Table 1.
RT-PCR primers.
Table 2.
Primary antibodies for immunohistochemistry.
Figure 1.
Antibody array on both conditioned medium (A) and cell lysates (B) of hDPSC.
48 hour conditioned medium and cell lysates were prepared from hDPSC. Angiogenesis-related proteins present were identified with an antibody array (A, conditioned medium; B, cell lysates). Graph C indicates the relative expression of each factor while table D shows the location of the antibodies spotted on the human angiogenesis antibody array. In red the proteins that were detected in the CM or in the cell lysates. Numerous pro-angiogenic factors such as VEGF and MCP-1 but also anti-angiogenic factors such as endostatin were found in both CM and cell lysates while FGF-2 was only present in the cell lysate. This assay was performed twice with CM and cell lysates of two different donors.
Figure 2.
A) RT-PCR confirmation of the expression various angiogenesis related molecules.
mRNA was isolated from hDPSC of 6 different donors and RT-PCR was performed. Housekeeping genes β-actin, GusB and β2-microglobulin were used as a control for the PCR reaction. All donors expressed VEGF, IGFBP-3, uPA, PAI-1 and TIMP-1, while endostatin, IL8 and MCP-1 where found in 5 of the 6 donors. B) Immunofluorescence (IF) on whole pulp tissue. Dental pulp tissue of 4 different donors were fixed in 4% PFA, embedded in paraffin and 7 µm sections were made. Subsequently, immunofluorescent stainings were performed with antibodies against the angiogenic factors (VEGF, MCP-1 and FGF-2, red) and against the endothelial cell and hDPSC marker CD146 (green). Nuclei were counterstained with DAPI (blue). Pictures show a representative staining of one donor. It is very clear that the dental pulp cells are slightly positive for CD146, while the endothelial cells that form the walls of the blood vessels express high amounts of CD146. Furthermore, almost all cells present within the human dental pulp tissue were found to express VEGF, MCP-1 and FGF-2 in situ. Scale bars = 50 µm.
Table 3.
Concentration of various angiogenesis related factors in the conditioned medium of hDPSC (as determined by ELISA).
Figure 3.
Cell proliferation (MTT) assay: MBEC or HMEC-1 endothelial cells were seeded in 96-well plates and 24 hours later the CM of hDPSC was added.
αMEM containing 10% FBS was included in the study as a positive control. 72 h later, the media were removed and cell proliferation was assessed. Conditioned medium of hDPSC was not able to increase the cell growth of neither MBEC nor HMEC-1 compared to control medium (with 0.1% FBS). Incubation of αMEM with 10% FBS resulted in a 3-fold increase in cell growth in both MBEC and HMEC-1. This assay was repeated 3 times with a total number of at least six different donors; *p<0.05; **p<0.01.
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.
Figure 5.
Chorioallantoic membrane (CAM) assay.
At day 9 of embryonic development, matrigel droplets with or without 50,000 hDPSC were applied onto the CAM of a chicken embryo (black arrow, Figure 5a). 72 hours later, pictures were taken (figure 5c–e). Figure 5c is a control matrigel droplet, while Figure 5d is a droplet containing 50,000 hDPSC. White scale bars represent 1 mm. To assess angiogenesis, two circles (with a radius of 1.5 and 2 mm, see figure 5e), were digitally positioned around the matrigel droplets and the blood vessels intersecting these circles were counted double blind. Graph 5b: hDPSC were able to significantly increase the number of capillaries intersecting both circles. This assay was repeated 4 times with hDPSC of 4 different donors to gain a database of at least 27 individual eggs. Values are represented as means ± S.E.M.