Of the small number of genes whose transcription

Of the small number of genes whose transcription differed by ≥3-fold between the two treatment groups (39, 51, and 93 out of 48,701 probe sets for E+G−, E+G+, and E−G+ fractions) (Figure 3G), most were upregulated in the WNT3A/BMP4-treated EBs (37, 43, and 61 probe sets, respectively). In the GFP+ fractions, these included transcripts for the WNT8 inhibitor RGS4 and for genes associated with anterior lateral and paraxial mesoderm, such as ACTC1, CDX2, LHX8, C6ORF32, and LUM, consistent with the role of WNT signaling in cardiac and somitic differentiation (Cohen et al., 2008; Geetha-Loganathan et al., 2008) (Tables S3 and S4). We compared the frequency of hematopoietic blast colony forming purchase IWR-1-endo (Bl-CFCs) in d4 EBs cultured in BMP4 alone and in WNT3A/BMP4. We have previously demonstrated that these mesodermally derived precursors displayed the capacity to differentiate into erythroid cells, endothelium, and smooth muscle (Davis et al., 2008; Yu et al., 2012), similar to results in mouse and human ESCs reported by other laboratories (Choi et al., 1998; Kennedy et al., 2007) and to hemangioblasts isolated from the mouse embryo (Huber et al., 2004). Examination of their hematopoietic potential revealed that d4 EBs formed in WNT3A/BMP4 medium generated Bl-CFCs at a slightly higher frequency than those differentiated in BMP4 alone (49 ± 12 and 27 ± 11 Bl-CFC/104 d4 EB cells, respectively), although the differences were not statistically significant (Figures 4A and 4B). Since they were cultured in MC supplemented with erythropoietin (EPO), most blast colonies contained a predominance of primitive erythroid cells (Figure 4A). Given their larger size, these data suggested that d4 EBs formed in WNT3A/BMP4 generated ∼5-fold greater number of Bl-CFCs per input hESC than those differentiated in BMP4 alone. Most Bl-CFCs were found in the MIXL1-GFP+ fractions as expected (Davis et al., 2008), with the highest frequency and greatest proportion in the E−G+ population (Figures 4B and 4C). In d4 WNT3A/BMP4 cultures, nearly 20% of the Bl-CFCs were localized to the most differentiated MIXL1-GFP dim (E−Gd) cells, a population not present in BMP4-only-treated cultures at that time. To test the hypothesis that the combination of WNT3A/BMP4 accelerated the generation of hematopoietic mesoderm, we performed experiments to examine Bl-CFC frequency at d2 and d3 of differentiation. While the maximal frequency of Bl-CFC in BMP4-induced cultures was seen at d3 of differentiation, inclusion of WNT3A accelerated the generation of these precursors such that maximal frequency was observed earlier, after just 2 days (Figure 4D). To determine whether the WNT3A/BMP4 combination ultimately generated a greater frequency of more mature hematopoietic cells and progenitors, we analyzed d4 EBs that had been cultured for a further 7–9 days in medium supplemented with BMP4, vascular endothelial growth factor (VEGF), stem cell factor (SCF), interleukin (IL)-3, and EPO (Figures 5A–5C). In cultures initiated in either no growth factor or WNT3A alone, only small numbers of hematopoietic or endothelial cells were generated, as evidenced by a low percentage of cells expressing CD31, CD34, and CD45 and the detection of infrequent hematopoietic CFCs in methylcellulose (Figures 5A–5C). Conversely, a much higher percentage of hematopoietic cells were generated in cultures initiated in BMP4, and the inclusion of WNT3A further enhanced hematopoietic differentiation. Although the proportion of CD31+ and CD34+ was similar in cultures initiated in the presence of WNT3A/BMP4 or BMP4, the frequency of CD45+ cells and CFCs were consistently higher in WNT3A/BMP4-treated EBs (Figures 5B and 5C). Thus, the synergy between WNT3A and BMP4 during the first 4 days of EB differentiation and mesoderm formation resulted in an augmented generation of hematopoietic cells a week later, underscoring the importance of WNT signaling at the earliest commitment steps during hESC-derived hematopoiesis.