Previous reports have suggested that WNTs modulate BMP signa

Previous reports have suggested that WNTs modulate BMP signaling by promoting C-terminal phosphorylation of SMAD1/5, key components of the BMP signal transduction pathway (Fuentealba et al., 2007). Therefore, we compared the phosphorylation of SMAD1/5 in EBs formed in the combination of WNT3A/BMP4 to those differentiated with either factor alone (Figures 1F and S1G). This analysis confirmed that WNT3A led to SMAD1/5 phosphorylation and the combination of 100 ng/ml WNT3A and 10 ng/ml BMP4 resulted in levels of SMAD1/5 phosphorylation that exceeded those seen in EBs treated with either factor alone and was comparable to the SMAD1/5 phosphorylation observed in EBs formed in 30 ng/ml BMP4. Collectively, these results suggested that enhanced BMP4 signaling might be in part responsible for the synergistic activity of WNT3A during BMP4-induced hESC differentiation. However, the fact that WNT3A alone was unable to efficiently induce MIXL1 expression also indicated that signaling involving SMAD1/5 intermediates was not sufficient for robust mesoderm induction. Given that transforming growth factor (TGF)-β family proteins also signal via MAPK pathways in a SMAD-independent fashion (Derynck and Zhang, 2003), we examined the effects of ERK and p38 inhibition on the induction of MIXL1-GFP by BMP4 and WNT3A/BMP4. Interestingly, while inhibition of ERK by U0126 reduced the frequency of MIXL1-GFP+ cells, differentiation was augmented by inhibiting p38 MAPK with SB203580 (Figure 1G). A recent report suggested that treatment with WNT3A primed Cy3.5 maleimide to primitive endoderm in mouse ESCs and enhanced visceral endoderm differentiation (Price et al., 2012). In order to determine whether a similar situation existed in hESCs, we compared the transcriptional profiles of the small percentage of GFP+ cells that could be isolated from MIXL1-GFP hESCs differentiated in WNT3A for 4 days with the gene expression patterns of GFP+ cells sorted from parallel cultures stimulated by a combination of WNT3A/BMP4 (Figures S2A–S2C). Consistent with the reported mouse ESC data, we observed enhanced expression of endodermal genes such as FOXA1, FOXA2, SOX17, CCKBR, APOA2, and CXCR4 in the WNT3A-induced GFP+ cells, while the GFP+ cells purified from the WNT3A/BMP4 cultures expressed higher levels of primitive streak and mesoderm associated genes (Figure S2C; Tables S1 and S2). Time-course analysis indicated that MIXL1-GFP hESCs differentiated in APEL supplemented with the combination of WNT3A and BMP4 more rapidly gained GFP and PDGFRα expression and more rapidly lost E-CADHERIN expression compared with cells treated with BMP4 alone (Figures 2A, 2B, and S3A). In the examples shown, differentiation proceeded rapidly and the accelerated onset of PDGFRα and MIXL1 expression and the loss of surface E-CADHERIN were evident by days 2 and 3 of differentiation. These conditions did not induce significant expression of surface CXCR4, a marker often used as an early indicator of endodermal differentiation. In addition to the synergy observed between WNT3A and BMP4 in enhancing the rate of mesoderm formation, d4 EBs formed in WNT3A/BMP4 containing medium were 2-to 3-fold larger, suggesting that WNT3A contributed to increased proliferation or decreased cell death (Figures 2C and S3B). We disaggregated d4 EBs and flow-sorted populations on the basis of E-CADHERIN and MIXL1-GFP expression (Figures 3A, 3B and S4). Day 4 EBs formed in WNT3A/BMP4-containing medium included a larger percentage of cells with a more mature phenotype (E−G+) than was observed in EBs differentiated in BMP4. Comparison of transcriptional profiles of the sorted cell populations revealed that genes expressed by corresponding fractions were very similar in both the BMP4- and WNT3A/BMP4-treated EBs, consistent with the notion that WNT3A primarily accelerated the differentiation observed with BMP4 alone (Figures 3C–3E and S5A–S5H). Examination of the transcription factors whose expression was most highly upregulated in the WNT3A/BMP4 E−G+ cells revealed that they were primitive streak or posterior mesodermal genes that were frequently expressed at a similar level in the corresponding BMP4 sorted fraction (Figure 3F).