Fig. 2.

Highly efficient derivation of eye field phenotypes from hESCs. (A) RT-PCR analysis showing the onset of Pax6 and Rx gene expression and concomitant loss of Oct4. (B and C) qPCR analysis of Oct4 gene expression (B) and Pax6 and Rx gene expression (C). Values were expressed as fold change relative to undifferentiated hESCs. (D) Immunocytochemical analysis of cells at day 10 showing uniform coexpression of Pax6 and Rx (merged image includes ToPro-3 nuclear stain). (E) FACS analysis confirming the rapid loss of Oct4 expression and the onset of both Pax6 and Rx protein expression. Negative controls for FACS analyses are indicated by the white histograms. (F and G) qPCR (F) and Western analysis (G) demonstrating the endogenous expression of the BMP and Wnt antagonists Noggin and Dkk-1. (H) qPCR showing the near complete loss of Pax6 and Rx gene expression in cells treated with BMP4 and Wnt3A. (Scale bar, 40 μm.)