Abstract
Recent progress in mammalian intestinal epithelial cell culture led to novel concepts of tissue modeling. Especially the development of phenotypically stable cell lines from individual animals enables an investigation of distinct intestinal loci and disease states. We here report primary and prolonged culture of normal porcine epithelial cells from colon for cell line development. In addition, a novel primary three-dimensional intestinal culture system is presented, which generated organoids composed of a highly polarized epithelial layer lining a core of subepithelial tissue. Cellular characterization of monolayer cell lines revealed epithelial identity and pointed to a proliferative crypt cell phenotype. We evaluated both RNAi and chemical approaches to induce epithelial differentiation in generated cell lines by targeting promoters of epithelial to mesenchymal transition (EMT). By in silico prediction and ectopic expression, miR-147b was proven to be a potent trigger of intestinal epithelial cell differentiation. Our results outline an approach to generate phenotypically stable cell lines expanded from primary colonic epithelial cultures and demonstrate the relevance of miR-147b and chemical inhibitors for promoting epithelial differentiation features.
Highlights
Of either ectopic miRNA expression or inhibition can promote intestinal epithelial differentiation in vitro
Cell culture generation from large intestine is of particular interest for disease-related or toxicological studies, as the colon is most susceptible to cancer or inflammatory diseases
The presented protocol has the advantage that only a limited number of animals is required for primary culture generation, which is successively expanded for functional approaches
Summary
Of either ectopic miRNA expression or inhibition can promote intestinal epithelial differentiation in vitro. Small molecule inhibitors (SMI) have been shown to counteract TGF-β 1 signaling antagonizing mesenchymal transition of renal tubular epithelial cells[13]. To address these issues, we aimed at developing a large intestinal epithelial cell culture system combining features such as low-passage numbers, sustained proliferative capacity and absence of transformation. We hypothesized that a series of successive enzymatic digestion steps of intestinal tissue can systematically produce epithelial cell populations from deeper crypt regions that harbor proliferative progenitor and stem cells
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