Figure 4. Transwell migration assay: hDPSC significantly induced the migration of the endothelial cells HMEC-1.

A) Transwell system: hDPSC were seeded in the lower compartment. The next day, cells were put on α-MEM 0.1% FBS. 24 h later, the cell culture inserts with a filter (with a pore size of 8 µm) containing HMEC-1 were placed onto the wells with hDPSC. 24 h later, migration was assessed by fixing the cells in 4% PFA, staining with 0.1% crystal violet and pictures were taken. Scale bars = 200 µm. B) Graph showing the effect of different conditions on the HMEC-1 proliferation (Y-axis = the area occupied by violet stained HMEC-1, in %). hDPSC significantly (p<0.001) induced HMEC-1 migration; This migration could be partially inhibited by addition of anti-VEGF antibodies but not by addition of MCP-1 antibodies. C) Graph presenting the effect of PI3K-inhibitor LY294002 and the MEK-inhibitor U0126 (10 and 1 µM) on hDPSC-induced HMEC-1 migration. (HMEC-1 migration caused by hDPSC was set at 100%). Both LY294002 and U0126, were able to significantly decrease the hDPSC-induced HMEC-1 migration at a concentration of 10 µM. Addition of only 1 µM of these inhibitors, slightly reduced the migration, but this reduction was not statistically significant. When both LY294002 and U0126 at a concentration of 10 µM each were added, the transwell migration was completely inhibited. This experiment was performed 5 times with hDPSC of at least 5 different donors.