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A.-J. F. Carr, A. A. Vugler, S. T. Hikita, J. M. Lawrence, D. O. Clegg, P. J. Coffey; Reprogrammed Human Fibroblast Cells Yield Rpe Cells Capable of Phagocytosis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3219.
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© ARVO (1962-2015); The Authors (2016-present)
To analyse the potential of inducible pluripotent stem (iPS) cells to differentiate into retinal pigment epithelial (RPE) cells, and investigate their functional properties in culture and in vivo, after transplantation into the Royal College of Surgeons (RCS) dystrophic rat.
iPS cells derived from human fibroblast cells transduced with OCT4, SOX2, NANOG and LIN28 were cultured on MEFS in the presence of bFGF. Once confluent, bFGF was omitted to allow differentiation of cells. Pigmented colonies were dissected manually, trypsinised and expanded on gelatin-coated plates. The enriched pigmented cells (iPS cell-RPE) were characterised by RT-PCR, Western blot and immunocytochemistry. Cells were incubated with fluorescently labelled porcine rod outer segments (ROS) for up to 20hr and processed for immunocytochemistry. After staining with Na+/K+ ATPase the ingestion of ROS was analysed by confocal microscopy. iPS-RPE were injected into the subretinal space of 24-day old RCS dystrophic rats and phagocytosis was examined using the confocal microscope to image rhodopsin ingestion by grafted human cells labelled human-specific markers.
In culture, iPS cell-RPE form a pigmented cobblestone-like monolayer and express many classic RPE cell markers including Cytokeratin8, CRALBP, bestrophin, MerTK, RPE65, PEDF and MITF. Expression of Na+/K+ ATPase was observed at the apical surface of the cells, indicative of polarisation, whilst the absence of Ki67 staining suggested cells could exit cell cycle. Confocal microscopy of cells incubated with fluorescently labelled ROS provided evidence of adherence and ingestion of ROS over the 20hr period. Cell survival was observed 7 days post-graft in the subretinal space of the dystrophic RCS rat, with clear evidence of phagocytosis within the intracellular compartment of the transplanted human cells. The expression of RPE cell markers was maintained in vivo.
iPS cell-RPE are morphologically similar to RPE cells and express many markers associated with RPE. Furthermore, these cells are functional, being capable of phagocytosing ROS debris in vitro and in vivo following transplantation to the degenerating subretinal space of the RCS dystrophic eye.
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