HBA macrophages increase number of HBA EC-derived HPCs. (A) Schematic diagram of the coculture system, with a monolayer of OP9-DL1-B1 cells overlaid with HBA EC, HBA macrophage (MФ), or MIX. One ee of HBA ECs (CD31⁺CD41⁻CD45⁻) and/or 1000 HBA MФ (CD45⁺CD11b⁺F480⁺) were added to each coculture well. After 7 days, nonadherent cells were harvested, FACS sorted, and plated in methylcellulose to quantitate hematopoietic colonies (CFU-C). The genotype of each colony was determined by Cre PCR. (B) Bright field microscopic images show the round morphology and density of HCs after 7 days of OP9-DL1-B1 culture of EC, MФ, or MIX. The number of (C) CD45⁺ cells and (D) CFU-Cs per ee of E10.5 (31 to 35 sp) HBA cells after 7 days of OP9-DL1-B1 culture of EC, MФ, or MIX. n = 4; *P < .05. The number of (E) CD45⁺ cells and (F) CFU-Cs per ee of E10.5 HBA from OP9-DL1-B1 culture of EC and MIX with or without Csf1r inhibitor BLZ945 (6.7 nM). BLZ945 (BLZ) is diluted in DMSO. EC^& and MIX^& have the DMSO^& diluent added as a vehicle control. SEM is shown. n = 3, *P < .05. Colony types indicated by color bars.