GM1 expression in caco-2 cells: characterisation of a fundamental passage-dependent transformation of a cell line.

Caco-2 cells, which are known to spontaneously differentiate in cell culture, adopt typical epithelial characteristics and are widely used as a model to study cellular uptake, transport and metabolism processes. However, groups of flat and undifferentiated cells have been observed amid differentiating Caco-2 cell monolayers. In this study, we isolated and characterised these morphologically distinct, flat and island-forming Caco-2 cells. We visualised the undifferentiated cell islands with the aid of optical and electron microscopy and identified mono-sialo-ganglioside one (GM1) as their unique marker. Furthermore, two distinct subpopulations of morphology and GM1 expression were dilution cloned (Caco-2(GM1-) and Caco-2(GM1+) ), leading to the first documented Caco-2 clone that does not show differentiation characteristics. Caco-2(GM1+) cells were flat, non-polarising with extremely low transepithelial electrical resistance (TEER), whereas Caco-2(GM1-) cells showed typical epithelial features and high TEER. Importantly, the proportion of Caco-2(GM1+) cells in a culture increased with passage number and eventually dominated the cell culture. The novel GM1 passage-dependent cell transformation described here shows that careful monitoring is required when performing in vitro cell studies. Therefore, to guarantee consistent and valid experimental data, GM1 expression and the loss of differentiation characteristics should be carefully monitored and the use of fresh cultures should be standard practice.