Isolation and Characterization of Bone Marrow Pericytes and Stromal Cells

Human BM pericytes (BM-PCs) and stromal cells (BM-SCs) were obtained as previously reported [24 (link), 25 (link)]. Once approximately 90% confluent, BM-MSCs were trypsinised and sorted for isolation of PCs. Around 1 × 10⁷ BM cells were resuspended in pre-cooled column buffer containing 0.5% (w/v) BSA and 2 mM EDTA in DPBS, labelled with CD45-conjugated microbeads (Miltenyi) for 15 min at 4℃ before subsequently being sorted by magnetic separation. The CD45-negative cells were then labelled with CD34-conjugated microbeads (Miltenyi), followed by magnet sorting. CD45^neg CD34^neg cells were labelled with CD146-conjugated microbeads (Miltenyi). The CD34^neg CD45^neg CD146^pos population, collected through immunomagnetic sorting, was considered BM-PCs. The remaining cells (CD45^negCD34^pos and CD45^negCD34^negCD146^neg populations) were pooled and considered BM-SCs. The purity of BM-PCs was assessed by immunocytochemistry staining (data not shown). Cell lines between passage 3 to passage 6 were used for subsequent experiments.