The isolation and culture of BM-EPCs were performed as described11 (link). Rat bone marrow cells were obtained by flushing femurs and humeri with
Dulbecco’s modified Eagle medium (DMEM)/F12 medium (Gibco, New York, NY, USA).
BMSCs were then isolated by density gradient centrifugation with the Histopaque
1077 (Sigma-Aldrich, St. Louis, MO, USA) from bone marrow cells. After washing
with red blood cell lysis buffer, bone marrow mononuclear cells were seeded into
culture flasks in DMEM/F12 medium supplemented with 10% fetal bovine serum
(Gibco). After 24 h, the plastic-adherent cells were removed, and the
nonadherent cells were collected, washed, and replated into fibronectin-coated
(10 μg/ml; BD Biosciences, San Jose, CA, USA) culture flasks with the inducing
medium containing DMEM/F12 medium supplemented with 10% fetal bovine serum, 20
ng/ml VEGF, 5 ng/ml basic fibroblast growth factor, 5 ng/ml epidermal growth
factor, 10 ng/ml insulin-like growth factor-1 (Peprotech, New Jersey, NJ, USA),
and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin).
Dulbecco’s modified Eagle medium (DMEM)/F12 medium (Gibco, New York, NY, USA).
BMSCs were then isolated by density gradient centrifugation with the Histopaque
1077 (Sigma-Aldrich, St. Louis, MO, USA) from bone marrow cells. After washing
with red blood cell lysis buffer, bone marrow mononuclear cells were seeded into
culture flasks in DMEM/F12 medium supplemented with 10% fetal bovine serum
(Gibco). After 24 h, the plastic-adherent cells were removed, and the
nonadherent cells were collected, washed, and replated into fibronectin-coated
(10 μg/ml; BD Biosciences, San Jose, CA, USA) culture flasks with the inducing
medium containing DMEM/F12 medium supplemented with 10% fetal bovine serum, 20
ng/ml VEGF, 5 ng/ml basic fibroblast growth factor, 5 ng/ml epidermal growth
factor, 10 ng/ml insulin-like growth factor-1 (Peprotech, New Jersey, NJ, USA),
and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin).
