BULK RNAseq - SV40 large T antigen immortalization of primary mouse intestinal epithelial stem and progenitor cells

Developing new tools for studying the intestinal epithelium is essential for addressing unan-swered questions in development, physiology, and disease pathology. Primary intestinal epitheli-al stem cell (ISC) cultures are an invaluable in vitro model system. However, their cultivation re-mains technically demanding and costly, limiting their accessibility and use. Though commonly utilized, most extant intestinal epithelial cell lines were derived from colon adenomas from vari-ous animals, are largely uncharacterized, and possess a plethora of genetic abnormalities, thus restricting rigor, reproducibility, and physiological relevance. Here we derived immortalized intestinal epithelial cell lines (iIECs) from six jejunal and six colonic stem cell-enriched spheroid cultures from wild type C57BL/6 mice. We transduced ISCs with a lentiviral vector encoding the SV40 large T antigen, and subsequently selected lines through adaptation to growth on plastic. Additionally, we weaned these lines off conditioned medium including the growth factors Wnt3a, Rspo3, and Noggin. We found that iIECs have growth characteristics similar to conventional plastic adapted cell lines. While we observed transcriptional signatures of epithelial-mesenchymal transition (EMT), these cells still exhibited epithelial characteristics including ex-pression of junctional proteins, mounted an innate immune response to viral infection, and se-creted cytokines in response to LPS stimulation. We further demonstrated the utility of iIECs through transient and stable genetic manipulation. iIECs occupy an important experimental niche through offering a scalable, practical, and physiologically relevant model system that can func-tion as a discovery platform before transitioning to primary spheroid cultures and/or animal models. We propose that they will be a valuable tool for advancing understanding of intestinal epithe-lial biology.