Fn coated plates

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 described^{30 (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 CD14^{3 (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 CD14^{3 (link), 31 (link)}.

ECFCs were isolated from the adherent mononuclear cell (MNC) fraction as described [39 (link)]. Then, ECFCs were expanded on fibronectin (FN)-coated plates (1 μg/cm²; Millipore, Billerica, MA, USA) using EGM-2 medium (without hydrocortisone; Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). ECFCs were used at passages P3–5 and at day <30. Endoglin-specific siRNA (Eng-siRNA; sc-35302, Santa Cruz Biotechnology, CA, USA) was used to silence human Eng. Briefly, 10 µM siRNA was mixed with the Dharmafect reagent (SO-2511539G Dharmacon, USA) to obtain transfection complexes, which were added to ECFCs in EGM2 medium in six-well plates. ECFCs transfected with scrambled siRNA (Scramble, Allstars Neg. control siRNA, Qiagen, Cambridge, MA, USA) were used as control (ctr-siRNA). To determine the efficiency of Eng suppression, immunofluorescence microscopy, flow cytometry, and Western blot analyses were used.