Endothelial cell attachment factor

For endothelial differentiation, CSC were plated at a density of 50.000 cells/well into 6-well culture plated coated with Endothelial Cell Attachment Factor (Sigma-Aldrich), in EndoGRO (Merck Millipore) and maintained in hypoxia (1% O₂ and 5% CO₂) in hypoxia chambers (Stem Cells Technologies) for 14 days, as previously described [31 (link)]. CSC-derived TEC were maintained in culture for further 20 passages without observing any loss of phenotype.

Epithelial differentiation was obtained in the presence of RPMI plus 10% FCS, without the addition of growth factors, as previously described [15 (link), 16 (link)]. For endothelial differentiation, B-CSC and R-CSC were plated into 6-well culture plated coated with Endothelial Cell Attachment Factor (Sigma-Aldrich), in Endogro (Merck Millipore) without growth factor supplement and maintained in hypoxia (1% O₂ and 5% CO₂) in hypoxia chambers (Stem Cells Technologies) for 14 days. The anti-angiogenic drugs (Sunitinib 1 μM, Bevacizumab 100 μg/ml) and the anti-VEGFR1 or anti-VEGFR-2 blocking polyclonal Ab, or an irrelevant rabbit serum (all from R&D Systems), all at 0.2 μg/ml, were added to cell cultures under hypoxic differentiation at day 0 and every three days thereafter.