Study of differentiation of hippocampal stem cells into rod photoreceptors in rat

Vision is one of the most important organs in the body. Damage to this organ causes a severe disability in humans. In retinitis pigmentosa, the degeneration of photoreceptors causes blindness. So far, more than 100 mutations have been detected in photoreceptors, which they result in opsin malfunction. The aim of present study is to differentiate the hippocampal stem cells of rat to the rod photoreceptors. Stem cells of the hippocampus were obtained from rat embryos, 18 days of age (E18). Pregnant female rats were killed and the head of their embryos were separated. Then, the embryos’ hippocampus was removed according to Banker’s method. Hippocampal cells were dissociated by Fish Bach’s method. The cells were cultured in flasks (25cm2). After 3 days, the cells were isolated by trypsin, counted using trypan blue and hemocytometer. Cell suspensions were prepared in two cell concentrations; high (2×105cells/l) and low (2×104 cells/l) concentration, then, cultured using DMEM/F12 supplemented with fetal bovine serum 10% (FBS) in six wells plates. Prior to culture of the cells, the first and second row of plates were coated with poly L-lysine and inactivated astrocytes, respectively. Following incubation of the plates at 37C for 4 days, different concentrations of All Transe Retinoic Acid (ATRA) and 9–CIS RA were added daily for 6 days, and finally immunocytochemistery was carried out using anti-opsin monoclonal antibody. The results of current study showed that the plates, which are respectively treated with ATRA and 9-CIS RA in a concentration of 500nM and 100nM for 6 days had the most differentiated cells. In addition, maximum differenced cells were observed with 100nM of 9-Cis RA. The differentiated cells were detected in wells, which were only coated with inactivated astrocytes in either a high or low concentration of cell suspension. These findings indicated that inactivated astrocytes as a feeder layer and extrinsic factors such as (ATRA) and 9–Cis RA increase differentiation of hippocampal stem cells into rod photoreceptors.