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  • In this study we employed APEL based differentiation to

    2018-11-08

    In this study, we employed APEL-based differentiation to assess the contribution of WNT signaling to hematopoietic development from human embryonic stem cells. Although mesoderm formation was dependent upon WNT signals, WNT3A alone was an inefficient inducer of mesoderm. However, WNT3A synergized strongly with BMP4 to generate MIXL1+ mesoderm from which hematopoietic blast colonies were generated in semisolid media. Surprisingly, the inclusion of WNT3A in the methylcellulose abrogated blast colony development and led instead to the formation of mesenchymal colonies, denoted mesospheres. These studies highlight the importance of the timing of growth factor exposure during development and show how WNT signaling during different temporal windows promotes either hematopoietic or mesenchymal differentiation.
    Results We examined the role of WNT signaling during the differentiation of human ESCs (hESCs) in APEL medium toward hematopoietic cells. To evaluate the role of WNT during mesoderm formation, hESCs were differentiated as spin embryoid bodies (EBs) (Ng et al., 2008), supplemented with WNT3A and/or BMP4. To facilitate the analysis, we employed two hESC lines (HES3 MIXL1GFP/w and MEL1 MIXL1GFP/w) in which GFP reports expression of MIXL1, a homeobox gene whose expression is restricted to mesoderm and endoderm precursors in the primitive streak (Davis et al., 2008). In contrast to the results of previous studies that employed BSA-, KOSR-, or N2B27-containing media (Gadue et al., 2006; Nakanishi et al., 2009; Sumi et al., 2008; Wang and Nakayama, 2009), we found that WNT3A alone was a poor inducer of mesendoderm, with few order SAR405 expressing MIXL1-GFP at day 4 (d4) (Figures 1A and 1B; Figures S1A and S1B available online). Instead, WNT3A-only-treated EBs resembled those formed in absence of growth factors, with approximately 80% of cells retaining high levels of the undifferentiated hESC/epiblast marker, E-CADHERIN (Figures 1B, 1C, and S1C). Indeed, transcriptional profiling indicated that EBs formed in WNT3A alone displayed a very similar pattern of gene expression to EBs formed in the absence of growth factors (Figure S1D). Nevertheless, in line with previous data from mouse and human ESC studies (Gadue et al., 2006; Jackson et al., 2010; Nostro et al., 2008; Sumi et al., 2008; Wang and Nakayama, 2009; Woll et al., 2008), we found that mesoderm induction by BMP4 was not only antagonized by NOGGIN and dependent on NODAL signaling, but was also WNT dependent, since inclusion of either FZD8 or DKK1 significantly reduced MIXL1-GFP expression (Figures 1D and S1E). Analysis of d4 EBs revealed a strong synergy between WNT3A and BMP4, with 82% ± 2% of cells treated with both growth factors expressing MIXL1-GFP, compared to 32% ± 5% and 2.5% ± 0.8% GFP+ cells in cultures treated with 10 ng/ml of BMP4 alone or 100 ng/ml of WNT3A alone, respectively (Figure 1C). Induction of MIXL1-GFP was associated with a parallel increase in expression of the primitive streak and early mesoderm marker, platelet-derived growth factor receptor (PDGFR)α (Davis et al., 2008), and a reciprocal reduction in the proportion of E-CADHERIN+ cells. Similar responses were observed in both independent MIXL-GFP hESC lines (Figure S1C). Inhibitor studies confirmed that mesoderm induction in hESCs by the combination of WNT3A and BMP4 was also inhibited by antagonists of BMP, NODAL, and WNT signaling (Figures 1D and S1E). In agreement with this finding, microarray analysis demonstrated that BMP4- or WNT3A/BMP4-induced EBs expressed BMPs, NODAL, and WNT genes (Figure 1E), consistent with our observations that endogenously produced growth factors in differentiating mouse ESCs provided paracrine or autocrine mesoderm inducing signals (Jackson et al., 2010). The increased expression of these genes in WNT3A/BMP4-treated hESCs was explicable by the higher proportion of MIXL1-GFP+ cells generated under this condition. Microarray analysis indicated that this subpopulation expressed growth factor genes at the highest levels (Figure S1F).