Pluripotent stem cells (PSCs) derived cardiomyocytes provide an invaluable cell source for numerous important applications. PSC-cardiomyocytes may be transplanted into injured hearts to repair the tissue damage caused by myocardial infarction. Patients-specific induced PSCs (iPSCs) derived cardiomyocytes can serve as platforms for personalized cardiotoxicity test and drug screening. Further, in vitro cardiac differentiation process may be employed as a model for studying heart development. To achieve efficient and consistent cardiac differentiation, a wide range of biochemical (e.g., growth factors and small molecules) and physical stimuli (e.g., mechanical stress and electrical stimulation) have been explored with various degrees of success. Reactive oxygen species (ROS) are known to be critical in cell signaling, mainly via their modifications of protein activities. ROS have been shown to be indispensable for cardiac differentiation. In this dissertation, we seek to utilize ROS as a potential new tool in the control of cardiac differentiation, maturation and removal of undifferentiated cells. Specifically, we modulated ROS by changing glucose level and/or applying antioxidants. Our results showed that differentiation of mouse embryonic stem cells (mESCs) in high glucose medium without any antioxidants resulted in high level of cellular ROS, and also increased cardiac differentiation efficiency from about 15% to over 40%, along with increased expression of cardiac markers such as NKX2.5 and MESP1. More interestingly, when mESCs were differentiated in the presence of commonly used thiol-containing antioxidants such as beta-mercaptoethanol (BME) or monothiol glycerol (MTG), the resulting cardiomyocytes showed lower TNNI3/TNNI1 expression ratio, lower beating frequency and slower contraction velocity, suggesting delayed cardiac maturation. In contrast, Trolox, a vitamin E derivative and a non-thiol antioxidant, did not have these effects despite its strong antioxidant efficacy. These results suggest that redox regulation of cardiac differentiation is not only dictated by overall cellular ROS, but fine-controlled on a subcellular level. Further, ROS may also be utilized to promote desired cellular apoptosis. When co-treated with GSK3 inhibitors and antioxidants such as N-acetyl cysteine (NAC), human iPSCs underwent rapid and extensive apoptosis. This unique effect might be leveraged to remove iPSCs from their differentiated descendants to minimize the future risk of tumorigenesis.Biomedical Engineerin
