RPE has been derived both from human embryonic and induced pluripotent stem cells, and is currently being used to develop in vitro disease models for retinal degenerative diseases and for a potential cell-based therapy. However, a thorough molecular and physiological characterization of ES or iPS cell derived RPE has not been performed. Furthermore, iPS cells can be derived from any somatic tissue using a number of different methods that involve viruses or integration of pluripotency factors into the genome. It is not clear how variable RPE is when differentiated from iPS cells that are derived from different tissue sources using different reprograming methods. To address some of these questions, in collaboration with Sally Temple (NYSCF), we generated iPS cells from genetically identical but epigenetically distinct tissue sources using lentiviral (integrative) or sendai-viral (integration-free) technologies. RPE cells were differentiated from several of these iPS cells lines (8 different iPS cell lines;16 clones) using published methods and analyzed for thorough molecular and functional authentication. Our characterization includes immunostaining for a set of RPE-specific genes, gene expression (mRNA &miRNA) analysis of RPE-signature genes and RPE-enriched miRNAs, electron microscopy, physiological measurements (transepithelial resistance, transepithelial potential, intracellular pH and calcium recordings, fluid transport, polarized cytokines secretion, and membrane properties of polarized RPE monolayers). Our preliminary results suggest that iPS cell derived RPE exhibit several key features of primary RPE cells including expression of key genes, vectorial secretion of key cytokines, transepithelial resistance and potential, ability to regulate potassium concentration in the apical bath, and ability to transport fluid from apical to basal sides of the tissue. RPE derived from multiple different iPS cell lines exhibit these properties, but some lines show variation when tested with these assays. When completed, this study will likely provide a basis for functional variation seen in RPE derived from multiple different iPS cell lines. It will likely also lead to a set of assays that can be used to authenticate RPE derived from any pluripotent stem cell source and improved release criteria for clinical grade RPE derived from iPS cells.
|Ou, Jingxing; Bharti, Kapil; Nodari, Alessandro et al. (2013) Vax1/2 genes counteract Mitf-induced respecification of the retinal pigment epithelium. PLoS One 8:e59247|