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

Jaakko Saari,Elina Mäntylä,Kati Juuti-Uusitalo,Teemu O Ihalainen,Katri Lindfors,Katri Kaukinen,Sanna Korpela,Fatima Siddique,Katriina Aalto-Setälä

doi:10.3390/ijms23031312

Jaakko Saari, Elina Mäntylä + Show 7 more

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https://doi.org/10.3390/ijms23031312

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Abstract

The small intestinal epithelium has an important role in nutrition, but also in drug absorption and metabolism. There are a few two-dimensional (2D) patient-derived induced pluripotent stem cell (iPSC)-based intestinal models enabling easy evaluation of transcellular transport. It is known that animal-derived components induce variation in the experimental outcomes. Therefore, we aimed to refine the differentiation protocol by using animal-free components. More specifically, we compared maturation of 2D-cultured iPCSs toward small intestinal epithelial cells when cultured either with or without serum, and either on Geltrex or on animal-free, recombinant laminin-based substrata. Differentiation status was characterized by qPCR, immunofluorescence imaging, and functionality assays. Our data suggest that differentiation toward definitive endoderm is more efficient without serum. Both collagen- and recombinant laminin-based coating supported differentiation of definitive endoderm, posterior definitive endoderm, and small intestinal epithelial cells from iPS-cells equally well. Small intestinal epithelial cells differentiated on recombinant laminin exhibited slightly more enterocyte specific cellular functionality than cells differentiated on Geltrex. Our data suggest that functional small intestinal epithelial cells can be generated from iPSCs in serum-free method on xeno-free substrata. This method is easily converted to an entirely xeno-free method.

Highlights

Proper functionality of small intestinal epithelium is important in nutrition, and in drug absorption and metabolism
We assessed whether fetal bovine serum (FBS) or knock-out serum replacement (KO-SR) were any different in their capacity to induce differentiation of induced pluripotent stem cell (iPSC) toward definitive endoderm (DE)
When the functionality of multi-drug resistance protein 1 (MDR-1) pumps was assessed in calcein retention assay, the iPSC-small intestinal epithelial cell types (SIECs) on laminin evinced clearer retention than iPSC-SIECs on Geltrex. Taken together, these findings suggest that small intestinal epithelial cells with typical small intestinal epithelial gene and protein expression and functionality can be generated when iPSCs are differentiated toward SIEC on recombinant laminin substrata

Summary

Introduction

Proper functionality of small intestinal epithelium is important in nutrition, and in drug absorption and metabolism. Cell-based intestinal models, an alternative to animal models, have been widely utilized to investigate human small intestine functionality and in drug screening studies [1,2]. From among the currently used models, Caco-2, a colonic adenocarcinoma-derived immortal cell line, has been the gold standard for intestinal research for decades [3,4]. Caco-2 has been valuable in high throughput screening of drug permeability and the identification of various crucial transporters, inhibitors, and inducers of the small intestinal epithelium [5]. The Caco-2 cell line displays major limitations in the expression of some transporters such as peptide transporter 1 (PEPT1), and drug metabolizing enzymes such as cytochrome P450 CYP3A4, in comparison to intestinal mucosa in vivo [3]

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