ESCs were differentiated using hanging drop method. Degree of cardiac differentiation was determined by the appearance of beating embryoid bodies (EBs) and by the expression of cardiac genes using real-time PCR and Western blot. Intracellular ROS level was examined by confocal imaging.
H2O2-treated EBs were found to have enhanced cardiac differentiation in the long run as reflected by, firstly, an earlier appearance of beating EBs, and secondly, an upregulation in cardiac structural protein expression at both mRNA and protein levels. Also, ROS upregulated the expression of several cardiac-related transcription factors, and increased the post-translationally-activated transcription factors SRF and AP-1. IL-1¦Â, IL-10, IL-18 and TNF-¦Á upregulated the expression of cardiac structural proteins and increased the ROS level in differentiating EBs. In addition, ROS scavenger reversed the cardiogenic effect of IL-10 and IL-18.
These results demonstrated that ROS enhance cardiac differentiation of ESCs through upregulating the expression and activity of multiple cardiac-related transcription factors. IL-1¦Â, IL-10, IL-18 and TNF-¦Á enhance cardiac differentiation and ROS may serve as the messenger in cardiogenic signaling from these cytokines.