Conditions and fluorescence activated cell sorting (FACS) parameters were based on work of Yuan et al.36. Conjugated antibodies were purchased from BD Biosciences (San Diego): CD184-APC (clone 12G5), CD271-PerCPy5.5 (clone C40-1457), CD24-PE (clone ML5), CD15-FITC (clone HI98). FACS buffer consisted of 1× Hank’s buffered solution, 2% fetal bovine serum, 20 mM glucose, 1× penicillin/streptomycin, 1 mM EDTA and was filtered and made fresh for each experiment. Accutase (Invitrogen, A11105-01), DNase I (Sigma, D4263), 1 unit /µL, DAPI (Invitrogen, D3571) was made at 2 mg/mL in H2O. To obtain CD184+, CD271−, CD24+, CD15+ cells, mixed NPC populations were either immunoselected using EasySep CD184 (Stem Cell Technologies) according to the manufacturer’s instructions and FACSed or directly FACSed. For preparing cells for FACS, 0.5–2×107 cells were washed once with DMEM/F12 and dissociated with Accutase at 37°C for 5 minutes. FACS buffer was added to terminate digestion and cells were triturated to form a single cell suspension. Cells were then washed twice with FACS buffer and resuspended at a concentration of 1×107 cells/mL. DNAse I was added to a final concentration of 100 units/mL
