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J. M. Lawrence, A.-J. Carr, A. A. Vugler, A. Ahmado, L. Chen, P. Andrews, J. Walsh, L. da Cruz, P. J. Coffey; A Novel in vitro Assay to Assess the Phagocytic Activity of Human Embryonic Stem Cell-Derived RPE. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5551.
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© ARVO (1962-2015); The Authors (2016-present)
To assess the potential of retinal pigment epithelial cells derived from human embryonic stem cells (HESCRPE) to phagocytose photoreceptor outer segments (OS) from porcine and human retinae.
HESCRPE were derived from the Sheff1 human embryonic stem cell line by superconfluent growth on mouse embryonic feeder cells. Foci of pigmented cells arose spontaneously which were manually transferred to filters coated with matrigel. Fluorescently tagged porcine rod outer segments (ROS) were added to the medium and HESCRPE were examined through time in order to quantify ROS ingestion. These preparations were processed for immunocytochemistry and confocal imaging. In a second, novel approach, HESCRPE were prepared as above and small pieces of human neural retina (surplus tissue from macular translocation surgery ongoing at Moorfields Eye Hospital) were abutted onto the RPE, placing photoreceptor OS adjacent to apical regions of the RPE. These were processed for immunocytochemistry or electron microscopy.
Increasing numbers of OS were associated with HESCRPE with time, so that by 20h an average of 10 ROS were counted in a 150µm2 area. Confocal microscopy showed that fluorescently tagged ROS were attached to, and had been ingested by, the HESCRPE. Ultrastructurally, OS from human retinal explants were enwrapped by HESCRPE apical microvilli and the cytoplasm contained lipid/lysosome inclusions which were absent from control HESCRPE unapposed to retina. Additionally, basolateral membrane infoldings became more pronounced following co-culture of HESCRPE with human retina. Immunocytochemically, HESCRPE adjacent to human retina showed increased levels of lecithin:retinol acyltransferase (LRAT), an enzyme necessary for the recycling of the visual pigment chromophore.
HESCRPE are able to phagocytose photoreceptor material. Additionally, exposure of HESCRPE to human retinal explants results in the upregulation of LRAT and a maturation of RPE structure.
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