Fig. 3

IL10 inhibition reduces mesoderm induction and decreases vascular cell differentiation from hiPSCs. (A) Experimental protocol for IL10 inhibition. Day 1 differentiating hiPSCs were cultured in mesoderm induction medium supplemented with 1 μg/ml IL10 functional neutralizing antibody (Anti-IL10) or IgG control (IgG) for 3 days, followed by incubation in EC or SMC differentiation medium for additional 4 days. Differentiating cells were collected at day 4 (for mesodermal gene detection) and 8 (detecting EC/SMC marker genes), and subjected to RT-qPCR (B-D) and Western blot (E-F) analysis, receptively. (B-D) RT-qPCR analysis of gene expression at mesoderm (MESO, day 4) stage (B), and EC/SMC differentiation stage (day 8) (C & D), respectively. (E-F) Western blot analysis of EC & SMC marker proteins at day 8 of differentiation. (G-J) EC functional assays. Equal number of day 8 differentiating ECs were subjected to Matrigel tube formation (G-H) as well as ac-LDL uptake and lectin binding (I-J) assays, respectively. MFI, mean fluorescence intensity. The data presented here are representative (E, G, I) or mean±S.E.M (B-D, F, H, J) of three or six independent experiments (n=3 or 6). *P<0.05 (vs IgG).