The majority of the CD45+ cells also express CD31, as well as c-kit (Figure 1A). in an accelerated differentiation and higher percentage of CD34brightCD31+Flk1+ cells at earlier stages of differentiation. These studies effectively demonstrate the importance of canonical Wnt signaling to mediate development of early hematoendothelial progenitors during human development. Introduction Hematopoietic and endothelial cells are mesoderm-derived lineages that demonstrate a close spatial, temporal, and genetic relationship during vertebrate embryogenesis.1 These CMK properties have led to the hypothesis that these cell lineages originate from a common precursor, so-called hemangioblasts or hematogenic-endothelial cells.2,3 Mouse embryonic stem cells (mESCs) have been instrumental to define the phenotypic and developmental pathways that regulate endothelial and hematopoietic development.4C8 For example, blast colony-forming cells (BL-CFCs) are thought to represent the functional equivalent of a common progenitor cell for both endothelial and hematopoietic cells after differentiation of mESCs.5 Importantly, similar cells with hematoendothelial potential have been identified in the posterior region of the primitive streak in mouse embryos, effectively translating in vitro differentiation from mESCs to in vivo embryonic development.9 However, recent in vivo lineage tracing studies of the developing yolk sac suggest that other mechanisms may also be involved. 10 Continued studies are therefore needed, especially in a human model system where the relationship between hematopoietic and endothelial cells remains poorly characterized Several reports of hematopoietic differentiation from human ESCs (hESCs) demonstrate that comparable strategies used to study development of hematopoietic and endothelial lineages from mESCs can be transposed to the hESC system.11C16 This allows analysis of early cell fate specifications of endothelial and CMK hematopoietic cells in a model system that is more directly relevant to human development. As with mESCs, there are 2 routine methods used to facilitate differentiation of hESCs: embryoid body (EB) formation and stromal cell coculture. Although the general kinetics of differentiation suggests a conserved pattern for development of endothelial and hematopoietic precursors between different methods of hESC differentiation, there are differences in the requirement for defined growth factors for development of hematopoietic precursors when hESCs are cocultured with stromal cells compared with EB differentiation.17 One study identified progenitor cells within hESC-derived EBs that express CD31, Flk1, and VE-cadherin but not CD45 (termed CD45negPFV cells), after approximately 7 to 10 days of differentiation, capable of generating both endothelial and hematopoietic cells. 13 A YWHAB similar study also identified hematogenic potential of endothelial cells from CD34+CD31+CD45? human EB-derived cells also after 10 days of differentiation.14 Another recent report demonstrates development of a cell CMK populace during EB-mediated differentiation of hESCs that express Flk1 (also termed KDR or VEGF-R2) and generate BL-CFCs similar to what has been observed for mESCs.15 Development of these human hemangioblast cells was observed earlier in the culture and was dependent on presence of bFGF, VEGF, and BMP4 during EB differentiation.15 Yet another group characterized a similar functional hemangioblast cell population, although these cells did not express CD34, CD31, or Flk1.16 The importance of VEGF and BMP4 for development CMK of hematopoietic and endothelial cells has been shown previously.17C19 However, the role of other exogenous factors regulating early cell fate specification of hematopoietic and endothelial precursors during human development still remains unclear. Wnt proteins have many important functions during development including maintenance and/or proliferation of rare stem and progenitor cell populations, cell fate specification, segmentation, and dorsal-ventral patterning.20 In the mouse, canonical Wnt signaling is required for formation of the primitive streak, where distinct mesoderm subpopulations including the hemangioblast are generated.9 Mice lacking canonical Wnt ligands, the Wnt coreceptors Lrp5/6, or -catenin do not develop primitive streak and fail to generate mesoderm from the epiblast.21C23 A recent study has also demonstrated the requirement of Wnt signaling for the generation CMK of in vitro primitive streak during mESC differentiation.24 Here, we characterize the phenotype of a hematogenic endothelium cell populace derived from stromal cellCmediated hESC differentiation. These cells identified as CD34brightCD31+Flk1+ differentiate into both hematopoietic and endothelial cells. Development of these cells is largely dependent on functional canonical Wnt signaling, as inhibition of this signaling pathway results in a decreased generation of these cells. Complementary analyses of hESCs cocultured with stromal cells that overexpress Wnt1 demonstrates accelerated development of this human hematoendothelial cell populace. Methods Cell culture The hESC lines H1and H9 (obtained from Wicell, Madison, WI) were maintained as undifferentiated cells as previously described.11,25 Murine S17 stromal cells (kindly provided by Dr K. Dorshkind, University of California,.