Abstract
Purpose: :
RPE damage or dysfunction can lead to severe visual loss in many ocular disorders such as Leber's congenital amaurosis and AMD. Differentiation of embryonic stem cells into RPE may become a useful therapeutic modality for selected individuals. The purpose of this study is to identify genes critical to RPE development, and to develop a database of the sequential gene expression that occurs within RPE prior to inducing stem cells to differentiate into RPE.
Methods: :
Total RNA was isolated from C57BL/6 mice RPE at embryonic days 8 and 11 (E8, E11), and post-natal days 5, 8, 12, 45 (P5, P8, P12 and P45) using Invitrogen’s RNAEasy kit. Affymetrix microarray 430A 2.0 chips (mouse genome) were used to probe RPE gene expression levels at these different developmental stages. Genes were identified as being transcription factors or as factors that were important in RPE development using Gene Ontology molecular and biological function data, respectively. Genesifter and several other microarray analysis software systems were used to identify transcription factors whose time course was similar to the time course of the known RPE transcription factor MITF and other genes peaked at different developmental stages. Expression constructs of selected genes were made and delivered into embryonic stem cells. RPE markers, including bestrophin and CRALBP, were used to monitor cell differentiation.
Results: :
Cluster analysis demonstrated one group of genes with a similar expression pattern that were enriched for visual perception with a p-value of 10-16, for phototransduction with p-value of 0.0002, and for RPE development at p =0 .0043. These genes share a peak expression level at time point P5, which was previously characterized as crucial to retinal development. Genes in this cluster include PDE6B, Nr2E3, RPB3, NeuroD1, RARG, Recoverin, Mitf and Rrh, which are known to play important roles in RPE development. Transfection of selected genes including RARG and Mitf induced phenotypical changes in stem cells demonstrated by positive immunocytochemical staining for bestrophin.
Conclusions: :
Mouse RPE gene expression patterns at different embryonic and postnatal stages reveal transcription factors important for retina development. Artificially introducing MITF and RARG into stem cells may direct pluripotent stem cells to differentiate into RPE cells.
Keywords: retinal pigment epithelium • age-related macular degeneration • gene transfer/gene therapy