Human endothelial colony forming cells were generated from umbilical cord blood collected at the time of delivery as previously described [23 (link)]. Cells were routinely cultured in endothelial cell growth medium (EGM-2) complete culture medium (Lonza, Switzerland) on rat tail collagen type 1-treated plates. The cells were used between the 6th and 11th passages.
Human mesenchymal stromal cells were obtained from bone marrow aspirates of patients undergoing clinical investigations upon written informed consent for the collection, analysis, storage, and reuse. Mononuclear cells were isolated using a Ficoll density gradient and cultivated on gelatin-treated cell culture dishes, in 1 g/L glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% MSC-qualified fetal bovine serum (Gibco, Waltham, MA, USA). Cells were used between the 6th and 11th passages. For in vivo studies, the cells were trypsinized and re-seeded at confluence for 24 h before use in the cellular transplant. On the transplantation day, the cells were detached with accutase (Sigma-Aldrich, St. Louis, MO, USA), and suspensions were prepared containing 40 × 106 cells/mL. Five microliters of cell suspension, containing 2 × 105 cells (for the MSC group), or a mixture of 1.8 × 105 MSC and 2 × 104 ECFC (for the MSC + ECFC group) was used per mouse.
Human mesenchymal stromal cells were obtained from bone marrow aspirates of patients undergoing clinical investigations upon written informed consent for the collection, analysis, storage, and reuse. Mononuclear cells were isolated using a Ficoll density gradient and cultivated on gelatin-treated cell culture dishes, in 1 g/L glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% MSC-qualified fetal bovine serum (Gibco, Waltham, MA, USA). Cells were used between the 6th and 11th passages. For in vivo studies, the cells were trypsinized and re-seeded at confluence for 24 h before use in the cellular transplant. On the transplantation day, the cells were detached with accutase (Sigma-Aldrich, St. Louis, MO, USA), and suspensions were prepared containing 40 × 106 cells/mL. Five microliters of cell suspension, containing 2 × 105 cells (for the MSC group), or a mixture of 1.8 × 105 MSC and 2 × 104 ECFC (for the MSC + ECFC group) was used per mouse.