Transcriptomics of mouse ileal stem cell-derived epithelium grown on transwells

Purpose: The goal of this study was to identify transcriptional changes that occur in mouse ileal stem cell-derived epithelium plated on transwells after the establishment of an air-liquid interface (ALI). Methods: Mouse ileal stem cell spheroids were plated on transwells and grown in L-WRN conditioned medium (top and bottom compartment) for 7 days, at which point the top medium was removed to establish ALI. Samples were collected for sequencing on the day of ALI initiation (non-ALI Day 0) and three days post top medium removal (ALI Day 3). Control samples were also collected from transwells submerged for all 10 days of the experiment (non-ALI Day 3) and from the initial stem cell spheroid cultures. Results: Non-ALI Day 0 and non-ALI Day 3 samples had similar transcriptional profiles, whereas ALI transwells showed upregulation of transcriptional factors that induce secretory cell lineages, as well as a general upregulation of metabolic genes involved in oxidative phosphorylation and downregulation of those involved in glycolysis. Conclusions: Removal of L-WRN conditioned medium from the top compartment of transwells plated with stem cell-derived intestinal epithelial cells establishes an air-liquid interface that changes the metabolic profile of the cultures and initiates a differentiation cascade for intestinal secretory cells such as goblet cells and paneth cells. mRNA profiles of ileal stem cell-derived epithelium on transwells in an undifferentiated or differentiated state (4 replicates each) or undifferentiated ileal stem cell spheroids as a control (3 replicates)