phalloidin Because many growth factors function in a context

Because many growth factors function in a context-dependent manner, we explored the effects of adding WNT3A to the MC on the growth of Bl-CFCs. In cultures containing a combination of hematopoietic growth factors (VEGF, SCF, IL-3, IL-6, thrombopoietin [TPO], EPO, and FLT3L), 0.2%–0.6% of phalloidin derived from EBs differentiated with BMP4 or WNT3A/BMP4 generated Bl-CFC colonies at d4 (Figures 6A–6D). Strikingly, addition of WNT3A to the methylcellulose dramatically reduced the frequency of hematopoietic blast colonies and instead supported the growth of compact, mesodermal colonies, termed “mesospheres,” at a similar frequency (0.4%–0.6%) to that observed for Bl-CFCs (Figures 6A, 6B, 6E–6G, S6A, and S6B). However, because mesospheres and blast colonies required mutually exclusive culture conditions, it was not possible to determine if they arose from common progenitor. We also demonstrated that the effects of adding WNT3A to the methylcellulose could be replicated with (2′Z,3′E)-6-bromoindirubin-3′-oxime (BIO), a canonical WNT signaling agonist that inhibits GSK3 (Figures S7A and S7B). Gene profiling revealed that the WNT3A- and BIO-induced mesospheres contained cells that expressed early mesodermal genes such as FOXF1, MEOX1, PDGFR, HAND1, and SNAI2 and were highly enriched for transcripts associated with chondrocyte and bone differentiation, such as DLK1, LUM, MGP, COL15A1, COL3A1, FRZB, ITGA5, and CDH11 as well as many other extracellular matrix (ECM) proteins (Figure 7A; Table S5). Notably, several ECM proteins upregulated in WNT and BIO mesospheres (TNC, DCN, and FMOD) were recently shown to be highly expressed in OP9 cells engineered to constitutively express WNT3A (Ichii et al., 2012). The gene expression profiles of WNT and BIO mesospheres were very similar, with 689/979 (∼70%) of the probe sets upregulated in BIO mesospheres also upregulated in WNT mesospheres (Figures S7C and S7D; Table S5). These gene expression profiles suggested nonhematopoietic mesodermal lineage potentials for the mesospheres. Indeed, we subsequently demonstrated that the mesospheres could differentiate toward adipocyte (marked by oil red O droplets and expression of FABP-4), osteoblast (marked by the formation of alizarin red aggregates and OSTEOCALCIN expression), and smooth muscle (marked by smooth muscle actin [SMA] expression) lineages (Figures 7B–7G). Whether the mesospheres contained multipotent progenitors or lineage restricted precursors for additional mesenchymal lineages remains to be determined.
Discussion We explored the role of WNT signaling during hematopoietic differentiation from hESCs in APEL medium, identifying distinct activities at different stages of differentiation. First, WNT was required for the induction of mesoderm, an outcome anticipated from prior published literature, including our own studies in differentiating mouse ESCs (Gadue et al., 2006; Jackson et al., 2010; Lengerke et al., 2008; Murry and Keller, 2008; Woll et al., 2008). We confirmed the requirement for BMP and NODAL signals in mesoderm differentiation and demonstrated that inhibition of ERK and p38 MAP kinases mediated inhibitory or stimulatory signals, respectively, downstream of BMP4 that impacted on MIXL1-GFP expression. Our observation that ERK inhibition reduced mesoderm induction by BMP is consistent with the finding of Zhang and colleagues, who demonstrated BMP4-mediated ERK activation suppressed neural differentiation of mouse ESCs (Zhang et al., 2010). Studies have shown that BMP4-induced ERK phosphorylation was required in other cellular contexts, including BMP4-dependent capillary sprouting in HUVECs and stabilization of Runx2 expression during osteoblast differentiation of C2C12 cells (Jun et al., 2010; Zhou et al., 2007). Similar to our findings, opposing actions of ERK and p38 MAPK were reported for other mesodermal lineages. For example, ERK inhibition enhanced chondrogenesis in chick limb bud mesenchyme, while p38 inhibition had the opposite effect (Oh et al., 2000). Similarly, BMP4-stimulated generation of VEGF in mouse osteoblast-like MC3T3-E1 cells was reduced in the presence of p38 inhibition but was unaffected by blocking ERK (Tokuda et al., 2003).