Attachment of R28 Stem Cells to RCS Eyecups Treated With MAP Derived Peptides

Abstract

Abstract: : Purpose:This research is a part of ongoing studies of cellular attachment with mussel adhesive protein (MAP) derivatives, examining the potential of R28 cells to attach and differentiate as retinal stem cells in the Royal College of Surgeons (RCS) rat model. It has been shown that R28 cells can be successfully transplanted by the del Cerro method into the RCS rat in which cells were localized in the retina to the outer nuclear layer, ganglion cell layer and along the posterior layer of the lens. It has also been shown that Mussel Adhesive Protein (MAP) derived peptides are capable of specifically attaching these R28 cells to TCPS in vitro. In this study, adhesion of R28 cells to dissected posterior eyecups of dystrophic RCS rats with and without MAP derived attachment peptides (DOPA–K–A–P; KAK and DOPA–G–G–C–K–A–K–G–C; cycDOPA) were examined. Methods:Three 2 month old RCS rats were sacrificed and globes were removed. The anterior eyecup was dissected off, lens and retinas removed, and one posterior eyecup placed in phosphate buffered saline (PBS) in each cell of a tissue culture treated 6 cell well plate. Six eyecup surfaces were examined. Two eyecups were treated with cycDOPA, two with KAK (1mg/ml 5% acetic acid and neutralized with 0.1M sodium bicarbonate), and two were untreated in PBS. Each well received 50,000 green fluorescent protein (GFP) labeled R28 cells in DMEM, 10% calf serum. They were incubated for 24 hours, fluid was aspirated, and eyecups were fixed with 4% paraformaldehyde. Eyecups were examined with fluorescence microscopy, and fluorescent cells in 4 high power fields (HPF) immediately superior, inferior, medial, and lateral to the optic disk were counted. Results: R28 cell adhesion was observed in all eyecups. Totaling the HPF counts, over 50% more cells attached to the eyecups treated with cycDOPA than to those receiving no treatment, and nearly three times as many cells attached to the eyecups treated with KAK as compared to the eyecups receiving no treatment. Conclusions:The treatment of the posterior eyecup with either cycDOPA or KAK enhances the number of R28 cells that attach to the posterior eyecup in vitro. In this study, treatment of the posterior eyecup with KAK attached more R28 cells than cycDOPA. Antibody labeling studies will determine if any treatment influences the possible differentiation of R28 cells on the RCS subretinal eyecup surface.

Keywords: transplantation • retinal degenerations: hereditary • cell adhesions/cell junctions