Fig 1: Isolation and characterization of EC clones from CD117+ ECs. (A) Isolation and clonal enrichment flow diagram for EC clones from the lungs of EGFP+ rats. (B) Representative differential interference contrast (DIC) image shows the typical endothelial cobblestone morphology of EC clones. (C) Representative DIC image of 2D matrigel assay. (D) Representative optical section (confocal microscopy) demonstrate binding of Griffonia simplicifolia lectin (red pseudo colour) in EC clones, indicating a microvascular phenotype (scale bar 25 μm). (E) 24 h 3D tube formation assay in fibrin shows formation of tube networks by EC clones (scale bar 200 μm). Cells were visualized by phalloidin staining of actin filaments (red pseudo colour). (F) confocal imaging of Matrigel plug at day 14 with EC clones stained for GFP (green pseudo colour) demonstrating GFP+ blood vessels. The red autofluorescence demonstrates blood and indicates active perfusion of the GFP+ vascular structures after 14 days. Scale bar: 25 μm. Counterstaining in (D–F) with DAPI. (G,H) Representative images demonstrate that EC clones form free-floating spheroids in low adhesion cell culture wells. When overlaid with a 50Vol%/50Vol% EGM2/Matrigel mixture, EC clone spheroids underwent angiogenic sprouting. The image (G) shows green fluorescence channel of EC clone spheroids (green pseudo colour), whereas image (H) shows DIC brightfield image of an EC clone spheroid after 7 days of sprouting. Scale bars: 50 μm. (I) Representative flow cytometry analysis of EC clones shows expression of endothelial markers (vWF, CD144, VEGFR2, CD105, CD34) and CD117, but not of typical hematopoietic and myeloid markers (CD45, CD11b/c and CD133). Note that vWF is an intracellular marker and required permeabilization of the cells during the staining process. (J) The fraction of clonally expandable wells increases from clonal generation 1 to 3. n = 3 (1st) and 6 (2nd, 3rd, 4th). (K) 4th generation EC clones proliferate faster than non-expanded CD117+ ECs (n = 3–4). *P < 0.05, **P < 0.01.