Fig. 4

IL10 promotes mesoderm induction and vascular cell differentiation from hiPSCs through activating STAT3 signaling. (A) IL10 increases STAT3 phosphorylation and nuclear accumulation in mesoderm cells. Day 1 differentiating hiPSCs were cultured in the absence (ctrl) or presence of 20 ng/ml IL10 for 3 days. Cells were fixed and subjected to immunofluorescence (IF) staining with phospho-STAT3 (p-STAT3) antibody. (B) IL10 increases the transcriptional activation of STAT3. Day 1 differentiating hiPSCs were cultured in the absence (ctrl or IgG) or presence of 20 ng/ml IL10 or 1 µg/ml anti-IL10 antibody for 3 days. Nuclear proteins were isolated and subjected to STAT3 transcription factor activation analysis using a commercially available kit (Abcam, ab207229). (C) Luciferase activity analysis of gene promoters. Upper panel, schematic diagram of wild-type (pGL3-huT/huTAGLN/huCD144/hueNOS-WT) and STAT3 binding site mutated (pGL3-huT/huTAGLN/huCD144/hueNOS-Mut) gene promoter reporters. Bottom panel, luciferase activity assays with the indicated reporters. Day 1 differentiating hiPSCs were transfected with individual gene promoter vectors and treated with Ctrl or 20 ng/ml IL10 for 3 days. Cell lysate were harvested and subjected to dual-luciferase activity analysis. (D) Chromatin immunoprecipitation (ChIP) assays showing IL10 promotes the direct binding of p-STAT3 to the indicated gene promoters. Day 1 differentiating hiPSCs treated with vehicle control or 20 ng/ml IL10 for 3 days were collected and subjected to ChIP assay using 5 µg IgG control or p-STAT3 antibody. Immunoprecipitaed DNAs were quantified by qPCR using specific primers for indicated gene promoters. (E-F) STAT3 inhibition abolish IL10-mediated gene regulation. Day 1 differentiating hiPSCs were incubated with vehicle control (DMSO) and 2 μM STAT3 inhibitor (S3I-201) in the absence or presence of 20 ng/ml IL10 during mesodermal stage. (E) Cells were harvested at the end of mesoderm induction stage for detecting STAT3 transcription factor activity as detailed in (B). (F) In additional sets of experiments, cells were collected at day 4 or day 8 and subjected to RT-qPCR analysis for detecting T gene (day 4) and vascular cell genes expression (SM22α, CD144, and eNOS), respectively. The data presented here are mean±S.E.M of five independent experiments (n=5). *P<0.05 (vs Ctrl), #P<0.05 (vs WT or Vehicle).