Expression of Dll4 ligand on vascular feeder cells specifically promotes myeloid differentiation of HPCs. (A-B) The CD43⁺ population was isolated from hESC differentiation cultures at day 12 and cultured for an additional 7 days in either the absence (plastic) or presence (hESC EC) of VPr⁺ EC feeders; scatter plots of the derivative populations (A) are shown at day 3 and 5 after isolation, and the distributions of CD41a⁺CD43⁺, CD41a⁻CD43⁺, and CD43^dimCD45^bright populations are quantified in panel B. (C) RNA sequencing was performed on the CD41a⁺CD43⁺, CD41a⁻CD43⁺, and CD43^dimCD45^bright populations; expression levels of NRARP, HES1, and HEY1 are shown. (D) RNA sequencing was performed on undifferentiated hESCs, crude non-EC derivatives of hESC differentiation, VPr⁺CD31⁺ ECs, primary fetal liver ECs from aborted human fetus, neonatal umbilical cord ECs (HUVEC), and E4ORF1 ECs; a heat map describing the expression of vascular specific markers and Notch ligands is shown. (E-G) VPr⁺ ECs were isolated from hESC differentiation at day 14 and incubated with lentivirus encoding control (scrambled) or Dll4 shRNA. Crude non-EC derivatives were also isolated and incubated with lentivirus encoding Dll4 cDNA. Flow cytometric analysis defined the expression of the VPr-mOrange transgene, CD31, and Dll4 in cultured derivatives after 6 days. (H) The mean fluorescence intensity (MFI) value of Dll4 surface expression was plotted for cells described in panels E-G at 2, 4, and 6 days after addition of lentivirus. (I) At 48 hours after addition of lentivirus, cells described in panels E-H were washed with culture medium, and GFP⁺CD43⁺ cells isolated from day 16 of differentiation were added at 10 cells per well of a 48-well dish. After 4 days, the total number of GFP-derivatives (black bar), as well as the percentage of CD43^dimCD45^bright, CD15⁺CD33^dim, CD41a⁺CD43⁺, and CD71⁺CD235a^bright/Lineage⁻ cells was measured. (I) The numbers indicate the concentration of lentivirus particles added to cells in ng/mL. (B,H) Error bars represent SD among 3 replicates. (I) Error bars represent SD among 8 replicates. *P < .05.