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Jason Silver, Daniel Riordon, Fang Hua, Omar Memon, Arvydas Maminishkis, Rebekah Gundry, Sheldon Miller, Kenneth Boheler, Kapil Bharti; A Surface Proteome of Primary Fetal Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6092. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) cells have been successfully differentiated from pluripotent stem cells. Because the efficiency of differentiation is not 100%, several non-RPE cells are present in these differentiation cultures. Impure cultures of RPE will likely not provide an efficacious cell-based therapy and may not be safe for transplantation. The aim of this study is identify a canonical set of RPE-specific surface markers that can be used to further purify RPE cells differentiated from stem cells.
Primary fetal RPE cells obtained from 16-18 week fetuses were grown as polarized, confluent monolayers for 6-8 weeks. Extracellular, N-linked glycoproteins were labeled and captured using the Cell Surface Capturing (CSC) Technology. CSC technology uses meta-sodium periodate to oxidize sugars on intact cells followed by covalent chemical labeling of oxidized carbohydrate-containing proteins with biotin for affinity enrichment. After biotinylation, cells are lysed, membrane fractions isolated, peptides released by trypsin, and Biotin labeled peptides captured and subjected to mass spectrometry to unequivocally identify labeled proteins based on peptide sequences.
To validate that the CSC technique works well on RPE cells, we initially looked for the presence of previously identified surface proteins. Our analysis identified several previously known proteins including aquaporin-1, interferon gamma receptor 1, insulin like growth factor 1 receptor, tyrosinase protein kinase MER, membrane frizzled related protein, sodium bicarbonate cotransporter 3, platelet-derived growth factor receptor beta, and transferrin receptor 1. In addition, we have identified previously unreported members of the ATP-dependent transporter family, several CD receptors, integrins, ephrins, sodium/potassium transporting ATPase subunits, and members of the cadherin super-family present on RPE cells.
Cell Surface Capturing successfully identified previously known and unknown surface markers on primary fetal human RPE cells. In addition, the newly identified cell surface proteins provide a basis for the elucidation of previously unrecognized signaling pathways that could mediate significant RPE functions and pathology.
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