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. 2017 Jan 30;5:2. doi: 10.3389/fcell.2017.00002

Figure 4.

Figure 4

Functional characterization of prevascularized CCCs by Dil-Ac-LDL uptake assay. hCMVECs cultured or hCMVECs/hMSCs co-cultured onto CCCs in vasculogenic culture conditions were incubated with 10 μg/ml of Dil-Ac-LDL for 4 to 6 h. Fluorescence microscopic analysis of hCMVECs only CCCs revealed abundant punctate perinuclear bright red fluorescence of differentiated and matured endothelial cells (day 7, B,C; day 14, E,F). These labeled vascular cells were organized into a delicate network of capillaries (A–C) or into a discrete cluster (D–F). Similarly, fluorescence microscopic analysis of hCMVECs/hMSCs co-cultured CCCs demonstrated typical abundant punctate perinuclear bright red fluorescence of the differentiated and matured endothelial cells (day 7, H,I; day 14, K,L). These Dil-labeled endothelial cells were structured into solid cohesive columns of VE-cadherin positive cells, mimicking functionally competent larger caliber vessels (J–L). Cells were also stained for nuclei (blue, DAPI) and endothelial cells (green, VE-cadherin, A,C,D,F,G,I, J,L). Merged Images (A–L). (A–L, scale bar 100 μm).