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

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.

iPSC-derived cells for whole liver bioengineering

“…Firstly, the use of complex supplements (e.g., KOSR) and coating solutions (e.g., Matrigel and Geltrex) simplifies cell culture routines and reduces experimental costs. However, these may compromise the precise tuning of cell fate determination–while complex supplements exhibit batch-to-batch variation ( Bushweller et al, 2022 ) -, and animal-derived reagents reduce the efficiency of hPSCs differentiation ( Saari et al, 2022 ). Secondly, classical monolayer (2D) cell culture and, consequently, hPSC differentiation inadequately mimic embryonic and tissue development, as gene regulation and morphogenesis critically depend on cell-cell and cell-ECM three-dimensional interactions ( Jensen and Teng, 2020 ).…”

Differentiating Human Pluripotent Stem Cells to Cardiomyocytes Using Purified Extracellular Matrix Proteins

“…While collagens used in complex, 3D scaffold or other synthetic fibrous matrix systems have been found supportive of CM differentiation and growth [ 69 , 70 , 71 ] and even maturation [ 72 ], and 2D, plate-based collagen coatings have been found supportive of iPSC differentiation into non-CM cell types [ 73 , 74 , 75 , 76 ], less has been reported on using 2D, plate-based collagen coatings to support CM differentiation specifically [ 4 , 5 ]. These limited, CM-focused, plate-based studies will be explored in the remainder of this section.…”