Optimization of Two-dimensional Feeder and Serum Free IPSC – Small Intestinal Epithelial-like Cell Differentiation Protocol

Abstract

Background and aims: Epithelium of the small intestine is comprised of six different cell types called small intestinal epithelial cells. These cells have functions in selective absorption of nutrients, secretion of mucus and gastric hormones, replenishing the cell population, and as a part of immune system. In celiac disease dietary gluten elicits an autoimmune response, that severely damages the epithelium. Currently the most used in vitro models for intestinal research are tumor-derived cell lines, explants, and primary cells. These methods have problems associated with them, such as poor in vivo resemblance, poor viability in vitro or challenges in availability. The aim for this study was to optimize a protocol to produce small intestinal epithelial cell, especially enterocytes, from human induced pluripotent stem cells (IPSCs). The use of IPSC-based in vitro models allows the creation of patient/disease-specific models and could produce more in vivo -like cells with better availability.

Methods: Two IPSC lines were used in this study. First, we optimized the differentiation of IPSCs into definitive endoderm cells and studied how serum or serum replacements and different protein substrata affect the differentiation. Then three different pre-reported conditions were tested to induce the differentiation of definitive endoderm cells to posterior definitive endoderm. Lastly, we tested two modified versions of earlier reported methods to induce differentiation of small intestinal epithelial cells from posterior definitive endoderm cells. The produced cells were characterized using quantitative polymerase chain reaction and indirect immunofluorescence staining and microscopy.

Results: We found that using serum replacement instead of fetal bovine serum in cell culture medium increased the differentiation efficiency of IPSCs to definitive endoderm. It was also shown that both Geltrex and human laminin 111 are suitable protein substrata for efficient definitive endoderm differentiation. From three tested posterior definitive endoderm differentiation conditions, all three could produce differentiated cells. Testing was continued with two protocols, that used either fibroblast growth factor 2 or glycogen synthase kinase inhibitor LY2090314, as these two options allowed the cells to survive the differentiation. In the last stage both tested medium compositions induced the differentiation of small intestinal epithelial cells, but with different morphologies and epithelial structure.