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.

Suspension culture is superior to planar culture in efficiently growing large numbers of cells. It also greatly improves cell functions by mimicking the in vivo environment. Although several methods are available to differentiate human induced pluripotent stem (iPS) cells into hepatocyte-like cells, their differentiation by suspension culture by using functional polymers remains unreported. We developed a suspension culture system using the functional polymers FP001 and FP003 to generate hepatocyte-like cells with superior phenotypes than human iPS cell-derived hepatocyte-like cells generated in planar culture. Specifically, optimizing the initial spheroid size and utilizing FP003 effectively maintained appropriate spheroid structures, resulting in higher albumin production compared to FP001. In 3D sphere culture, the mRNA expression of albumin (ALB), drug-metabolizing enzymes and transporters, and the activity of CYP1A2 were increased in the presence of 0.01% FP003. Additionally, immunofluorescence analysis confirmed uniform hepatic differentiation throughout the spheroids. These findings suggest that differentiation in 3D sphere culture with the functional polymer FP003 improved the function of human iPS cell-derived hepatocyte-like cells. Therefore, we conclude that this system may be an effective strategy to generate human iPS cell-derived hepatocyte-like cells with enhanced functions.