Figure 5.

Spatial expression of NR2F2, OCT4 and Pax6 during hESC differentiation. (A–L′) Undifferentiated and (A–D) differentiated (7 days in non-conditioned medium as a monolayer; E–L′) RUES2 hESCs colonies were stained for the indicated proteins. (C, G, K, K′) Nuclear counterstain with Sytox Orange. I′–L′ are magnifications of white boxes in I–L, respectively. Scale bars represent 200 μm in A–L and 50 μm in I′–L′. (M) Real-time PCR analysis of the temporal expression of NR2F2 (blue line), Pax6 (red line) and Sox1 (green line) in RUES2 cells induced to differentiate into neural ectoderm. The protocol of differentiation is depicted below the histogram and described in detail in the Materials and methods section. Note the logarithmic scale. All measurements, except Sox1 D4, are statistically different compared with the undifferentiated (D0) sample (Student's t-test; P<0.01). (N–Q) RUES2 cells differentiated in N2M for 10 days with the protocol showed in M were stained for NR2F2 (N) and Pax6 (O), and counterstained with Sytox Orange (P). Scale bars represent 50 μm.