The effects of inflammatory cytokines on iPSC-derived small intestinal epithelial cells

Abstract

Background: Celiac disease (CD) is an autoimmune disease that commonly primarily affects the small intestinal epithelium of the genetically predisposed individuals, triggered by dietary gluten. Up to date, the only treatment option is a life-long gluten free diet. Celiac disease research is typically conducted using short lived intestinal biopsies or intestinal in vitro models that harness either non-CD patient -derived immortalized cell lines or CD patient intestinal biopsy -derived small intestinal stem cells. While both models offer advantages, they are both restricted to epithelial lining cell types, among other constraints. Celiac disease is not only limited to intestinal epithelial lining. Thus, there is a need for a celiac disease -specific in vitro model that could later be elaborated to autologous multiculture, to understand the onset and progression of celiac disease, for the eventual goal of additional treatment options. Aims: The aim of this study was to validate the functionality of CD patient -specific induced pluripotent stem cell (iPSC) -derived small intestinal epithelial cells (SIECs) with two well characterized inflammatory cytokines active in CD, namely TNF-α and IFN-γ, on a 2-dimensional, apically and basally easily accessible transwell based intestinal in vitro model. Methods: Two iPSC lines, healthy control and CD patient, were differentiated and matured on planar surface on transwell inserts for a 2-dimensional intestinal in vitro model. The iPSCs were differentiated towards SIECs according to the in-house optimized differentiation protocol. The mature cell cultures were then exposed to cytokines, TNF-α or IFN-γ, for 48 hours. The effects of the cytokines were assessed with multiple functionality assays; Transepithelial resistance (TEER) measurements and FD4-assay were conducted to assess the changes in paracellular permeability. AMCA-assay was conducted to assess the changes in dipeptide transporter PEPT1 uptake activity. Lactate dehydrogenase (LDH) assay was conducted to observe overall cytotoxicity of the cytokines. Immunofluorescence staining and confocal microscopy were utilized for assessing the cells’ differentiation status and changes in overall morphology. RNA was extracted for further RNA sequencing analysis. For a baseline reference, Caco-2, which is a well-validated commercial cell line for intestinal in vitro models, underwent similar cytokine inductions, characterization and functionality testing. Results and conclusions: The results of this study indicated that the cytokines, TNF-α and IFN-γ, affect the cells in a cell type and cell line -specific manner, although similarities also exist; The cytokines seem to elevate PEPT1 activity. The results on the cytotoxicity were in turn more controversial and cell-line dependent. The differentiation and maturation of the iPSC-SIECs strongly varied between replicates. The results obtained on iPSC-SIECs were not corresponding with the results obtained on Caco-2, which was foreseeable based on existing literature. Further optimization to the iPSC-SIEC differentiation protocol is still needed to minimize the heterogeneity between cell lines and replicates. This study serves a great basis for upcoming research, with the eventual goal of creating a physiologically relevant intestinal in vitro model for celiac disease research.