iECs derived from the C1-2 hiPSC line were analyzed for sprouting abilities. a Schematic is outlining the experimental workflow. b Representative confocal images of HUVEC (left) and iEC (right) sprouting spheroids after 24 h in collagen gel in media supplemented with or without VEGF. Arrows indicate disconnected cells. c Quantification of the average number (#) of sprouts per spheroid and d average sprout length (N = 3, n = 25–30). e Extracellular Acidification Rate measured via Seahorse Assay in C1-2 iECs and HUVECs (red, non-glycolytic acidification; green, glycolysis; purple, glycolytic max; N = 3, n = 9). f Western blot analysis of glycolysis enzymes and g quantification, N = 3. Statistical significance levels are set at *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 by two-tailed Student’s t-test and Sidak’s multiple comparison test. For bar graphs: data are presented as mean ± SD. For box and wisker plots: centerline, median; box limits, upper and lower quartiles; wiskers, minimum to maximum value. Scale bar: 100 μm.

a Filopodia formation in C1–2 iECs and HUVECs was assessed using F-actin and Myosin X stain followed by confocal microscopy of dispersed cells. b Quantification of filopodia was analyzed using an F-actin stain with the Image J plug-in FiloQuant. (N = 3, n = 45). Graph plotted without outliers. c Schematic of the microfluidic device with amplification of the 2D cell culture and migration channels region and d image of C1-2 iEC migration in the channels with and without a VEGF gradient at t = 6 h. Arrows indicate iECs migrating through the channels. The boxed images show cells at t = 0. e Quantification of cell entry and velocity in the channel of individual cells (N = 3, n = 105–120). Statistical significance levels are set at *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 by two-tailed Student’s t-test and Tukey’s multiple comparison test. Data are presented as mean ± SD. Scale bar: 100 μm.

a RT-qPCR data for VEGFR2 normalized to HUVECs. (N = 3; n = 9). b Representative flow cytometry data for VEGFR2 in HUVECs (blue, 6.63%) and C1-2 hiPSC-ECs (green, 99.05%) (N = 3). c Western blot analysis of VEGFR2 phosphorylation at tyrosine 996 and 1175 and d quantification. (N = 3). e RT-qPCR data for VEGF normalized to C1-2. (N = 3, n = 9). f VEGFR2 expression in HUVECs cultured with and without VEGF (50 ng/ml) and the TGFβ inhibitor, SB431542. (N = 3) Statistical significance levels are set at *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 by two-tailed Student’s t-test and Tukey’s multiple comparison test. Data are presented as mean ± SD. Scale bar: 100 μm.

a Representative confocal images of iEC spheroids cultured in ECGM (left), ECGM supplemented with the VEGFR2 inhibitor, ZM323881 (middle), and ZM323881 with VEGF (50 ng/ml; right). b Quantification of spheroid sprout number and length in spheroids cultured in culture media supplemented with VEGF (solid), and spheroids cultured in culture media supplemented VEGF and ZM323881 (striped). (N = 3, n = 15). c Extracellular Acidification Rate measured via Seahorse Assay in C1-2 iECs with or without ZM323881 pretreatment (N = 2, n = 6). d Western blot for HK1 and HK2 in hiPSC-ECs cultured in ZM323881 or control conditions and e quantification, N = 2. f Heat map for RT-qPCR results of mRNA expression displayed as ddCT normalized to iECs N = 2. Statistical significance levels are set at *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 by two-tailed Student’s t-test and Tukey’s multiple comparison test. For bar graphs: data are presented as mean ± SD. For box and whisker plots: centerline, median; box limits, upper and lower quartiles; whiskers, minimum to maximum value. Scale bar: 100 μm.

RT-qPCR for VEGFR2 in C1-2 iECs treated with a TSA, b CPTH6, and c C646. (N = 3, n = 9). d Schematic is outlining of P300 HAT VEGFR2 regulation and C646 inhibition in iECs. Schematic created with BioRender.com. e Flow cytometry for VEGFR2 on iECs treated with C646 for varying periods (C-Control, ECGM; VC-Vehicle Control, DMSO; 1,3,18, and 24 h) N = 3. f ChIP qPCR data are shown as binding events per 1000 cells to the +57 K location at the VEGFR2 transcriptional start site. N = 2. g Representative confocal microscopy images of C1-2 iECs spheroids cultured in media supplemented with VEGF or VEGF and C646. (N = 2, n = 10) (N = 2, n = 10). Statistical significance levels are set at *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 and Tukey’s multiple comparison test by two-tailed Student’s t-test and Tukey’s multiple comparison test. Data are presented as mean ± SD. Scale bar: 100 μm.

a Schematic outlining zebrafish caudal fin regeneration assay. Schematic created with BioRender.com. b Representative images of caudal fin regeneration of control (top) and iEC injected (bottom) at 6 dpf and 9 dpf and c quantification including controls (n = 35–64). d Representative images of C646 iEC injected zebrafish at 6 dpf and 9 dpf and e quantification including controls (n = 28–33). Statistical significance levels are set at *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 by Tukey’s multiple comparison test. Data are presented as mean ± SD. Scale bar: 100 μm.