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Alejandro Morales Martinez, Vladimir Khristov, Andrea Li, Ruchi Sharma, Omar Memon, Daniel Riordon, Rebekah Gundry, Kenneth Boheler, Sheldon S Miller, Kapil Bharti; Identification of Surface Proteome for Human Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2336.
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© ARVO (1962-2015); The Authors (2016-present)
The degeneration of the retinal pigment epithelium (RPE) plays a major role in photoreceptor cell death and vision loss in age-related macular degeneration (AMD). Induced pluripotent stem (iPS) cell derived RPE has provided hope for an autologous therapy against AMD. However, the potential presence of undifferentiated cells in transplantable tissues increases the risks of tumor or teratoma formation. Here we propose to identify RPE surface proteome to discover a canonical set of membrane biomarkers that can be used to validate and purify iPS cell derived RPE.
Cell surface capturing (CSC) technology was used to identify N-glycosylated proteins present specifically on the apical and/or basal surfaces of primary human fetal RPE monolayer. Bioinformatics analysis based on in silico gene expression, the number of hits in CSC technology, predicted protein transmembrane domains, and predicted subcellular localization was used to select a sub-set of proteins for further validation. Gene expression was use to determine relative expression of selected genes in iPS cells, iPS cell derived RPE, fibroblasts, and primary RPE. Immunostaining was performed to confirm relative apical or basal localization of selected sub-set of proteins.
1,800 protein tags were identified using CSC technology. Approximately, 200 genes were selected based on the number of predicted transmembrane domains (more than one) and the number of times they were identified in the CSC technology (more than one). These 200 genes were analyzed using EST profiles and in silico subcellular localization, and a list of 61 genes was identified as preferentially localized on the membrane in adult cells. mRNA expression analysis for these 61 genes in iPS cell derived RPE, fibroblasts, hfRPE, and iPS cells identified 36 candidates that were preferentially expressed in iPS cell derived RPE as compared to fibroblasts and iPS cells. Immunostaining on many of these proteins has validated the increased expression in the iPS cell-derived RPE and hfRPE.
We have successfully identified previously known and unknown surface markers on primary human fetal RPE cells. This specific set of proteins is being used as surface markers to validate iPS cell derived RPE and to generate pure population of RPE cells for transplantation in AMD patients. These results will lead to safer and likely more effective cell-based therapy for AMD.
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