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Bruce A Pfeffer, Chengjian Tu, Jun Li, Jun Qu, Steven J Fliesler; Comparative proteomic profiling of cultured RPE cells derived from human embryonic stem cells vs. rhesus monkey. Invest. Ophthalmol. Vis. Sci. 2016;57(12):232.
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© 2017 Association for Research in Vision and Ophthalmology.
Cultured retinal pigment epithelial (RPE) cells from non-human primate, and those derived from human embryonic stem cells (hesc), each with their own strategic and translational merits and deficiencies, are potential substitutes for RPE cell lines and cultures established from human donor eyes. A fair consensus with regard to human RPE signature gene and protein expression has emerged from analysis of native and cultured RPE in published reports. We generated proteomic profiles to compare cultured hesc-derived and rhesus macaque RPE cells in terms of correlation with human RPE signature genes and proteins.
Human (hesc-derived) and rhesus RPE cells (4th and 3rd passage, respectively) were maintained as stable, confluent monolayers for 8 weeks. Both strains displayed a polarized, pigmented epithelial phenotype in vitro. Triplicate sets of cell lysates were subjected to on-pellet proteolytic digestion and separations by nano-reverse-phase LC/ion-current-based tandem mass spectroscopy [Tu et al., Mol Cell Proteomics 2013]. Identifications from the UniProt human protein database were made on the basis of at least two unique peptides for each protein. Proteins detected were quantified by AUC using SIEVE, and ranked by relative abundance.
Approximately 1100 – 1300 proteins per sample were identified. Intensity vs. retention time plots showed consistent alignment for replicates within each species (87.6% and 95.1% for hesc- and rhesus RPE, respectively), but only a mean of 41.3% between species. For the 110 most abundant proteins detected in either hesc- or rhesus RPE, 70.0% vs. 77.3% of these overlapped with the other subset, respectively. Of the 50 most abundant proteins detected, 35 from either hesc-derived or rhesus cultured RPE corresponded to an entry from at least one comprehensive, recently published list of human RPE signature genes or proteins [Strunnikova et al., Hum Mol Genet 2010; West et al., Mol Cell Proteomics 2003]. Cytoskeletal, retinoid-processing, ion and small molecule transport, and antioxidant/pro-survival functional categories were highly represented for both species. RPE65, an exemplary signature protein for native RPE, was detected in both hesc- and rhesus RPE, with comparative normalized abundance values of 0.041 and 0.350, respectively.
Proteomic profiling may be used to authenticate cultured hesc-RPE and non-human primate RPE.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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