Establishment of a novel culture method for maintaining intestinal stem cells derived from human induced pluripotent stem cells

Abstract

The small intestine plays an important role in the pharmacokinetics of orally administered drugs due to the presence of drug transporters and drug-metabolizing enzymes. However, few appropriate methods exist to investigate intestinal pharmacokinetics. Induced pluripotent stem (iPS) cells can form various types of cells and represent a potentially useful tool for drug discovery. We previously reported that differentiated enterocytes from human iPS cells are useful for pharmacokinetic studies; however, the process is time and resource intensive. Here, we established a new two-dimensional culture method for maintaining human iPS-cell-derived intestinal stem cells (ISCs) with differentiation potency and evaluated their ability to differentiate into enterocytes exhibiting appropriate pharmacokinetic function. The culture method used several factors to activate signalling pathways required for maintaining stemness, followed by differentiation into enterocytes. Functional evaluation was carried out to verify epithelial-marker expression and inducibility and activity of metabolic enzymes and transporters. Our results confirmed the establishment of an ISC culture method for maintaining stemness and verified that the differentiated enterocytes from the maintained ISCs demonstrated proper pharmacokinetic function. Thus, our findings describe a time-and cost-effective approach that can be used as a general evaluation tool for evaluating intestinal pharmacokinetics.

Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells

“…Efflux transporters such as multi-drug resistance protein 1 (MDR-1)/P-glycoprotein (P-gp), and multi-drug transport protein (MRP1) are abundantly expressed in intestinal enterocytes [26,27] and serve to protect cells against xenobiotics [22]. Thus, in the final step we studied the efflux pump functionality in the iPSC-SIECs differentiated and cultured on Geltrex or laminin, which was performed with Calcein retention assay.…”

“…In addition to the organoids, induced pluripotent stem cells (iPSCs) can be cultured in 2D and differentiated to generate all small intestinal epithelial cell types (SIECs) [17][18][19][20][21][22]. In 2D cultures, the cells are grown on flat porous surface, which allows for the handling of the culture both from apical and basal sides of the cells.…”

“…In addition, the possible genetic disease predispositions are present in the resulting SIECSs and allow for studying of the possible effects of genetic factors in the function of the iPSC-differentiated cells in vitro. All published methods are based on the developmental pathway of embryo-and organogenesis in vitro starting from the establishment of definitive endoderm (DE), followed by posterior definitive endoderm (PDE) induction, and finally differentiating cells toward SIECs [17][18][19][20][21][22].…”

In Vitro Human Cell–Based Experimental Models for the Evaluation of Enteric Metabolism and Drug Interaction Potential of Drugs and Natural Products

“…Technological advancement continues to be made to improve the efficiency of the differentiation of iPSCs to various differentiated cell types including three-dimensional beating cardiomyocytes (Sasano et al, 2020), hepatocytes (Takayama et al, 2018), neurons (Cheng et al, 2017), and threedimensional kidney organoids (Takasato et al, 2016). Successful differentiation of iPSCs into enterocytes has also been reported (Iwao et al, 2014(Iwao et al, , 2015Negoro et al, 2016;Blutt et al, 2017;Nadkarni et al, 2017;Uchida et al, 2017;Kondo et al, 2018;Macedo et al, 2018;Lees et al, 2019;Kondo et al, 2020), including three-dimensional enterocyte organoids (Onozato et al, 2018). The efficiency of enterocyte differentiation from human iPSCs was significantly improved by advancements in culture medium formulation and differentiation protocol (Ozawa et al, 2015).…”

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

“…Because it is difficult to obtain and culture primary human IECs, many researchers have tried to establish a differentiation method for generating IECs from hiPSCs. [1][2][3][4][5][6][7] Although the application of hiPSC-derived intestinal epithelial cells (hiPSC-IECs) to pharmacokinetic studies has been attempted, these cells have not been sufficiently applied to regenerative medicine.…”