107 TGF-β IS RESPONSIBLE FOR THE FIBROBLAST-INDUCED DIFFERENTIATION OF THE INTESTINAL EPITHELIAL CELL LINE T84

Abstract

The small bowel mucosal crypt-villus axis is an interesting model for epithelial cell biology. There has been a need for an in vitro method where crypt-like gut epithelial cells differentiate to absorptive enterocytes. T84 (ATCC CCL 248) is a human intestinal epithelial cell line with crypt-like secretory functions. When T84 cells were cultured three-dimensionally within type I collagen gels, cell clusters showing no organised orientation were formed. When growing T84 cells with fibroblasts (IMR-90, ATCC CCL 186), but separated with a cell-free gel, 76% of the T84 colonies were organised to vesicles. Two types of vesicles rounded either with a one-cell layer of cuboidal (32% of the total colony counts) or columnar (44% of the total colony counts) cells were formed. Electron microscopy and histochemical analyses revealed that 77% of the cells in the columnar type of vesicles were highly differentiated showing well formed microvilli with high alkaline phosphatase activity. When T84 cells were cultured three-dimensionally and feeded with culture medium containing TGF-β (20 μg/ml) 56% of the colonies were organised to vesicles with highly differentiated columnar type cells and 44% remained unorganised. The fibroblast-induced differentiation of the T84 cells was due to TGF-β as neutralising antibodies against human TGF-β and against the latent form of TGF-β abolished the formation of the differentiated type of vesicles. Hepatocyte growth factor was not a morphogen for T84 cells. We conclude that organisation and differentiation of crypt-like T84 intestinal epithelial cells to absorptive enterocytes cells in the present three-dimensional co-culture is induced by fibroblast secretion products, especially TGF-β. The present in vitro model may prove to be the method for studying the gene products that regulate the extracellular matrix-mesenchymal cell-epithelial cell cross-talk occurring in the human jejunum on the mucosal crypt-villus axis, where the chloride-secreting crypt cells proliferate and differentiate to absorptive villus tip enterocytes.