The small intestine plays an important role in all aspects of pharmacokinetics, but there is no system for the comprehensive evaluation of small-intestinal pharmacokinetics, including drug metabolism and absorption. In this study, we aimed to construct an intestinal pharmacokinetics evaluation system and to generate pharmacokinetically functional enterocytes from human induced pluripotent stem cells. Using activin A and fibroblast growth factor 2, we differentiated these stem cells into intestinal stem cell-like cells, and the resulting cells were differentiated into enterocytes in a medium containing epidermal growth factor and small-molecule compounds. The differentiated cells expressed intestinal marker genes and drug transporters. The expression of sucrase-isomaltase, an intestine-specific marker, was markedly increased by small-molecule compounds. The cells exhibited activities of drug-metabolizing enzymes expressed in enterocytes, including CYP1A1/2, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, UGT, and sulfotransferase. Fluorescence-labeled dipeptide uptake into the cells was observed and was inhibited by ibuprofen, an inhibitor of the intestinal oligopeptide transporter solute carrier 15A1/PEPT1. CYP3A4 mRNA expression level was increased by these compounds and induced by the addition of 1a,25-dihydroxyvitamin D 3 . CYP3A4/5 activity was also induced by 1a,25-dihydroxyvitamin D 3 in cells differentiated in the presence of the compounds. All these results show that we have generated enterocyte-like cells that have pharmacokinetic functions, and we have identified small-molecule compounds that are effective for promoting intestinal differentiation and the gain of pharmacokinetic functions. Our enterocyte-like cells would be useful material for developing a novel evaluation system to predict human intestinal pharmacokinetics.

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The evaluation of drug pharmacokinetics in the small intestine is critical for developing orally administered drugs. Caucasian colon adenocarcinoma (Caco-2) cells are employed to evaluate drug absorption in preclinical trials of drug development. However, the pharmacokinetic characteristics of Caco-2 cells are different from those of the normal human small intestine. Besides this, it is almost impossible to obtain primary human intestinal epithelial cells of the same batch. Therefore, human iPS cell-derived enterocytes (hiPSEs) with pharmacokinetic functions similar to human intestinal epithelial cells are expected to be useful for the evaluation of drug absorption. Previous studies have been limited to the use of cytokines and small molecules to generate hiPSEs. Dietary fibers play a critical role in maintaining intestinal physiology. We used gellan gum (GG), a soluble dietary fiber, to optimize hiPSE differentiation. hiPSEs cocultured with GG had significantly higher expression of small intestine- and pharmacokinetics-related genes and proteins. The activities of drug-metabolizing enzymes, such as cytochrome P450 2C19, and peptide transporter 1 were significantly increased in the GG treatment group compared to the control group. At the end point of differentiation, the percentage of senescent cells increased. Therefore, GG could improve the differentiation efficiency of human iPS cells to enterocytes and increase intestinal maturation by extending the life span of hiPSEs.