Fig. 3
A xeno-free, feeder-free differentiation strategy led to efficient production of high-quality retinal pigment epithelial (RPE) cells. a Schematic illustration of the RPE differentiation pipeline. b Images of pigmented patches after 35 days of differentiation (upper row; scale bars = 1 cm) with and without initial neuroectodermal induction (+/– ind.) for hESC1-RPE. Images of cell culture inserts (middle row; scale bar = 6.5 mm) and DIC confocal images (lower row; scale bars = 10 μm) after 9 weeks of final maturation on inserts, illustrating the difference in pigmentation. c Quantification of pigmentation using image analysis after 9 weeks on inserts presented as mean pixel intensity relative to spontaneous differentiation (– ind.; n = images from two individual differentiation experiments), and (d) quantification of cell size after 9 weeks on inserts presented as mean cell area (μm2; n = number of cells examined). e Mean TEER values after 9 weeks on inserts, differentiated with or without induction (n = inserts analyzed from four individual differentiation experiments). f Phalloidin labeling for filamentous actin and vertical confocal sections of junctional proteins zonula occludens-1 (ZO-1) and Claudin-19 (Cl-19). Scale bars = 20 μm. Error bars denote standard deviation. Mann–Whitney U test was used for assessing statistical significance; ***p < 0.01. Blebb. blebbistatin, EB embryoid body