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Fig. 4 | Stem Cell Research & Therapy

Fig. 4

From: Development of a surrogate potency assay to determine the angiogenic activity of Stempeucel®, a pooled, ex-vivo expanded, allogeneic human bone marrow mesenchymal stromal cell product

Fig. 4

Functional in vitro HUVEC migration, proliferation and tube formation with rhVEGF and phBMMSC CM, followed by inhibition of VEGF activity. a Transwell migration of HUVECs in response to rhVEGF serially diluted from 8 ng/ml to 31.2 pg/ml in SFM. Significant inhibition of HUVEC migration was observed in 2 ng/ml rhVEGF with anti-VEGF mAb (10 μg/ml) compared to migration observed with 2 ng/ml rhVEGF concentration, **p = 0.001. b Transwell migration of HUVECs in response to phBMMSC CM serially diluted with SFM from 100% (4 ng/ml of secreted VEGF) to 0.19% (7.8 pg/ml of secreted VEGF). The maximum migration of HUVECs was observed at 50% dilution comprising 2 ng/ml secreted VEGF which was significantly inhibited by anti-VEGF mAb (10 μg/ml) in comparison with phBMMSC CM + IgG isotype, *p = 0.02, and phBMMSC CM, **p = 0.0009. Threshold for HUVEC proliferation was observed at 3.125% CM dilution with 125 pg/ml VEGF. c Proliferation of HUVECs in response to rhVEGF serially diluted starting from 8 ng/ml in SFM. rhVEGF did not effectively induce proliferation and therefore no measurable effect was observed with the anti-VEGF mAb. d HUVEC proliferation with phBMMSC CM serially diluted from 100% to 0.19% in SFM. Addition of anti-VEGF mAb to the 50% CM significantly reduced proliferation, **p = 0.001, in comparison with CM containing IgG isotype, *p = 0.04. Proliferation in HUVECs initiated at 0.78% CM dilution with 31.2 pg/ml VEGF. eq HUVEC tube formation in response to phBMMSC CM serially diluted from 100% to 0.19%. Maximum tube formation was observed at 50% dilution containing 2 ng/ml secreted VEGF. Significant reduction in tube formation was observed when anti-VEGF mAb was used in 50% diluted CM, while IgG isotype did not alter the tube formation of HUVECs. r Graphical representation of tube formation induced by phBMMSC CM at various concentrations. Tube formation seemed to initiate at 0.78% CM dilution. Statistical significance was determined using Student’s t test. Data presented as mean ± SEM. SEMs shown as error bars. CM conditioned medium, phBMMSC pooled human bone marrow derived mesenchymal stromal cell, SFM serum-free medium, VEGF vascular endothelial growth factor, HPF high-power field

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