Figure 5. Morphology and gene expression analysis of manually selected and long-term cultured human embryonic stem cell (hESC)-retinal pigment epithelium (RPE; Regea 08/023) and hiPSC-RPE (FiPS 5–7) cells. A: Bright-field micrograph of hESC-retinal pigment epithelium (RPE) and human induced pluripotent stem cell (hiPSC)-RPE cells cultured for 136 days on Collagen IV. The cells have acquired a cobblestone morphology and a high degree of pigmentation, which is typical of RPE cells. Low magnification images were captured with a Nikon Eclipse TE2000-S phase contrast microscope (Nikon Instruments Europe B.V. Amstelveen, The Netherlands) and higher magnification images with an Olympus BX60 microscope (Olympus, Tokyo, Japan) using a 60× oil immersion objective. Scale bar 20 μm. B: Reverse transcription (RT)–PCR analysis showing the expression of optic vesicle, optic cup, RPE, neural crest melanocyte, pluripotent stem cell, mesoderm and endoderm marker genes by undifferentiated cells (D0), human foreskin fibroblast (hFF) feeder cells, and putative RPE cells (D196) from Regea 08/023 and FiPS 5–7 cells. N/A=not analyzed. C: Relative OCT3/4 expression between undifferentiated FiPS 5–7 and putative hiPSC-RPE after 196 days of differentiation.