Multiplexed Optical Sensors for Redox Profiling in Human iPSC Models of Disease and Drug Response

About This Grant

ABSTRACT / SUMMARY Reactive oxygen species (ROS) and regulation of redox pathways are critical to human health and disease, as they influence cellular metabolism, signaling, and stress responses. Disruptions in redox homeostasis contribute to the pathophysiology of numerous disorders, including neurodegenerative diseases, muscular degeneration, and drug-induced cardiotoxicity. However, the tools for monitoring redox dynamics in living human cells remain limited in dimensionality, sensitivity, and applicability to disease-relevant models. To overcome these challenges, my research program aims to develop a next-generation, multiplexed optical platform for quantitative redox phenotyping and apply it to disease modeling and drug screening in human induced pluripotent stem cell (iPSC)- derived systems. Over the past five years, my lab has engineered two advanced genetically encoded hydrogen peroxide (H₂O₂) sensors, oROS-G and oROS-HT, exhibiting improved dynamic range, kinetics, and spectral flexibility. We established a high-throughput optical screening platform and integrated machine learning approaches to accelerate protein sensor engineering. These sensors have been applied in diverse host systems, including iPSC-derived neurons and cardiomyocytes, and have revealed new aspects of redox signaling in cell health. Building on this foundation, our future research will continue along three complementary directions. First, we will complete the development of a fully multiplexed, intensity-based TreDox sensor suite to simultaneously monitor oxidative pressure and antioxidant capacity with single-cell resolution in real time. Second, we will engineer lifetime-resolved redox biosensors and use fluorescence lifetime imaging microscopy (FLIM) to enable robust, expression-independent quantification of intracellular redox states. Third, using single-cell optical phenotyping, we will apply these tools to profile redox imbalances and early cytotoxicity signals in human iPSC- derived cardiomyocytes, neurons, and skeletal muscle cells. We aim to detect subtle cellular imbalances in redox pathways that precede cellular dysfunction and are often missed by traditional high throughput assays. This research program will fill critical gaps in our ability to study redox biology in human-derived host systems by integrating state-of-the-art protein engineering, advanced imaging, and human stem cell models. The tools and knowledge generated will improve our understanding of redox-linked disease mechanisms, enhance the predictive power of preclinical drug testing, and establish a flexible, generalizable platform for functional phenotyping at single-cell resolution.