Fig 1: Addition of BMP9 to differentiation media results in efficient and stable generation of c-KIT+FOXF1+ EPCs. (A) The schematic shows the directed differentiation protocol starting with line 4 ESC maintenance, mesoderm induction, and endothelial lineage promotion with the modification of added Bone morphogenic protein 9 (BMP9) to promote efficient and stable expression of FOXF1. (B) Flow cytometry analysis shows the result of line 4 ESC differentiation. Differentiated line 4 ESCs produce mainly endothelial cells as marked by CD31+CD45− cell surface expression. Within the endothelial cell compartment, a greater percentage of cells become c-KIT+FOXF1+ EPCs after addition of BMP9. This experiment was repeated 5 time. (C) Further FACS analysis shows that in vitro-generated c-KIT+FOXF1+ EPCs exhibit appropriate endothelial marker expression such as Cluster of differentiation 146 (CD146), Endoglin (ENG, CD105), and Angiopoietin-1 receptor (CD202b). (D) Immunostaining reveals that undifferentiated ESCs lack expression of FOXF1 as compared to nuclear expression found in differentiated and FACS-sorted EPCs. Image magnification is × 10. This experiment was repeated 4 times with similar results. Values are shown as mean ± SD. **p < 0.01, ***p < 0.001, ns is not significant.