[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 [25cm[2]]. After 3 days, the cells were isolated by trypsin, counted using trypan blue and hemocytometer. Cell suspensions were prepared in two cell concentrations; high [2x10[5]cells/ micro l] and low [2x10[4] cells/ micro 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

Effects of retinoic acid and astrocyte as a feeder layer in differentiation of hippocampal stem cell

Adult stem cells are pluripotent cells conventionally isolated from some part of body by different methods. From developmental stand point, murine neural stem cells represent an accessible and important system for studies of basic stem cell property such as self- renewal and multipotency. In this study hippocampal stem cells obtained from embryonic day 18 [E18]. Pregnant female rats were killed, embryos heads separated and then hippocampus isolated by the method of Banker, then their cells dissociated by the methods of Fishbach, and plated in flask 25cm, after 3 days cells separated by tripsin, counted with trepan blue and hemocytometer, divided into two density [high 200000] and [low 20000 cells]. Before transplantation of cells, six well plates coated with poly L lysin and inactivated astrocyte. Then isolated cells transplanted into 6 well plate for 4 days with medium DMEM/F12 supplemented with FBS10%. After 4 days different doses of ARTA and RA cis-9 added per well for 6 days, and then immunocytochemistery were done. After 6 days of treatment with above factors, doses of 100nM RA cis-9 and 500 nM ATRA have the more staining cells with monoclonal antibody. But in 100 nM RA cis-9, we saw maximum differentiated cells. All of differentiation were done on wells with inactivated astrocyte layer in high and low density. Inactivated astrocyte as a feeder layer and extrinsic factors such as All Transe Retinoic Acid [ATRA] and RA cis-9 can cause differentiation in hippocampal stem cells into photoreceptor like cell