FIG. 1.

Induction of hESCs to the mesodermal lineage by combined treatments with BIO, Activin A, and BMP4. (A) Expression patterns of mesoderm-lineage genes (T, MIXL1, and WNT3), ectoderm-lineage genes (SOX1 and ZIC1), and endoderm-lineage genes (SOX17 and CXCR4) in ABB-treated hESCs (ABB stands for a combination of Activin A, BIO, and BMP4). Real-time PCR was used to estimate and compare the expression levels of early developmental genes in untreated hESCs (control) and ABB-treated hESCs. Statistical significance between samples was evaluated by paired t-test (*P<0.05). (B) Protein level expression of the mesoderm-lineage gene (T) and pluripotent gene (OCT4) in ABB-treated hESCs (bottom) and untreated hESCs (top). In this figure, immunostaining was used to detect the presence of T and OCT4 in hESCs and ABB-treated hESCs (scale bar is 200 μm). (C) Activation status of signaling pathways: WNT, bone morphogenetic protein (BMP), and TGF-β in ABB-treated hESCs. In this figure, the increases in phosphorylation of SMAD1/5/8 and SMAD2/3 represent the activation of BMP and TGF-β, respectively; the decrease in phosphorylation of β-catenin represents the activation of the WNT signaling pathway; α-tubulin was used as housekeeping gene. The expression of T and OCT4 at the protein level in ABB-treated cells and untreated hESCs was confirmed by western blotting. hESCs, human embryonic stem cells; BIO, 6-bromoindirubin-3′-oxime; BMP4, bone morphogenetic protein 4; PCR, polymerase chain reaction; TGF-β, transforming growth factor β.