Extended Data Fig. 3: Analysis of structures derived from other hiPSC lines and factors impacting neuro/mesodermal differentiation.

a, Representative bright field images of day 7 structures generated from SOX2-GFP, WTC11 and KUTE4 cell lines (n = 3, n = 3, n = 2). b-d, Immunostaining of sections of day 7 structures for neural progenitors (SOX1/3) and somitic (PAX3) markers, n = 3 for SOX2-GFP and WTC11, n = 2 for KUTE4. e-h, Representative bright field images (e, f; n = 3 and n = 4) and sections immunostained for neural progenitors (SOX2) and somitic (FOXC2) markers (g, h; n = 3 and n = 4) of day 7 structures generated by aggregating different cell numbers at day 0 (120, 160 and 300 cells). i, Representative bright field images (left panel) and sections immunostained for SOX2 and FOXC2 markers (right panel) of day 7 structures generated by aggregating different cell numbers at day 0 (300 or 600 cells) in presence of CHIR or with the addition of LDN and SB (n = 3). j, Representative bright field images (left panel) and sections immunostained for SOX2 and FOXC2 markers (right panel) of day 7 structures in presence of CHIR or with the addition of bFGF (20 ng/ml) for the first 2 days of differentiation (n = 3). k, Immunostaining of plated day 3 aggregates for neural progenitor (SOX2) and PSM (TBX6) markers. Aggregates obtained with different initial cell numbers were cultured using different B27 supplement (percentage of aggregates with TBX6 staining at day 3: B27 control, 50 ± 13% (120 cells) versus 83 ± 8% (300 cells), n = 2; B27 batch #2450484, 14 ± 9% (120 cells) versus 32 ± 21% (300 cells), n = 2; B27 batch #2450486, 2% (120 cells) versus 8% (300 cells), n = 1). l, Representative bright field images of day 7 structures obtained with the different initial cell numbers and different B27 supplements (n as in k). Scale bars, 200 µm (a, e, f, g, h, i, j, l), 100 µm (b, c, d), 50 µm (k).