Human umbilical cord blood was obtained from the Brigham and Women’s Hospital in accordance with an Institutional Review Board-approved protocol and according to the Declaration of Helsinki. Informed consent was not obtained as cord blood was excess human material, normally discarded, and was obtained without identifiers. ECFC were isolated from the adherent cell fraction using CD31-coated magnetic beads (Invitrogen) as described30 (link). ECFC were expanded on fibronectin (FN)-coated plates (1 μg/cm2; Millipore, MA) using EGM-2 (without hydrocortisone; Lonza) supplemented with 20% fetal bovine serum (FBS; Hyclone) and 1x glutamine-penicillin-streptomycin (GPS; Cellgro). ECFC between passages 5 and 8 were used for all experiments. ECFC express CD31, VE-cadherin, VEGFR-2, but not CD90, CD45 or CD143 (link), 31 (link).
MPC were isolated from the MNC fraction of human adult bone marrow (Lonza). MNC fraction isolated using Ficoll-Paque (GE Healthcare) was seeded on 1% gelatin-coated plates using MSCGM medium (Lonza) supplemented to 10% FBS, 1x GPS. Unbound cells were removed at 48 hours, and the adherent cell fraction maintained in culture until 70% confluent. MPC were expanded in MSCGM medium supplemented to 10% FBS, 1x GPS. MPC between passages 5 and 8 were used for all experiments. MPC express CD90 and CD105 but not CD31, VE-cadherin, VEGFR-2, CD45 or CD143 (link), 31 (link).
MPC were isolated from the MNC fraction of human adult bone marrow (Lonza). MNC fraction isolated using Ficoll-Paque (GE Healthcare) was seeded on 1% gelatin-coated plates using MSCGM medium (Lonza) supplemented to 10% FBS, 1x GPS. Unbound cells were removed at 48 hours, and the adherent cell fraction maintained in culture until 70% confluent. MPC were expanded in MSCGM medium supplemented to 10% FBS, 1x GPS. MPC between passages 5 and 8 were used for all experiments. MPC express CD90 and CD105 but not CD31, VE-cadherin, VEGFR-2, CD45 or CD143 (link), 31 (link).
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