Isolation, culture and identification of rat retinal progenitor cells in vitro

Abstract

To characterize embryonic rat retinal progenitor cells (RPCs) by flow cytometry (FACS), immunofluorescence and (3)H-Thymidine assay in vitro. RPCs were prepared from the retina of embryonic day 19 Sprague Dawley rats and were cultured in DMEM: F12 medium with N2-supplement, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) for 0 to 4 days. Cell proliferation and cluster formation were quantified by (3)H-Thymidine assay and morphometric analysis, respectively. Antibodies used to characterize the RPCs included markers for neural progenitors (Nestin and Sox-2), astrocytes (glial fibrillary acidic protein [GFAP]), horizontal cells (Calretinin), ganglion cells (CD90), rods (Rhodopsin), biopolar cells (PKC) and amacrine cells (Syntaxin) by the use of FACS. In primary cells, 22.23%, 16.04% and 19.66% of cells were found to express Nestin, Sox-2 and Calretinin, respectively. GFAP, CD90, Rhodopsin, PKC, Syntaxin were not detected or trail. Meanwhile, more than 90% cells were Nestin positive by immunofluorescence. After 6 days culture in vitro, 43.36% cells express Nestin by FACS. Nestin and GFAP were expressed by use of immunofluorescence. There was significant proliferation of RPCs within the first 4 days as evaluated by (3)H-Thymidine assay. The results demonstrate that combine FACS, immunofluorescence and (3)H-Thymidine assay together could characterize RPCs. RPCs proliferate well at the early period of cell culture.