Fig. 3

Characterization of ectodermal and neuronal properties of in vitro induced RNLCs. a PAX-6, an ectodermal and a pan-neuronal/retinal markers, showed expression in RNLCs as shown by both western blotting and ICC, and flow cytometric analysis suggested that around 90% of the RNLCs were PAX-6 positive. The expression was analysed by flow cytometry, western blotting and ICC. b qPCR analysis of retinal markers in RNLCs, RM and retinoblastoma cell line Y79 (positive control) (n = 5). *p < 0.05; **p < 0.01. c RNLCs expressed Rhodopsin indicating that these cells had an induced retinal neuron-like properties. The images were captured at × 63 magnification. The western blot analysis also confirmed that RNLCs expressed Rhodopsin after retinal growth factor induction. d Potentiometric analysis for the changes in membrane potential during light and dark conditions were analysed by using DiBAC4(3) dye. An only dye-treated control was an auto-fluorescence control. Cells treated with valinomycin and gramicidin-D were used as depolarization (DP) and hyperpolarization (HP) controls. The changes in fluorescent intensity were monitored when the cells where dark-adapted and light-exposed for 15 min and 30 min respectively. It was observed that RM did not exhibit any noticeable change between dark- (81.5%) and light-exposed (82.9%) conditions while RNLCs exhibited an increase in fluorescence intensity (depolarization) (91.5%) in dark conditions and a significant decrease in fluorescence (Hyperpolarization) (33.9%) when the cells were exposed to light and further hyperpolarized (14.6%) on continual light exposure