Figure 1

Differentiation occurs at the edge of hESC colonies.

(A) Phase and (B) immunostaining images of hESC colonies when they are undifferentiated (top) and after 3 days BMP4 treatment (bottom). (C) Analysis of expression of pluripotency marker, SOX2 and differentiation marker, AP2α, after 3 days BMP4 treatment. Fluorescent intensity is plotted as a function of distance from the colony edge and normalized to the maximum intensity of each colony [n = 20 colonies, ****p < 0.0001 and represents statistics for AP2α (green) and SOX2 (red) levels between distance 35 μm and 175 μm from the edge using a two-tailed paired t-test]. Error bars represent S.D. from the mean. (D) The differentiation band width of hESC colonies plated on matrigel-coated glass coverslips or plastic dishes (red data points) reveals a constant average band width of differentiation of 149 μm (blue line) ± 59 μm (S.D.) (dotted cyan lines). The black line indicates a differentiation band width equal to the colony radius (n = 175 colonies). (E) The number of AP2α-positive cells quantified per circumference in a number of different colonies (red data points) shows a constant average number of cells per mm of circumference of 43 (maroon line) ± 12 cells (S.D.) (dotted magenta lines) (n = 11 colonies).