Isolation of Porcine Retinal Stem Cells

Abstract

Abstract: : Purpose: Retinal stem cells are pluripotential cells that can differentiate into an array of neural retinal cell types. Previous work on adult mice has suggested that the retinal pigment epithelium from the ciliary body is homologous to the ciliary marginal zone found in amphibians and is a source of retinal stem cells. The purpose of this study is to isolate retinal stem cells from adult pig eyes and propagate these cells in vitro. Methods: Eyes from 3 month old pigs were dissected and treated with 2% dispase for 90 minutes at 37^oC. The pigment epithelium was then peeled from the ciliary body using fine jewelers forceps under a dissecting microscope. The sheets of pigment epithelium were collected in HBSS and dissociated with 0.25% trypsin for 5 minutes at 37^oC. The cells were then grown in serum-free defined medium (DMEM/F12 + N2 + FGF) at a density of 1 x 10⁴ per ml. After 3-4 days in culture, the suspended cells were collected and grown on laminin-coated chamber slides in defined medium + 1% FBS. After 3-4 days, the cells were fixed in 4% paraformaldehyde and fluorescent immunocytochemistry was performed for nestin, NSE, and vimentin. Results: Pigment epithelial cells were harvested from the ciliary body with an average yield of 7.67 x 10⁵ cells per eye. In serum-free defined medium, most of the cells remained suspended and grew as clusters. Laminin and FBS promoted adhesion and dispersion of the previously suspended cell clusters. A great majority of these cells were positive for vimentin, but a small group of cells were immunoreactive for nestin and NSE. Conclusions: Retinal stem cells are present in the pigmented epithelium of the ciliary body of adult pig eyes and can be grown successfully in vitro. ACKNOWLEDGEMENTS: This research was made possible by the generous support of the Illinois Society for the Prevention of Blindness, Fight for Sight/Prevent Blindness America, and The Richard A. Perritt Charitable Foundation. DISCLOSURE CODE: N

Keywords: retinal culture • retinal pigment epithelium • plasticity