Abstract
Abstract: :
Purpose: To determine the expression profiles of RPE cells grown on different matrix proteins with morphologically normal RPE cells in vivo. Method: Morphologically normal RPE cells overlying nonthickened Bruch’s membrane (n=5000 cells) from 3 eyes (ages 63, 71, 74) were laser capture microdissected (LCM). ARPE-19 cells were seeded on plastic, Matrigel, laminin, collagen I, collagen IV, and fibronectin at high density, grown for 7 days in DMEM/F12+10%FBS, then DMEM/F12+1%BSA for an additional 3 days. The RNA from 5000 cells was extracted by column purification, and first and second strand cDNA probes were radiolabeled with 33P-dATP and 33P-dCTP. The radiolabeled probes were hybridized to ResGen’s known human membrane array (n=4325 genes), and signal was visualized by phosphorimager analysis. The TIFF images were analyzed by ResGen’s Pathways 3 software. Experiments were repeated twice (n=3) for each condition. The data were normalized to a mean signal reference array, and subsequently analyzed using Cluster/Treeview and SAM. Real time PCR was used to confirm differential expression of selected genes. Results: 69% of 165 genes that were identified from a recent RPE specific library were expressed by laser captured cells on this array while a range from 63 to 99% of these genes were expressed by RPE cells grown on different matrices. Unsupervised cluster analysis revealed that RPE cells grown on plastic clustered closest to laser captured cells, followed by cells grown on collagen 1, Matrigel, then collagen IV, fibronectin, and laminin. The data set was subjected to 2 class unpaired SAM analysis (LCM cells versus in vitro cells). With a false discovery rate of 5% and 2 fold differential expression, 950 genes had differential expression between LCM and in vitro cells. Supervised cluster analysis using this data set showed a similar cluster pattern. Genes with identified roles in cell differentiation (n=510) and cell-matrix interaction (n=308) were then cluster analyzed. Both of these supervised approaches also showed that cells grown on plastic had the closest expression profile to morphologically normal RPE cells. Conclusions: RPE cells grown on plastic showed an expression signature that was most similar to morphologically normal RPE cells that were laser capture microdissected. This approach will be useful in establishing the gene clusters that are important in determining the effect of cell-matrix interaction on cell differentiation of the RPE cell.
Keywords: extracellular matrix • gene microarray • laser