Figure 3. In vitro studies of cocultured cells demonstrating features of photoreceptor precursor cells. A: Immunofluorescence staining of human embryonic stem cell (ES)-derived photoreceptor precursor cells expressing rhodopsin (lower row) compared to that of spherical neural mass (SNM)-derived single cells that were not cocultured with human ES-derived RPE cells (upper row). B: Expression of red opsin, blue opsin, recoverin, and phosphodiesterase 6 beta (PDE 6B) in human embryonic stem cell–derived photoreceptor precursor cells. C: PCR results for the expression of rhodopsin to evaluate the amount of expression according to the coculture period. The expression of rhodopsin was most increased on day 7 of coculture, and the expression of the early neural marker nestin decreased over time. D: Microarray heatmap showing the expression of markers representative of different stages of retinal photoreceptor cell differentiation (ES, embryonic body (EB), SNM, and 3 weeks after coculture). E: In vitro functional evaluation by measuring the level of cGMP hydrolysis in ES-derived photoreceptor precursor cells kept in the dark or maintained in ambient daylight for 24 h. A decrease in the cGMP level was observed when ES-derived photoreceptor precursor cells were kept in the light condition compared to when they were kept in the dark condition (n=5 in each group, *p<0.05, Mann-Whitney U test). When a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (50 mM), was added 48 h before the determination of cGMP levels, the difference between the cGMP levels in the light and dark conditions was abolished.