Although WNT A in isolation was a poor mesoderm

Although WNT3A in isolation was a poor mesoderm inducer in hESCs differentiated in APEL medium, it synergized with BMP4 to efficiently induce MIXL1-GFP+ mesoderm, increasing the yield of Bl-CFCs and hematopoietic CFCs. Our data complement studies in which specific roles for BMP and WNT signaling during the formation of hematopoietic mesoderm from mouse ESCs were identified (Gadue et al., 2006; Jackson et al., 2010; Lengerke et al., 2008; Nostro et al., 2008). In contrast to our findings, a number of studies have demonstrated that primitive streak induction in differentiating mouse ESCs could be mediated by WNT3A alone (Gadue et al., 2006; Jackson et al., 2010; Nostro et al., 2008). While there may be differences between mouse and human ESC differentiation, we speculate that this discrepancy may reflect the influence of components of mouse ESC differentiation medium that are absent from APEL (such as BMP-like activity associated with serum or BSA). This hypothesis is consistent with the observed reduction in the hematopoietic-inducing activity of WNT3A by the BMP antagonist NOGGIN when human ESCs were differentiated in BSA-based medium (Wang and Nakayama, 2009). The synergy between WNT3A and BMP4 in the generation of human hematopoietic Pirfenidone was consistent with the observations that BMP4 induced ventral-posterior mesoderm and subsequently directed mesodermal cells toward a blood fate by activating WNT3A signaling and Cdx gene expression in mouse ESCs (Lengerke et al., 2008). Similarly, Nostro et al. showed that BMP4 had a strong posteriorizing effect on mesendodermal progenitors (Nostro et al., 2008), an effect that was likely to promote the generation of hematopoietic progenitors. Our study complements this work, showing that synergy between WNT3A and BMP during mesoderm induction enhanced the subsequent formation of hematopoietic progenitors through an increased rate of differentiation toward MIXL1+ mesendoderm and by an increase in cell numbers within EBs. Other studies have examined the role of noncanonical and canonical WNT signaling during different stages of hematopoietic development from human pluripotent stem cells. Woll and colleagues examined the influence of WNT1, WNT5, and the WNT inhibitor, DKK1, on hematopoietic development from hESCs (Woll et al., 2008). Using stromal cell coculture, they demonstrated that hematoendothelial precursors were suppressed by DKK1, while their development was accelerated following exposure to WNT1, but not WNT5. The results of their study suggested that canonical but not noncanonical signaling supported the development of a hemogenic precursor, but did not specifically address the stage of differentiation at which the WNT signaling was required (Woll et al., 2008). Our results, indicating synergy between canonical WNT and BMP4 signaling in the generation of hemangioblasts, are consistent with these data. Conversely, results from a second study examining hematopoietic development from hESCs argued that noncanonical signaling by WNT11 was important in guiding hESC differentiation toward mesoderm, while canonical signaling mediated by WNT3A acted only later in differentiation to promote proliferation of hematopoietic progenitors (Vijayaragavan et al., 2009). This latter observation is consistent with studies demonstrating a regulatory role of canonical WNT signaling on hematopoietic stem and progenitor cells in mouse bone marrow (Luis et al., 2011; Trowbridge et al., 2010). Indeed, recent studies examining factors regulating hematopoietic regeneration in the zebrafish and mouse demonstrated the requirement for both BMP and WNT signals (Trompouki et al., 2011). Although it is difficult to readily reconcile all the findings from these studies, the data could be interpreted to suggest that there may be several windows during which WNT signaling differentially affects hematopoietic development. First, we have shown that the early addition of WNT3A synergistically expanded and accelerated the development of BMP4-induced hematopoietic mesoderm, but that the clonal growth of hemangioblast colonies required the removal of WNT3A. This is analogous to the sequential roles of WNT during cardiogenesis elucidated in vertebrate embryos and differentiating mouse and human ESCs (Mummery et al., 2012; Tzahor, 2007). Second, canonical WNT signaling promotes the growth of hematopoietic stem cells and committed hematopoietic progenitors, both during development and in the adult. Finally, the complexity of WNT interactions influencing hematopoiesis is increased by recent data that indicate that WNT signaling also impacts on the bone marrow stromal niche, inducing secretion of a range of mediators that influence hematopoietic stem and progenitor activity (Ichii et al., 2012).