Abstract
BackgroundIEC-18 cells are a non-transformed, immortal cell line derived from juvenile rat ileal crypt cells. They may have experimental advantages over tumor-derived gastrointestinal lineages, including preservation of phenotype, normal endocrine responses and retention of differentiation potential. However, their proclivity for spontaneous differentiation / transformation may be stereotypical and could represent a more profound experimental confounder than previously realized. We hypothesized that IEC-18 cells spontaneously diverge towards a uniform mixture of epigenetic fates, with corresponding phenotypes, rather than persist as a single progenitor lineage.ResultsIEC-18 cells were cultured for 72 hours in serum free media (SFM), with and without various insulin-like growth factor agonists to differentially boost the basal rate of proliferation. A strategy was employed to identify constitutive genes as markers of divergent fates through gene array analysis by cross-referencing fold-change trends for individual genes against crypt cell abundance in each treatment. We then confirmed the cell-specific phenotype by immunolocalization of proteins corresponding to those genes. The majority of IEC-18 cells in SFM alone had a loss in expression of the adenomatous polyposis coli (APC) gene at the mRNA and protein levels, consistent with adenoma-like transformation. In addition, a small subset of cells expressed the serotonin receptor 2A gene and had neuroendocrine-like morphology.ConclusionsIEC-18 cells commonly undergo a change in cell fate prior to reaching confluence. The most common fate switch that we were able to detect correlates with a down regulation of the APC gene and transformation into an adenoma-like phenotype.
Highlights
IEC-18 cells are a non-transformed, immortal cell line derived from juvenile rat ileal crypt cells
We note that anti-amino IGF binding protein-2 (IGFBP-2) showed no staining in IEC-18 cells and that pretreatment with synthetic antigen abolished C2 staining, confirming that C2 is a carboxyl fragment of IGFBP-2
To further characterize the observed heterotypy, we examined IEC-18 cells with anti-actin antibody in IEC-18 cells without performing antigen retrieval and allowing the detection reaction to proceed until only a limited amount of staining is seen
Summary
IEC-18 cells are a non-transformed, immortal cell line derived from juvenile rat ileal crypt cells They may have experimental advantages over tumor-derived gastrointestinal lineages, including preservation of phenotype, normal endocrine responses and retention of differentiation potential. As we have worked with this lineage over the years, the rare but unmistakable morphology of an occasional neuroendocrine-like cell, the suspicious frothiness of a possible paneth-like cell and the rare giant vacuole of a goblet cell precursor have prompted us to wonder how prevalent spontaneous differentiation towards other lineages and/or transformants might be (especially in serum-restricted conditions) This is a fairly crucial experimental question because serum-restricted conditions allow the tightest experimental rigor but may drive IECs towards altered cell fates, potentially introducing uncontrolled experimental confounders. Our current investigation uses a novel strategy to demonstrate that spontaneous cell fate divergence is highly prevalent in IEC-18 cells, but it is fairly uniform and a predictable confounder for gene array studies in this cell line
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