Summary A hierarchical order of factors in the generation of FLK 1-and SCL-expressing hematopoietic and endothelial progenitors from embryonic stem cells

There has been great interest in identifying factors that control hematopoietic commitment, as they can provide molecular mechanisms involved in hematopoietic establishment. Previous studies implicate that endoderm-derived factors and mesoderm-inducing factors could play a role in hematopoietic and endothelial cell differentiation. For example, in Xenopus, the formation of erythroid cells from the animal cap is induced by BMP4 and basic fibroblast growth factor (bFGF) or by BMP4 and activin A, and the generation of erythroid cells by exogenously expressed GATA1 can be potentiated by bFGF (Huber et al., 1998). Studies of quail-chick chimeras have shown that the endoderm can induce the formation of hematopoietic cells from the somatopleural mesoderm, which normally does not have such potential (Pardanaud et al., 1996). In addition, the formation of blood islands from quail epiblasts is dependent on bFGF (Flamme and Risau, 1992). In this system, bFGF-mediated blood island formation correlates with the induction of the Flk1 gene (Kdr – Mouse Genome Informatics) (Flamme et al., 1995), suggesting that bFGF is crucial for the emergence of the hemangioblast, the common progenitor of hematopoietic and endothelial cells (Sabin, 1920; Murray, 1932; Wagner, 1980). Furthermore, in quail embryos, bFGF, vascular endothelial growth factor (VEGF) and transforming growth factor (TGF) β 1 can induce hematopoietic differentiation from the somatopleural mesoderm (Pardanaud et al., 1996). Similarly, in mice, the primitive endoderm can induce hematopoietic differentiation from the anterior epiblast, a tissue that cannot generate hematopoietic cells (Belaoussoff t al., 1998). Specifically, both activin A and BMP4 can induce hematopoietic differentiation from the anterior headfold region (Kanatsu and Nishikawa, 1996), and Indian hedgehog can promote hematopoietic differentiation from the anterior epiblast (Dyer et al., 2001). Although these studies support the notion that TGF β family members and bFGF can induce hematopoietic differentiation, The receptor tyrosine kinase FLK1 and the transcription factor SCL play crucial roles in the establishment of hematopoietic and endothelial cell lineages in mice. We have previously used an in vitro differentiation model of embryonic stem (ES) cells and demonstrated that hematopoietic and endothelial cells develop via sequentially generated FLK1+ and SCL+ cells. To gain a better understanding of cellular and molecular events leading to hematopoietic specification, we examined factors necessary for FLK1 + and SCL+ cell induction in serum-free conditions. We demonstrate that bone morphogenetic protein (BMP) 4 was required for the generation of FLK1 + and SCL+ cells, and that vascular endothelial growth factor (VEGF) was necessary for the expansion and differentiation of SCL-expressing hematopoietic progenitors. Consistently, Flk1-deficient ES cells responded to BMP4 and generated TER119 + and CD31+ cells, but they failed to expand in response to VEGF. The Smad1/5 and map kinase pathways were activated by BMP4 and VEGF, respectively. The overexpression of SMAD6 in ES cells resulted in a reduction of FLK1+ cells. In addition, a MAP kinase kinase 1 specific inhibitor blocked the expansion of SCL+ cells in response to VEGF. Finally, VEGF mediated expansion of hematopoietic and endothelial cell progenitors was inhibited by TGFβ1, but was augmented by activin A. Our studies suggest that hematopoietic and endothelial commitment from the mesoderm occurs via BMP4mediated signals and that expansion and/or differentiation of such progenitors is achieved by an interplay of VEGF, TGFβ1 and activin A signaling.