids. We evaluated the usefulness of the monolayers by comparing their functions with those of the original ELCs and the organoids. RESULTS: We established organoids from ELCs (ELC-org) that 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. Gene expressions of many drug-metabolizing enzymes and drug transporters in ELC-org-mono were enhanced, as compared with those in ELC-org, to a level comparable to those in adult human small intestine. The CYP3A4 activity level in ELC-org-mono was comparable or higher than that in primary cryopreserved human small intestinal cells. ELC-org-mono had the efflux activities of P-gp and BCRP. Importantly, ELC-org-mono maintained high intestinal functions without any negative effects even after long-term culture (for more than a year) or cryopreservation. RNA-seq analysis showed that ELC-org-mono were more mature as intestinal epithelial cells than ELCs or ELC-org. CONCLUSIONS: We have successfully improved the function and convenience of ELCs by utilizing organoid technology.

Vinblastine treatment decreases the undifferentiated cell contamination of human iPSC-derived intestinal epithelial-like cells.

Human induced pluripotent stem cell-derived intestinal epithelial cells (hiPSC-IECs) are expected to be utilized in regenerative medicine. To perform a safe transplantation without the risk of tumor formation, residual undifferentiated hiPSCs must be removed from hiPSC-IECs. In this study, we examined whether vinblastine (a multiple drug resistance 1 [MDR1] substrate) could remove residual undifferentiated hiPSCs in hiPSC-IECs and attempted to generate hiPSC-IECs applicable to transplantation medicine. We found that the expression levels of pluripotent markers were largely decreased and those of intestinal markers were increased by vinblastine treatment. The treatment of undifferentiated hiPSCs with vinblastine significantly decreased their viability. These results suggested that undifferentiated hiPSCs can be eliminated from hiPSC-IECs by vinblastine treatment. We hypothesized that MDR1-negative cells (such as undifferentiated hiPSCs) die upon vinblastine treatment because they are unable to excrete vinblastine. As expected, the cell viability of MDR1-knockout hiPSC-IECs was significantly decreased by vinblastine treatment. Furthermore, teratomas were formed by subcutaneous transplantation of hiPSC-IECs mixed with undifferentiated hiPSCs into mice, but they were not observed when the transplanted cells were pre-treated with vinblastine. Vinblastine-treated hiPSC-IECs would be an effective cell source for safe regenerative medicine.

Generation of Human iPSC-Derived Intestinal Epithelial Cell Monolayers by CDX2 Transduction.

BACKGROUND & AIMS: To develop an effective and safe orally administered drug, it is important to predict its intestinal absorption rate, intestinal first-pass effect, and drug-drug interactions of orally administered drugs. However, there is no existing model to comprehensively predict the intestinal pharmacokinetics and drug-response of orally administered drugs. In this study, we attempted to generate homogenous and functional intestinal epithelial cells from human induced pluripotent stem (iPS) cells for pharmaceutical research. METHODS: We generated almost-homogenous Villin- and zonula occludens-1 (ZO1)-positive intestinal epithelial cells by caudal-related homeobox transcription factor 2 (CDX2) transduction into human iPS cell-derived intestinal progenitor cells. RESULTS: The drug absorption rates in human iPS cell-derived intestinal epithelial cell monolayers (iPS-IECM) were highly correlated with those in humans (R2=0.91). The expression levels of cytochrome P450 (CYP) 3A4, a dominant drug-metabolizing enzyme in the small intestine, in human iPS-IECM were similar to those in human small intestine in vivo. In addition, intestinal availability in human iPS-IECM (the fraction passing the gut wall: Fg=0.73) was more similar to that in the human small intestine in vivo (Fg=0.57) than to that in Caco-2 cells (Fg=0.99), a human colorectal adenocarcinoma cell line. Moreover, the drug-drug interaction and drug-food interaction could be observed by using our human iPS-IECM in the presence of an inducer and inhibitor of CYP3A4, i.e., rifampicin and grape fruit juice, respectively. CONCLUSION: Taking these results together, we succeeded in generating the human iPS-IECM that can be applied to various intestinal pharmacokinetics and drug-response tests of orally administered drugs.

Efficient Generation of Small Intestinal Epithelial-like Cells from Human iPSCs for Drug Absorption and Metabolism Studies.

The small intestine plays an important role in the absorption and metabolism of oral drugs. In the current evaluation system, it is difficult to predict the precise absorption and metabolism of oral drugs. In this study, we generated small intestinal epithelial-like cells from human induced pluripotent stem cells (hiPS-SIECs), which could be applied to drug absorption and metabolism studies. The small intestinal epithelial-like cells were efficiently generated from human induced pluripotent stem cell by treatment with WNT3A, R-spondin 3, Noggin, EGF, IGF-1, SB202190, and dexamethasone. The gene expression levels of small intestinal epithelial cell (SIEC) markers were similar between the hiPS-SIECs and human adult small intestine. Importantly, the gene expression levels of colonic epithelial cell markers in the hiPS-SIECs were much lower than those in human adult colon. The hiPS-SIECs generated by our protocol exerted various SIEC functions. In conclusion, the hiPS-SIECs can be utilized for evaluation of drug absorption and metabolism.

Uterine changes during tamoxifen, toremifene, and other therapy for breast cancer: evaluation with magnetic resonance imaging.

PURPOSE: We have performed pelvic magnetic resonance imaging (MRI) in patients undergoing breast cancer surgery before and after adjuvant drug therapy. Our purpose was to detect any radiographic uterine changes induced by various types of adjuvant therapy on pre- and postmenopausal patients by evaluating prospectively performed MRI. MATERIALS AND METHODS: Between September 2004 and December 2007, a total of 41 women with breast cancer (11 premenopausal, 30 postmenopausal) were enrolled. All underwent MRI of the pelvis before and after drug therapy, and uterine changes were evaluated. Postoperative drugs used were selective estrogen receptor modulators (SERMs) including tamoxifen and toremifene (n = 18), aromatase inhibitors (n = 13), and anticancer drugs (n = 10). RESULTS: Only the postmenopausal patients receiving SERMs showed a significant increase in endometrial thickness: from 2.4 +/- 0.4 mm before therapy to 4.5 +/- 2.6 mm after therapy (P = 0.0485). No statistically significant endometrial change was evident in postmenopausal patients treated with aromatase inhibitors (P = 0.573) or anticancer drugs (P = 0.754). Also, in premenopausal patients treated with SERMs or anticancer drugs, the change in endometrial thickness was not statistically significant (P = 0.958, 0.370). CONCLUSION: This prospective study using MRI has demonstrated that uterine changes associated with adjuvant drugs for breast cancer occur exclusively in postmenopausal patients receiving SERMs.