Effective differentiation of hPSCs into DE is coupled with

Effective differentiation of hPSCs into DE is coupled with the suppression of E-CADHERIN/CDH1 and pluripotency. Whereas BMP signaling has been reported to suppress E-CADHERIN/CDH1 and pluripotency (Teo et al., 2011, 2012), we observed that WNT3A is not as effective as GSK-3 inhibitors in suppressing both E-CADHERIN/CDH1 and the pluripotency program. This could partly explain the continued requirement for FBS when deriving DE with WNT3A. Therefore, our study identifies the unprecedented need for an additional compound to suppress pluripotency when combining Activin and WNT3A to generate DE p97 without serum.
Results
Discussion We report the use of a chemically defined differentiation medium to compare DE-inducing effects by Wnt or BMP signaling in the presence of Activin signaling. This direct comparison demonstrates that both Wnt and BMP can independently synergize with Activin signaling to comparably and effectively induce DE cells from hPSCs in the absence of FBS. Numerous reports have established that 25 ng/ml WNT3A is optimal for inducing DE in the presence of Activin and FBS (Table S1). However, 10–30 ng/ml WNT3A is insufficient to synergize with 100 ng/ml Activin to effectively generate DE cells in our serum-free system. The need for 100 ng/ml WNT3A for efficient DE formation in a chemically defined system suggests that FBS (which exhibits batch-to-batch variability) introduces additional differentiation-promoting factors to supplement 25 ng/ml WNT3A in inducing DE cells. Although Kunisada et al. (2012) and Illing et al. (2013) used CHIR99021 to induce DE differentiation from hPSCs, they also included FBS in their protocol. We now provide several alternative methods for effectively inducing DE cells, all via the activation of Wnt signaling pathway (in addition to the indispensable Activin signaling pathway). These include 100 ng/ml WNT3A, 3 μM CHIR99021, and 1.5 μM BIO, all of which exhibit similar effects in DE induction. This three-pronged approach confirms the ability of Wnt to synergize with Activin signaling to induce DE in a chemically defined medium. Our comparison with Kroon et al. (2008) (WNT3A-based) and Touboul et al. (2010) (BMP4-based) further demonstrates that these three Wnt signaling-based optimized conditions induce DE comparably and efficiently, and give rise to DE derivatives with equivalent potential. However, it is surprising to note that WNT3A is not as effective in downregulating E-CADHERIN/CDH1 and pluripotency factors as compared to GSK-3 inhibitors (or even BMP4) in a serum-free system. It is known from early mammalian development that the expression of Oct4, and in particular Sox2 and Nanog (Avilion et al., 2003; Hart et al., 2004) coincides with the in situ expression of Wnt3, Wnt3a and Wnt5a but not that of Bmp4 (Arnold and Robertson, 2009; Liu et al., 1999; Tam and Loebel, 2007; Teo et al., 2011; Yamaguchi et al., 1999). Thus, given the overlapping expression of Wnt and pluripotency genes but not that of Bmp, it is conceivable that Wnt does not play a major role in suppressing pluripotency in contrast to BMP. In this study, we reveal fundamental mechanistic differences between the two developmental signaling pathways: (1) BMP signaling suppresses pluripotency and simultaneously promotes DE formation, whereas (2) Wnt signaling mostly serves to synergistically promote DE formation and may require additional factors to suppress pluripotency. FBS, which is commonly added in presence of WNT3A, could be promoting both differentiation and suppressing pluripotency. Although the use of GSK-3 inhibitors results in a downregulation of E-CADHERIN/CDH1 and pluripotency factors SOX2 and NANOG, and GSK-3 inhibition activates downstream Wnt signaling, the inhibition of GSK-3 could potentially affect other signaling pathways in which GSK-3 is active, such as cellular proliferation and migration (Doble and Woodgett, 2003). This is in contrast to the specific WNT3A-induced Wnt signaling. In our experiments, we also observed that WNT3A (up to 200 ng/ml) was not as effective as the GSK-3 inhibitors in elevating β-catenin transcript expression during DE differentiation (data not shown). This could imply that WNT3A-induced Wnt signaling events occurred solely at the protein level. Further, this suggests that GSK-3 inhibitors potentiate the increased presence of β-catenin, thereby strengthening Wnt signaling (besides inhibiting the GSK-3-containing destruction complex). Therefore, the inhibition of multi-functional GSK-3 coupled with the increased β-catenin expression could partly account for the differences observed between WNT3A-induced and GSK-3 inhibition-induced Wnt signaling. Specific to CHIR99021 (a potent and highly selective GSK-3 inhibitor as compared to BIO), the strong inductive effects on mesendodermal (EOMES, MIXL1, BRACHYURY) marker gene expression (Kubo et al., 2004; Tada et al., 2005) and a marginal increase in mesodermal marker TBX6 indicate that excessive Wnt signaling via CHIR99021 (>3 μM) is likely to promote mesoderm while suppressing DE formation.