Figure 4. Pro-angiogenic potential of high density cultured cells in vitro and in vivo.

A, In vitro tube formation assay with Matrigel for the cells derived from high density (HD) and regular density (RD) groups with quantification of the number of branch points per field (n = 9). *p<0.05, Scale bars, 100 µm. B, An in vivo angiogenic assay was performed by transplantation of expanded cells into ischemic hind limbs of nude mice. Representative views of ischemic left hind limbs at 3 weeks after treatment with PBS, regular density cultured cells or high density cultured cells and percent distribution of outcomes after treatment (n = 10). C, Laser Doppler images of blood perfusion with mice in a supine position at day 1 after femoral artery ligation and at day 21 after cell transplantation or PBS injection. Restoration of blood flow was calculated by comparing the laser Doppler image data of ischemic left limbs with that of non-ischemic right limbs at day 21. A significant improvement of blood supply was achieved in the group treated with high density cultured cells (n = 4). *p<0.05. D, Blood vessels in hind limb adductor muscles were identified by ILB4 staining. A higher number of capillaries were observed in the group treated with high density cultured cells (n = 4). *p<0.05, Scale bars, 100 µm. E, Donor-derived cells were detected by CM-DiI labeling of hind limb adductor muscles. A higher number of CM-DiI⁺ cells were observed in the muscles treated with high density cultured cells (n = 5). *p<0.05, Scale bars, 100 µm.