Abstract
Compared with many induced pluripotent stem cell (iPSC) lines generated using retrovirus and other non-integrating methods, the utilization of human protein-induced iPSC (piPSC) lines may provide a safer alternative for the generation of retinal pigment epithelial (RPE) cells for transplantation in retinal degenerative diseases. Here we assess the ability of piPSCs to differentiate into RPE cells, and to perform native RPE cell behavior. piPSCs were seeded in 6-well low-attachment plates to allow embryoid body formation, and then analyzed for pluripotent stem cell markers NANOG, SSEA4 and TRA-1-60 by immunofluorescence. Following colony formation, piPSCs were assessed for confirmation of RPE cell differentiation by staining for zonula occludens (ZO-1), bestrophin, microphthalmia-associated transcription factor (MITF) and retinal pigment epithelium specific protein-65 (RPE65). To evaluate piPSC-RPE cell phagocytic ability, adult bovine photoreceptor rod outer segments (ROS) were fed to piPSC-RPE cells, which were analyzed by fluorescent microscopy and flow cytometry. Undifferentiated piPSCs expressed all pluripotent markers assessed and formed embryoid body aggregates after 7 days. Differentiated piPSC-RPE cells expressed ZO-1, bestrophin, MITF and RPE65, typical RPE cell markers. Flow cytometry revealed robust ingestion of fluorescently-labeled ROS by piPSC-RPE cells, which was over four-times greater than that of undifferentiated piPSCs and comparable to that of an immortalized RPE cell line. Phagocytosis activity by piPSC-RPE cells was significantly reduced after the addition of anti-integrin αVβ5. In conclusion, piPSCs can be differentiated toward an RPE cell fate, expressing RPE cell markers and resembling native RPE cells in behavior. These results demonstrate that piPSCs can be differentiated into RPE-like cells using a method that has an increased safety profile, a critical consideration for the development of better treatments for retinal degenerative diseases such as age-related macular degeneration (AMD).
Highlights
Age-related macular degeneration (AMD) is a leading cause of blindness in the United States and Western Europe, and it will become an increasing burden as the population ages [1, 2]
Results protein-induced Induced pluripotent stem cells (iPSCs) (piPSC) maintained pluripotency after subcultures were positive for pluripotency markers At day 0, immunocytochemistry was used to assess the expression of stem cell markers in the piPSC grown on MEF
In this study we have confirmed the capacity of piPSCs to differentiate into retinal pigment epithelial (RPE) cells using an RPE cell differentiation protocol established by Meyer and coworkers [25]
Summary
Age-related macular degeneration (AMD) is a leading cause of blindness in the United States and Western Europe, and it will become an increasing burden as the population ages [1, 2]. The exudative or “wet” type is characterized by neovascularization of the choroid and affects 10% of AMD patients [3]. This form of AMD can be controlled with intravitreal injections of vascular endothelial growth factor inhibitors. The dry type is more common, representing the majority of individuals with AMD [3]. In both types of AMD, the disease is characterized by dysfunction and eventual loss of retinal pigment epithelial (RPE) cells, a critical cell type in the maintenance of retinal function [4,5,6,7,8,9].
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