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. Author manuscript; available in PMC: 2016 Jan 20.
Published in final edited form as: Circulation. 2014 Oct 30;131(3):300–309. doi: 10.1161/CIRCULATIONAHA.113.007394

Figure 1.

Figure 1

Direct reprogramming of human fibroblasts to functional endothelial cells via activation of innate immunity and microenvironmental cues. (A) Protocol for direct reprogramming of human BJ fibroblasts to endothelial cells. The figure outlines the time course and sequential treatments: Human fibroblasts were treated with Poly I:C (30ng/ml) for 7 days in induction medium containing DMEM/FBS and 7.5% knockout serum replacement (KSR). Following 7 days, the medium was changed to transdifferentiation medium containing DMEM/FBS and 10% KSR containing 20ng/ml BMP4, 50ng/ml VEGF and 20ng/ml bFGF. After another 7 days, the medium was replaced with endothelial growth medium (EGM-2) containing 50ng/ml VEGF, 20ng/ml bFGF and 0.1 mM 8-Br-cAMP for another 14 days. The medium was changed every 2-3 days. Cells were then FAC sorted with CD31 and then expanded in EGM-2 medium containing SB431542, a specific TGFβ receptor inhibitor. (B) Fluorescent activated cell sorting (FACs) plot data obtained using CD31+ antibody to quantify iECs. (left panel) - vehicle control; (right panel) - Poly I:C. (C) Dot plot depicting FACs plot data obtained using CD31+ antibody to quantify iECs. All data represented as mean ± S.E.M., (n=3) **p<0.005 vs Vehicle control, standard unpaired student t test. (D) RT-PCR data showing iECs express endothelial markers CD31 compared to Fibroblasts (n=3) *p<0.05, 1-way ANOVA corrected with Bonferroni method. (E) Immunofluorescence staining of iECs for endothelial markers CD31. (F-G) iECs take up acetylated LDL and form capillary-like networks on matrigel. (H) iECs are capable of producing NO in response to acetylcholine and Ca2+ ionophore A23187 (n=3) *p<0.05, 1-way ANOVA corrected with Bonferroni method. (I) Angiogenic cytokines are produced by iECs when subjected to hypoxia (n=3, done in duplicates) *p<0.05, 1-way ANOVA corrected with Bonferroni method. (J) iECs form blood capillaries (arrows) in subcutaneous matrigel plugs. The numerous spaces in the figure are the areas of matrigel that are bounded by the capillaries.