The core functions of stem cells (SCs) are critically regulated by their cellular redox status. Glutathione is the most abundant non-protein
thiol functioning as an antioxidant and a redox regulator. However, an investigation into the relationship between glutathione-mediated
redox capacity and SC activities is hindered by lack of probe. Here, we demonstrate that cyanoacrylamide-based coumarin derivatives are
ratiometric probes suitable for the real-time monitoring of glutathione levels in living SCs. These probes revealed that glutathione levels
are heterogeneous among subcellular organelles and among individual cells and show dynamic changes and heterogeneity in repopulating
SCs depending on oxidative stress or culture conditions. Importantly, a subpopulation of SCs with high glutathione levels exhibited
increased stemness and migration activities in vitro and showed improved therapeutic efficiency in treating asthma. Our results indicate
that high glutathione levels are required for maintaining SC functions, and monitoring glutathione dynamics and heterogeneity can
advance our understanding of the cellular responses to oxidative stress.