Identification of a Canonical Set of Biomarkers to Obtain Pure Retinal Pigment Epithelial Cells from Differentiated Induced Pluripotent Stem Cells

Abstract

Purpose: Photoreceptor cell death and vision loss in age-related macular degeneration (AMD) is associated with degeneration of the retinal pigment epithelium (RPE). Differentiation of induced pluripotent stem (iPS) cells into RPE has provided hope for an autologous therapy against AMD. Current methods however do not achieve 100% differentiation efficiency. Presence of undifferentiated cells in transplanted tissue increases the risk of tumor or teratoma formation. Identifying a canonical set of surface proteins will allow for purification of iPS cell-derived RPE, thus providing a safer, well-characterized cell-based therapy for AMD.

Methods: Cell surface capturing (CSC) technology was performed to identify N-glycosylated proteins present on the apical and/or basal surfaces of RPE cells. Bioinformatics analysis based on the number of predicted transmembrane domains, the number of hits in CSC technology, and mRNA expression allowed for selection of a sub-set of proteins for further analysis. Immunostaining performed on human fetal RPE, fibroblasts, and iPS cells validated several surface proteins as unique to RPE.

Results: CSC technology yielded over 1,800 proteins expressed on the apical and/or basal surfaces of RPE. Bioinformatics analysis selected for 26 proteins of particular interest. Ingenuity Pathway Analysis (IPA) indicated that these proteins are likely involved in RPE-related functions, such as epithelial development and melanogenesis. Several of the 26 selected proteins have been validated by immunofluorescence as RPE-specific. Unique surface markers are being used to purify iPS cell-derived RPE using fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS).

Conclusions: CSC technology, combined with bioinformatics analysis and immunofluorescence, successfully identified previously known and unknown surface markers on primary human fetal RPE cells. FACS and MACS cell purification approaches are being used to obtain pure RPE cultures from differentiated iPS cells. This purification step is essential in ensuring a safer and more efficacious cell-based therapy to treat degenerative eye diseases.

Keywords: 701 retinal pigment epithelium • 412 age-related macular degeneration • 663 proteomics