Figure 3.
Vascular Factors Increase CBSC Endothelial Differentiation In Vitro
(A) Epifluorescent detection of GSA I (ECs; red) in adherent NS cultured on fibronectin in standard neural crest culture medium without mitogens (CTRL) and supplemented with vascular or neuronal factors. Scale bar, 200 μm.
(B) Quantification graph indicating the mean percentage of GSA I+ cells among total cells in different culture conditions. Data are represented as the mean ± SEM.; ∗p ≤ 0.05, ∗∗p ≤ 0.01; # p ≤ 0.001, one-way ANOVA Newman-Keuls post hoc test compared to CTRL measurement. n = from three to nine independent NS cultures.
(C and D) Graphs representing a significant increase in mRNA expression of two classical hypoxia-inducible genes, VEGF (C) and GLUT1 (D). n = 4 independent NS cultures. In vitro hypoxia experiments were performed at 3% O2. Data are represented as mean ± SEM.
(E) RT-PCRs revealing expression of mature endothelial markers in rat liver and in GSA I+ cells isolated from CB or adherent NS by flow cytometry.
(F) Agarose gel showing TH mRNA amplified by RT-PCR in GSA - cell fraction isolated from adherent NS. Three independent RNA extractions were tested for each gene expression analysis.
(G) Binding of FITC-GSA I and uptake of DiI-acetylated LDL (Ac-LDL) in ECs in vitro, visualized by fluorescence microscopy. Nuclei are counterstained with DAPI (blue). Scale bars, 50 μm (top) and 25 μm (bottom). n = 3 NS cultures.
See also Figures S3, S4, and S6.