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F. Wang, C. Zhi, J. Hammer, A. Maminishkis, S.S. Miller; Expression Profiles of Primary Human Fetal Retinal Pigment Epithelial (hfRPE) Cells Cultured on Flask and Transwell . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2855.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously developed and physiologically characterized a human fetal RPE primary culture system (Maminishkis, et al., ARVO, 2005). Here we examined the expression profiles of RPE cells grown on Primaria flask and human extracellular matrix coated transwells. We also determined the expression of proteins involved in the visual cycle, in melanin pigment synthesis and in the formation of the junctional complexes between cells.
Total RNA was extracted from four to eight week–old primary cultures of human fetal RPE and reverse transcribed. The biotinylated cRNA was hybridized to an Affymetrix Human Genome U133 Plus 2.0 Array containing 38,500 genes. The normalized gene expression levels were compared between cells grown on flask and transwell. Real–time RT–PCR was used to corroborate some of the differentially expressed genes. Immunofluorescence labeling was used to localize proteins involved in the visual cycle and the formation of junctional complexes.
Three key enzymes in melanin synthesis, silver homolog, tyrosinase related protein 1, and glycoprotein nmb, were expresses at high levels (in abundance all ranked in the top 100 transcripts) in cultured hfRPE. Retinol dehydrogenases 11, 5 and 8 were also highly expressed, as well as retinol binding protein 1, retinaldehyde binding protein 1 (CRALBP), and RPE65 (all ranked in top 2000 transcripts). Immunofluorescence labeling confirmed the intracellular localization of CRALBP and RPE65. Claudin 19 and ZO–1 message are expressed at a higher level than claudin 10 and occludin. Claudin 19, ZO–1, occludin and claudin 10 are all clearly co–localized on the junctional complexes. RPE cells grown on transwells have approximately 80 genes with lower expression levels and approximately 75 genes with higher expression level compared to cells grown on Primaria flasks. Genes with lower expression levels include K dependent Na/Ca exchangers, sodium bicarbonate cotransporters, K channels. Genes with higher expression levels include collagens, metallothioneins, decorins.
This hfRPE primary culture system expresses high levels of critical enzymes and proteins required for a host of RPE functions such as melanin synthesis, visual cycle activity, and the formation of junctional complexes. The value of this model system will be determined by its ability to mimic the normal physiology, function and structure of native RPE as well as its ability to serve as a vessel for studies of human pathophysiology.
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