CB samples from healthy full term newborns were obtained from the CB Bank of St Louis Hospital (Paris, France). Human APB from healthy adults was obtained from the French Establishment of Blood (EFS) (Rungis, France). Mononuclear cells (MNCs) were obtained by density gradient centrifugation using Pancoll human solution (Pan-Biotech).
For ECFC isolation, MNCs were seeded into rat-tail collagen type-I (BD-Bioscience) coated wells as previously described [26 (link)]. ECFC colonies appeared after 7–20 days of culture. From passage 1 (P1), cells were seeded at 5000 cells/cm2 and grew in EGM-2MV medium (Lonza).
Human pan T cell isolation kit (Miltenyi-Biotec) was used to isolate total CD3+ T cells from MNCs of peripheral adult blood. Furthermore, CD25+ cells were depleted from the CD3+ T cell population using anti-CD25 biotin conjugated antibody (Miltenyi-Biotec), followed by anti-biotin microbeads staining (Miltenyi-Biotec). Then, the magnetic-activated cell sorting (MACS) method was used in all cell isolations. The resulting CD3+CD25− T cells, more than 93% purity, were cultured in the presence of ECFCs. The isolation of T cells from co-culture in presence of ECs is based on the biological capacity of ECs to adhere to plastic plates, and T cells that stay in suspension; hence, they were collected with gentle resuspension and aspiration.
For ECFC isolation, MNCs were seeded into rat-tail collagen type-I (BD-Bioscience) coated wells as previously described [26 (link)]. ECFC colonies appeared after 7–20 days of culture. From passage 1 (P1), cells were seeded at 5000 cells/cm2 and grew in EGM-2MV medium (Lonza).
Human pan T cell isolation kit (Miltenyi-Biotec) was used to isolate total CD3+ T cells from MNCs of peripheral adult blood. Furthermore, CD25+ cells were depleted from the CD3+ T cell population using anti-CD25 biotin conjugated antibody (Miltenyi-Biotec), followed by anti-biotin microbeads staining (Miltenyi-Biotec). Then, the magnetic-activated cell sorting (MACS) method was used in all cell isolations. The resulting CD3+CD25− T cells, more than 93% purity, were cultured in the presence of ECFCs. The isolation of T cells from co-culture in presence of ECs is based on the biological capacity of ECs to adhere to plastic plates, and T cells that stay in suspension; hence, they were collected with gentle resuspension and aspiration.