Isolation of Pericyte Progenitor Cells

Pericyte progenitor cells were isolated from vein leftovers of patients undergoing coronary artery bypass graft surgery or varicose vein removal, as previously described55 (link). In brief, saphenous veins were carefully dissected from surrounding tissues using a sterile scalpel and then thoroughly washed in PBS. Veins were manually minced before 4-h incubation with 3.7 mg ml⁻¹ Liberase 2 (Roche) and filtered passing the cell suspension through a 30-μm cell strainer. Cells were depleted for ECs with anti-CD31-conjugated beads (Miltenyi), according to the manufacturer's instruction. The remaining cells were purified by selecting CD34+ cells by anti-CD34 beads (Miltenyi). Target cells were then plated on fibronectin (10 μg ml⁻¹)-coated plates in presence of differentiation medium (EGM-2—2% FBS, Lonza). Adherent colonies were passaged to new culture dishes once they reached 60–70% confluence and frozen stocks generated after Passage 2 (P2) for the experiments shown in this publication. Trypsin-EDTA (Life Technologies) was utilized to detach cells from the growth substrate.