Characteristic Analysis of Intestinal Transport in Enterocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells

Abstract

We previously demonstrated that differentiated enterocytes from human induced pluripotent stem (iPS) cells exhibited drugmetabolizing activities and cytochrome P450 CYP3A4 inducibility. The aim of this study was to apply human iPS cell-derived enterocytes in pharmacokinetic studies by investigating the characteristics of drug transport into enterocyte-like cells. Human iPS cells cultured on feeder cells were differentiated into endodermal cells using activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, epidermal growth factor and small-molecule compounds induced the maturation of the intestinal stem cell-like cells. After differentiation, we performed transepithelial electrical resistance (TEER) measurements, immunofluorescence staining, and transport studies. TEER values increased in a time-dependent manner and reached approximately 100 V 3 cm 2 .Efflux transport of Hoechst 33342, a substrate of breast cancer resistance protein (BCRP), was observed and inhibited by the BCRP inhibitor Ko143. The uptake of peptide transporter 1 substrate glycylsarcosine was also confirmed and suppressed when the temperature was lowered to 4°C. Using immunofluorescence staining, villin and Na + -K + ATPase were expressed. These results suggest that human iPS cell-derived enterocytes had loose tight junctions, polarity, as well as uptake and efflux transport functions. In addition, the rank order of apparent membrane permeability coefficient (P app ) values of these test compounds across the enterocyte-like cell membrane corresponded to the fraction absorbance (F a ) values. Therefore, differentiated enterocytes from human iPS cells may provide a useful comprehensive evaluation model of drug transport and metabolism in the small intestine.

Application of Intestinal Epithelial Cells Differentiated from Human Induced Pluripotent Stem Cells for Studies of Prodrug Hydrolysis and Drug Absorption in the Small Intestine

“…P-gp, BCRP, and PEPT1 play important roles in intestinal drug absorption. The functions of BCRP and PEPT1 in hiPSC-IECs have been previously reported (Iwao et al, 2015;Ogaki et al, 2015;Ozawa et al, 2015;Kodama et al, 2016;Uchida et al, 2017), whereas the present study is the first to reveal the functional expression of P-gp in hiPSC-IECs. The P app value of [ 3 H]digoxin (a P-gp substrate) in the basolateral-to-apical direction was greater than that in the apical-tobasolateral one (efflux ratio, 2.43), and the differences in P app in these two directions disappeared in the presence of cyclosporin A (efflux ratio, 0.743) (Fig.…”

“…In recent years, several researchers have succeeded in generating intestinal epithelial cells from human induced pluripotent stem cells (hiPSCs) (Spence et al, 2011;Kauffman et al, 2013;Ogaki et al, 2013;Forbester et al, 2015;Iwao et al, 2015;Ozawa et al, 2015;Uchida et al, 2017). Human induced pluripotent stem cell-derived intestinal epithelial cells (hiPSC-IECs) would be a good tool to predict the absorption of oral drugs in humans, because they are reported to have the functions of transporters, such as BCRP and PEPT1, as well as of metabolic enzymes, such as CYP3A4 and CES2 (Iwao et al, 2015;Ogaki et al, 2015;Ozawa et al, 2015;Kodama et al, 2016;Kabeya et al, 2017;Uchida et al, 2017). Unlike Caco-2 cells, hiPSC-IECs show an expression pattern of CES isozymes similar to the human small intestine; CES2 is expressed at levels higher than CES1 (Kabeya et al, 2017).…”

“…This suggests that hiPSC-IECs would be more useful than Caco-2 cells for investigating the intestinal hydrolysis of CES substrates, such as ester prodrugs, but it is unknown whether the hydrolysis in hiPSC-IECs reflects the in vivo function of intestinal hydrolysis. In addition, Kodama et al (2016) demonstrated that the permeabilities across hiPSC-IEC monolayers via passive diffusion were correlated with Fa values in humans, but it has not been clarified whether human intestinal absorption of substrates for transporters can be accurately predicted by using hiPSC-IECs.…”

Intestinal cells differentiated from human induced pluripotent stem cells for use in drug development studies

“…However, pharmacokinetics-related gene expression and pharmacokinetic functions were insufficient using that method [14]. We have since demonstrated that mitogen-activated protein kinase, DNA methyltransferase, and transforming-growth factor-β inhibitors are useful for inducing differentiation to intestinal epithelial cells [15][16][17]. In fact, the differentiated intestinal epithelial cells we generated exhibited a wide range of pharmacokinetic functions, including drug-metabolizing enzyme activities, CYP3A4 inducibility via 1α,25-dihydroxyvitamin D 3 , and transporter activities via PEPT1 and BCRP.…”

Rotational 3D mechanogenomic Turing patterns of human colon Caco-2 cells during differentiation

“…The formation of “rings of cells”/domes are also observed in functional enterocyte epithelium differentiated from human induced pluripotent stem (iPS) cells, which could be used to replace the Caco-2 cell model. Wound healing morphogen FGF2 treatment, the latter inhibits differentiation and prevents the formation of “ring of cells”/dome in Caco-2 cells [8, 41–43]. Knockdown extracellular matrix protein which stimulates differentiation in Caco-2 cells delayed the “ring of cells”/dome formation [44].…”