HEK293 fibroblasts and HUVECs were maintained on tissue culture polystyrene flasks in DMEM + 10% FBS. Immortalized hBMECs (77), a gift of K. S. Kim and M. Stins (Johns Hopkins University, Baltimore, MD), were maintained in RPMI 1640 + 10% FBS + 10% NuSerum + 1× minimum essential medium (MEM) nonessential amino acids on flasks that had been coated with a solution of 1% rat tail collagen in 70% ethanol that was allowed to evaporate. Eight-well glass chamber slides were coated with 200 μl of concentrated growth factor reduced Matrigel per well and incubated at least 1 hour at 37°C to set the Matrigel. HUVECs or hBMECs were plated at 2.2 × 104 cells per eight-well chamber slide in 500 μl of Endothelial cell growth medium-2 (EGM-2) medium (Lonza) alone or with 6.6 × 104 HEK293 fibroblasts, primary brain pericytes, or hPSC-derived mural cells at D22 of the differentiation. Cells were incubated for 24 hours at 37°C to allow cord formation, and bright-field images were taken on live cells at 24 hours following plating. Cords were subsequently fixed and stained according to the immunocytochemistry methods listed above. Matrigel-associated cords were mounted onto glass slips and imaged using Olympus epifluorescence and Nikon A1R-Si+ confocal microscopes. Cord length and number of cords per field were quantified by hand using the ImageJ ROI manager tool and averaged over at least three independent fields per condition per differentiation.

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