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Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular‐like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain‐containing receptor (KDR) and CD144/KDR. Because the CD133 + KDR +  population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144 + KDR +  population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144 + KDR +  peak was shifted to day 7. Most of these cells, however, were CD31 − , becoming CD31 +  in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4‐boosted day 7, CD144 + KDR + CD31 −  population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12. STEM CELLS 2009;27:1750–1759

A Boost of BMP4 Accelerates the Commitment of Human Embryonic Stem Cells to the Endothelial Lineage