Differential effects of short- and long-term BMP-4 treatments on hES cell differentiation. (A) Morphology of the differentiated hES cells at days 1, 2, 3, and 7 of BMP-4 treatment. (B) As determined by Q-PCR, the pan-mesoderm gene brachyury was highly expressed at 24 hours of hES cell differentiation by BMP-4 treatment (B25). However, the extra-embryonic lineage genes, GATA6, SOX7, AFP for extra-embryonic endoderm, and CDX2, hCG alpha, hCG beta for extra-embryonic ectoderm, were induced by long-term, but not short-term, BMP-4 treatment. And hES cells differentiated in SFM in the absence of BMP-4 (Null) showed little or undetectable brachyury expression. Numbers on the x-axis indicate days of differentiation; numbers on the y-axis indicate relative gene expression level normalized to that of GAPDH. Error bars indicate standard deviations. (C) Immunofluorescent labeling of differentiating hES cells demonstrated decreased expression of brachyury and increased expression of SOX7 with continuous BMP-4 treatment. BRACH indicates brachyury. Scale bars represent 50 μm.