In Vitro Angiogenesis Assay

Fifty thousand cells of transduced MSCs were plated in 1 mL of Endothelial Basal Media with 2% FCS. Conditioned media was collected two days later. Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (C2517A; Basel, Switzerland). Cells were maintained in culture in the supplier's recommended complete medium (EGM-2) at 37°C, 5% CO₂. Five to ten thousand HUVECs were plated in 100 mL of conditioned media on Matrigel (Millipore, Billerica, Massachusetts). Cells were incubated for 4 to 6 h at 37°C and were visualized by light microscopy. The amount of angiogenesis was quantified by counting the number of cells in branch point capillaries in five random fields per replicate.

Five thousand transduced MSCs were also co-plated with five thousand PKH26-labeled murine bEND.3 endothelial cells (American Type Culture Collection, Manassas, Virginia) on Matrigel. Ten thousand PKH26-labeled mouse bEND.3 endothelial cells were also plated alone under the same conditions. Experiments were performed in triplicates. Fluorescence microscopy was performed with a Leica DMI4000 B microscope, and the amount of tubulogenesis was quantified as described above.