Figure 1. Figure 1. Differentiation of photoreceptor precursors from human embryonic stem (ES) cells via embryonic body (EB) and spherical neural masses (SNMs). Human ES cells (A) are detached to generate EBs (B), which were then attached and cultured in neural precursor (NP) selection medium including 0.5% N2 supplement for 5 days. Then, the medium was switched to NP expansion medium with 1% N2 supplement and basic fibroblast growth factor (bFGF). Neural rosettes (C) and neural tube structures (C-I) were mechanically isolated and then cultured in suspension to form SNMs. Some SNMs in early culture show cystic structures (D) that balloon outward, and these cystic SNMs (D-I) were separated and cystic portions were fragmented with mechanical dissection under a stereomicroscope and were attached and cultured in RPE (retinal pigment epithelium) generation medium with 1% N2 supplement and 1% B-27 supplement, to form ES-derived RPE cells (ERPE, E), which are positive for zonula occludens 1 (ZO1, F), retinal pigmented 486 epithelium-specific 65-kDa protein (RPE65, G), and bestrophin (H). Other non-cystic SNM (I) were passaged and mechanically cut and plated as SNM-derived single cells (J), positive for neural stem cell markers musashi (K) and nestin (L). SNM-derived single cells (J) were cocultured with ES-derived RPE cells (ERPE, E) to differentiate photoreceptor precursor cells.