Short-term BMP-4 treatment of hES cells efficiently induces brachyury expression.(A) Undifferentiated H1, H7, and H9 hES cells express BMP-4 ligand, its receptors BMPR1a, BMPR1b, and BMPR2. (B) hES cells grown on matrigel retained alkaline phosphatase activity (hES cells), whereas the differentiated hES cells did not (Control). (C) hES cells grown on matrigel expressed the pluriopotency marker Oct3/4, but did not express brachyury. (D) As measured by immunocytochemistry, hES cells differentiated in the presence of 25 ng/mL BMP-4 for 24 hours (B25) showed a high level of brachyury expression. However, when the hES cells were differentiated without BMP-4 treatment (Null), no brachyury expression could be detected. BRACH indicates brachyury. Scale bars represent 200 μm (B) and 100 μm (panels C,D).