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S Wang, B Lu, R Lund; Tracking immortalized human RPE transplants pre-labeled with BrdU . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3451.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In the Royal College of Surgeons (RCS) rat, the retinal pigment epithelial (RPE) cell fails to phagocytose rod outer segments, due to a mutation of the receptor tyrosine kinase gene. This mutation leads to photoreceptor loss. This degeneration can be slowed by subretinal injection of normal RPE cells. However, it is unclear how the grafted cells settle and distribute in the host eye. This study is to examine the survive and distribution of grafted human RPE cells in the dystrophic RCS rat at different survival after transplant. Methods: A spontaneously derived line-ARPE19 obtained from the American Type Culture was culture to sub-confluence; 30µM BrdU was added into the culture medium for 48 hours before harvesting. A cell suspension of (2x105) in 2µl of D-MEM /F-12 medium was delivered trans-sclerally through a fine glass pipette into the subretinal space of 22-25day dystrophic pigmented RCS rats. All animals were maintained on oral cyclosporine A (210mg/liter of drinking water). Animals were perfused at one, two and four week post-transplantation. 10µm sections were cut on cryostat and stained for cresyl violet and anti-BrdU immunohistochemistry. Results: At one week post-transplant, cresyl violet stained sections showed a lump of cells in the subretinal space. Specific anti-BrdU antibody staining revealed most of the cells are BrdU positive. The typical granular products of BrdU staining in the nuclei were evident. The grafted cell bolus measured 100µm long and 30-50 µm high. At both edge of the bolus cells spread along the host RPE. After two week post-transplantation, well-formed cell layers (10-15µm high) close to the injection site were seen. The grafted cells spread along the host RPE cells up to 210µm. At either side of the bolus, 2-4 cell thick BrdU positive cells were seen spreading along the host RPE cell. At four week post-transplantation, a single layer of grafted cells, typically 1-3 cell thick were evident spreading from injection site along the host RPE cells (covering about 200µm length). These BrdU positive cells integrated with host RPE cells and formed contact with neuronal retina. Conclusion: This study revealed that human RPE cells prelabeled with BrdU can survive and integrate with host retina. Grafted cells formed a bolus of cells in the subretinal space at short survival, eventually spreading along and interact with the host RPE cells.
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