Stepwise retinal specification from human iPS cells. (A) Various stages of retinal differentiation were observed, beginning with Pax6+/Rx+ eye field cells by day 10. (B–D) Mitf+ and Chx10+ cells, indicative of the optic vesicle/optic cup stages, are evident by day 40. (E) By day 80, clusters were present containing Chx10+ retinal progenitors and Crx+ photoreceptor precursor cells. (F–H) Many Crx-expressing cells expressed the photoreceptor protein recoverin (F) and the cone-specific protein opsin (G and H). (I) RT-PCR analysis demonstrating the stepwise expression of retina- and photoreceptor-associated genes in differentiating iPS cells over time. (J and K) RPE cells derived from iPS cells acquired a typical hexagonal morphology and pigmentation (J) and expressed Mitf and ZO-1 (K). (Scale bars, 50 μm.) Blue stain in B and D is Hoechst nuclear dye; blue stain in F is To-Pro-3 nuclear dye.