Objective: To investigate the effectiveness of all-trans-retinoic acid (ATRA) at different concentrations on proliferation and differentiation of the rat embryonic neural stem cells (NSCs), and to find the optimal concentration of ATRA that promoting the differentiation of NSCs into neurons.
Methods: NSCs were isolated from cerebral cortex of rat embryos (embryonic day 12-16, average 15 days), and were cultured in serum-free medium (DMEM/F12 medium containing 20 ng/mL bFGF and 20 ng/mL EGF) at the concentration of 1 x 10(6) cells/mL. Subcultures were performed 7 days after the primary culture. The cell clusters of the 3rd passage were centrifuged and divided into 5 groups. In the experimental groups (groups A, B, C, D), the ATRA concentration was 0.5, 1.0, 5.0, 10.0 micromol/L in DMEM/F12 complete medium respectively, while in control group (group E), the ATRA concentration was 0 in DMEM/F12 complete medium. The proliferation rate of each group was analyzed by cell counting day by day till 7th day, and BrdU positive cell counting 1, 3, 5, 7, 9 days after culture. In addition, collecting the 3rd passage NSCs and divided into 5 groups. In the experimental groups (groups A, B, C, D), the ATRA concentration was 0.5, 1.0, 5.0, 10.0 micromol/L in DMEM/F12 medium containing 5% FBS respectively, while in control group (group E), the ATRA concentration was 0 in DMEM/F12 medium containing 5% FBS. The capacity of NSCs differentiation toward neurons was determined by immunofluorescence double-labelling and flow cytometry.
Results: Cell counting 1-7 days after culture in each experimental group (groups A, B, C, D) showed no significant differences (P > 0.05). Cell counting at each time point of all the experimental groups were less than those of control group (P < 0.05). BrdU positive cells were increased 1, 3, 5, 7, 9 days after culture in each experimental group (groups A, B, C, D), but there was no significant difference between each experimental group (P > 0.05). BrdU positive cells at each time point of control groups were more than those of all the experimental groups (P < 0.05). The differentiation ratio of neurons was enhanced in experimental groups and the optimal ATRA treatment concentration was 1.0 micromol/L (experimental group B). The differentiation ratio of neurons induced by ATRA in group B was 29.46% +/- 0.47%, 47.25% +/- 0.46% and 66.81% +/- 0.57% respectively after cultured 3, 5 and 7 days, whereas the differentiation ratio of neurons was 11.11% +/- 0.59%, 14.10% +/- 0.32% and 15.92% +/- 0.70% respectively in control group. The majority of NSCs differentiated into astroglial phenotypes in control group. By flow cytometry detection, the differentiation ratio of neurons after cultured 3 days and 7 days in experimental groups were more than those in control group (P < 0.05).
Conclusion: ATRA treatment remarkably promoted the differentiation of NSCs into neurons and the optimal concentration was 1.0 micromol/L.