TGFbeta- and IMR induced epithelial cell differentiation in a three-dimensional intestinal crypt-villus axis model

The three-dimensional epithelial differentiation model, in which T84 epithelial cells are induced to differentiate either with TGFbeta1 or IMR-90 mesenchymal cell-secreted soluble factors, is previously shown to model differentiation seen in jejunal crypt-villus axis (Halttunen et al. 1996). The gene expression changes were studied in fully undifferentiated and fully differentiated three-dimensional T84 cultures with cDNA microarray method. Keywords: developmental stage Human intestinal epithelial T84 cells (CCL 2´48, ATCC Rockville, MD, USA) were cultured in three-dimensional type I collagen gel as previously described Halttunen et al 1996 . T84 cells were induced to differentiate either by adding 20 ng/ml human recombinant TGFbeta1 (hTGF-beta1, R&D Systems Europe, Oxon, UK) or by soluble factors secreted by IMR-90 type human embryonic lung fibroblasts (CCL 186, ATCC). IMR fibroblasts, cultured on the top of the epithelial cells, were separated from the epithelial cells by cell-free collagen layer. T84 cells cultured within collagen gel supplemented with medium were used as undifferentiated control. We studied only fully undifferentiated and fully differentiated epithelial cells harvested after seven days of culturing. All experiments were carried out in triplicate. Isolation of RNA:The mRNA was extracted from the cell culture samples to ice-cold TRIzol reagent (Life Technologies, Inc. Frederick, MD, USA) according to the manufacturer’s protocol. All samples were subjected to DNAse I treatment (Roche Diagnostics GmbH, Mannheim, Germany). Total RNA was quantitated by spectrophotometry and quality checked by agarose gel electrophoresis. cDNA synthesis and array hybridization Gene expression was monitored using a Human GeneFilter GF200 (Research Genetics, Huntsville, AL, USA), consisting of 5188 test sequences, 96 control points and 192 housekeeping genes. Arrayed sequences contained both genes with known or predicted function and expressed sequence tags (ESTs) with unknown function. Probe preparation and microarray hybridization were performed following manufacturer’s (Research Genetics) protocol using 1.5 microg total RNA as template, 10microl (10mCi/ml) 33P dCTP (ICN Radiochemicals), 1,5microl dNTP mix containing dATP, dTTP, dGTP at 20 mM (Pharmacia ), 1,5 microl reverse transcriptase (Supercript II, Life Technologies), 1,0 microl DTT. Elongation for 90 min at 37oC. The label was purified with Bio-Spin 6 Chromatography column (Bio-Rad), and hybridized for 12 h at 42oC in roller bottle. After hybridization membrane was washed twice in 2 X SSC 1%SDS, 0.5XSSC. The hybridized membrane was let to expose Phosphoimager screens for 12 h. Hybridization signals were detected on a Storm 860 phosphoimager (Molecular Dynamics, Amersham Biosciences, Buckinghamshire, England) . Microarray data analysis:Filter images were aligned and spot intensities analysed with Pathways Software (Research Genetics). Raw data were imported to Microsoft Excel. The handling of the raw data, normalization and improvement of fidelity by setting a cut-off value were done as previously described in Juuti-Uusitalo et al. 2004. Intensity values were normalised in order to avoid possible differences in the amounts of RNA and differences in hybridisation efficiency. The normalisation factor was determined before background subtraction. The normalisation was done by the sum method (Kroll et al. 2002). The scaling factor was set to ensure that the sums of spot intensities were equal for all filters.