Figure 3.

Fibrocartilage stem cells (FCSCs) induce proliferation of human umbilical vein endothelial cells (HUVECs), and direct FCSC-HUVEC association increases vessel caliber. FCSCs’ ability to support angiogenesis was evaluated in vitro using a fibrin-induced bead angiogenesis assay (FIBA). HUVECs were transduced with a lentivirus encoding red fluorescence protein (RFP). HUVEC-coated beads were embedded in fibrin gel using either D551 fibroblasts or FCSCs as a cell feeder layer. (a) Representative images of beads with HUVEC sprouts from FIBA. Scale bar = 50 µm. CellSense Imaging Software was used to quantify the (b) number of sprouts per bead, (c) vessel length, and (d) vessel caliber. Data represented are mean ± SD; n = 4 experiments; paired Student’s t test. (e) Growth curve of HUVECs cultured in conditioned media (CM) derived from fibrocartilage stem cells (FCSC-CM), D551 fibroblasts (D551-CMs), or bone marrow stromal cells (BMSC-CM). Data are mean ± SD; n = 6 experiments; *P ≤ 0.03, **P ≤ 0.002, ***P ≤ 0.0002, ****P ≤ 0.0001; 2-way analysis of variance followed by Tukey’s post hoc test. (f, g) The effect of direct HUVEC-FCSC contact was investigated in a FIBA experiment where HUVEC-coated beads were embedded ± FCSCs within the fibrin clot. CellSense Imaging Software was used to quantify the (f) vessel caliber. (g) Sequential confocal microscopic images of FIBA experiment with direct HUVEC-FCSC contact showing GFP⁺ FCSC (green) localized to the luminal side of blood vessels derived from HUVECs (red).