Egm 2 culture medium

Human metastatic melanoma cell line A375 (ATCC, Manassas, VA, USA) was grown in high D-glucose DMEM, with 10% (v/v) heat-inactivated fetal bovine serum (Euroclone Milan, Italy), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mmol/L glutamine in a humidified atmosphere with 5% CO₂ in air. The culture medium was changed every 2 days. HUVECs (ATCC, Manassas, VA, USA) were grown in Endothelial Basal medium, supplemented with 10% FBS (Euroclone, Milan, Italy), on gelatin-coated dishes. Endothelial Colony Forming Cells (ECFCs) were isolated from >50 mL human umbilical cord blood (UCB) of healthy newborns after maternal informed consent as previously described [20 (link),46 (link)] The purification and use of stem cells from cord blood for research purposes is permitted by an Italian law after obtaining informed consent from the mothers (art. 2, paragraph 1, letter f, decree of 18 November 2009). ECFCs were grown in EGM-2 culture medium (Lonza, Lonn, Swiss), supplemented with 10% FBS (Euroclone, Milan, Italy) onto gelatin-coated dishes. ECFCs were grown in a humified atmosphere with 5% of CO₂ in air and the medium was refreshed every 2 days.

The human melanoma cell line A375 (MITF wild type, BRAF V600E, NRAS wild type) was obtained from American Type Culture Collection (Manassas, VA) and was grown in Dulbecco’s modified Eagle’smedium (DMEM, Euroclone, Milano, Italy) containing 2 mM glutamine, 100 UI/ml penicillin, 100 μg/ml streptomycin and 10% FBS (Euroclone, Milano, Italy). A375–M6 melanoma cells (M6) were isolated from lung metastasis of SCID bg/bg mice i.v. injected with A375 cells and grown in the same conditions of A375. A375 and M6 were independently validated by STR profiling by the DNA diagnostic centre BMR Genomics (Padova, Italy). Cells were amplified, stocked, thawed and were kept in culture for a maximum of 4 months. ECFCs were isolated from > 50 ml human umbilical cord blood (UCB) of healthy newborns as described previously [24 (link)], were selected as CD45⁻, CD34⁺, CD31⁺, CD105⁺, ULEX⁺, vWF⁺, KDR⁺ cells [24 (link)] and were grown in EGM-2 culture medium (Lonza), supplemented with 10% FBS. Human Microvascular Endothelial Cells (HMVECs) were purchased from Lonza and were grown in the same conditions of ECFCs.

Endothelial Colony Forming Cells (ECFCs) were isolated from > 50 ml human umbilical cord blood (UCB) of healthy newborns, as described previously [14 (link), 15 (link)], after maternal informed consent and in compliance with Italian legislation, and analyzed for the expression of surface antigens (CD45, CD34, CD31, CD105, ULEX, vWF, KDR, uPAR) by flow-cytometry [14 (link)]. ECFCs were grown in EGM-2 culture medium (Lonza), supplemented with 10% FBS (Euroclone) onto gelatin coated dishes. Human microvascular endothelial cells (HMVEC) were purchased from Lonza and were grown in the same conditions of ECFCs.

Peripheral blood samples (20 ml) from participants at 0 and 12 weeks were placed in heparin tubes and diluted (1:1 in volume) with phosphate-buffered saline (PBS). Then, each sample received 10 ml of Ficoll-Paque separation solution and was centrifuged at 2000g and 20 °C for 10 min. The mononuclear cell layer was separated, placed in EGM-2 culture medium (Lonza, Basel, Switzerland) in fibronectin-coated plates pre-lined with fibronectin, and incubated in 5% CO₂ at 37 °C. The morphological changes of the cells were dynamically observed under an inverted fluorescence microscope (CKX53, Olympus, Tokyo, Japan). After 2 weeks, adherent cells were incubated in 2.4 µg/ml of acetylated low-density lipoprotein solution for 1 h, which was fixed with 20 g/l paraformaldehyde, incubated in a lectin antibody solution for 1 h, and observed and photographed under the fluorescence microscope. The red and green double-stained cells were identified as EPCs (so-called ‘early EPCs’) [24 (link)].