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N. V. Strunnikova, J. Barb, F. Wang, C. Zhi, A. Maminishkis, J. Hammer, P. J. Munson, S. S. Miller; Analysis of Genetic and Functional Similarities Between Primary Human Fetal RPE (hfRPE) Cultures and Fetal or Adult Native RPE Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):789.
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© ARVO (1962-2015); The Authors (2016-present)
Previously developed primary cultures of hfRPE cells were studied as a model of RPE physiology and as a possible model of retinal disorders. Genetic and functional analysis was carried out to test the hypothesis that cultured human fetal RPE is an adequate model of native RPE cells.
Total RNA was isolated from three adult donors (59-79 yrs), four fetal donors (gestational age 17 weeks), and five primary hfRPE cultures (P1). The gene expression profiling of native adult, fetal and cultured RPE was done using HU133 Plus 2.0 chip from Affymetrix containing 54,675 probes. Data analysis was carried out using the MSL toolbox, GoMiner, and EASE software packages. A set of "RPE signature" genes were defined as those genes up-regulated 10 fold or more compared to the mean expression values of our RPE samples relative to the median gene expression value of the entire Novartis expression database (http://symatlas.gnf.org/ SymAtlas). RT-PCR, western blot and immunocytochemistry were used for validation.
Principal component analysis and hierarchical clustering of gene expression profiles from native and cultured hfRPE shows distinct grouping of cultured hfRPE samples, and donor to donor variation. Similarity between hfRPE and native RPE was evaluated by a novel approach that utilizes the common "gene signature" of the particular cell type. Using this methodology, the set of "RPE signature genes" was determined and includes 214 genes. Analysis of "RPE signature genes" using the GO database identified a set of significantly (p <0.05) overrepresented functional categories such as: "perception of light", "oxidoreductase activity", "pigment biosynthesis", "vitamin A metabolism", "transport." These groups contain 74 genes known to function in native RPE cells (eg, RPE65, RBP1, RBP11, RDH10, RHD11, RGR, CRABP, SLC16, SLC, TYRP1).
Using hfRPE, native RPE, and Novartis expression data we determine a set of RPE specific genes. Analysis of this data set confirmed the presence of a large number of RPE specific "functional markers" described in the literature. This "signature" approach is being used to compare and evaluate other RPE cell models
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