Differentiation potential of human ESCs grown in 3-D self-assembling scaffolds. Embryoid bodies (EBs) derived from 3-D grown ESCs (H9 cells) spontaneously differentiated into three germ layers and expressed specific proteins analyzed by immunocytochemistry. (A) Differentiated derivatives of 3-D grown ESCs expressed GATA4, BRACHYURY, and TUJ1 proteins representing the endoderm, mesoderm, and ectoderm, respectively, as shown by confocal images (20X). All scale bars represent 100 μm. (B) Differentiated derivatives of 3-D grown ESCs expressed germ layer-specific genes SOX7 and GATA6 (endoderm), BRACHYURY and MIXL1 (mesoderm), and PAX6 and NCAM (ectoderm) as determined by qRT-PCR. Results are expressed as the fold expression ± SE normalized to reference genes HMBS, GAPDH, and β-ACTIN (* p < 0.05 and ** p < 0.01).