CELL-DYNAMICS AND DIFFERENTIATION OF CONDITIONALLY IMMORTALIZED HUMANINTESTINAL EPITHELIAL-CELLS

Background & Aims: Intestinal epithelial cell lines capable of differe ntiating in monolayer culture have been difficult to obtain, prompting the use of novel approaches including immortalization with oncogenes or tumor viruses, The aim of this study was to obtain conditionally im mortalized human intestinal epithelial cells (temperature-sensitive fe tal human intestinal [tsFHI] cells) and study their growth and differe ntiation capabilities. Methods: Intestinal cells from human fetuses we re transformed with a temperature-sensitive simian virus 40 large tumo r antigen, cloned, and screened for expression of intestinal markers. Results: Establishment of tsFHI cells was crucially dependent on a gro wth medium based on OptiMEM. At 32 degrees C, the tsFHI cells prolifer ate and display crypt cell markers, Only a few scattered differentiate d cells are observed together with cells displaying the morphological signs of apoptosis, A shift to 39 degrees C results in irreversible gr owth arrest and acquisition of an enterocyte-like phenotype. Expressio n of the cyclin-dependent kinase inhibitor p21/WAF1/Cip1 is induced wi thin hours of a shift to 39 degrees C 1-2 days before corresponding in creases in brush border enzymes, suggesting a role in the induction ra ther than in the maintenance of a differentiated phenotype. Conclusion s: The tsFHI cells recapitulate key aspects of intestinal cell dynamic s in vitro and seem particularly suited for the study of early events in cell differentiation.