Supplementary Figure 2: Patterning of a human epiblast model by high BMP4 concentration.

(a) Population fractions of colonies at various BMP4 concentrations from IF staining and from live-cell imaging data, presented separately. The statistics was deduced from seven independent experiments for 1 ng/ml (four IF stained, three imaged live), from two independent experiments for 5 ng/ml (one IF stained, one imaged live), and from three independent experiments for 0.1 ng/ml (one IF stained, two imaged live). The data for each BMP4 concentration from independently run experiments was pooled, IF and live imaging separately, and the n indicated in each pie chart represents the number of total individual 3D colonies. (b) BRA and SOX2 intensity change of the majority population under pluripotency conditions and with BMP4 for 2 days at different concentrations. From left to right: n = 38, n = 8, and n = 23 individual 3D colonies. Thick line is mean, grey line is s.e.m. (c) Live-cell imaging of a human epiblast model stimulated with 0.1 ng/ml BMP4 added at time zero, representative of the majority population from 13 imaged 3D colonies. See also Supplementary Video 2. (d) IF staining of epiblast models stimulated for 2 days with 0.1 ng/ml BMP4, showing epiblast markers (representative of the majority population of 82 3D colonies). (e) Live-cell imaging of a model human epiblast stimulated with 5 ng/ml BMP4 added at time zero, representative of majority population from 28 imaged 3D colonies. See also Supplementary Video 3. (f) Left: IF of representative colonies after high (5 or 10 ng/ml) BMP4 dose for two days, showing the expression of germ layer and extra-embryonic markers (top, representative of 20 3D colonies; bottom, 30 3D colonies). Right: population fractions of colonies 2 days after 5 ng/ml BMP4 in terms of differentiation markers (n = 30 individual 3D colonies).