Intracellular Reactive Oxygen Species Associated with Embryonic Stem Cell Differentiation

Reactive Oxygen Species (ROS) generated from mitochondrial respiration and other metabolic processes are important signaling molecules and have been linked to stem cell differentiation. Embryonic stem cells (ESCs) have the capacity to give rise to almost all cell lineages in the body, serving as a great system to study the mechanisms regulating cellular differentiation. ESCs maintain low ROS levels as compared with differentiated cells through glycolytic metabolism and enhanced antioxidant defense capability. However, how the redox status affects lineage specification during ESC differentiation remains elusive. Here, we have established a biosensor system to monitor the intracellular hydrogen peroxide (H₂O₂) level, a major form of ROS, in ESCs and cell aggregates. In this system, the cytoplasmic protein HyPer is stably expressed in ESCs and displays different spectral properties in presence of reducing and oxidizing agents. Therefore, ratiometric analysis of oxidized and reduced HyPer signals can be performed for individual cells in fluorescence microscopy images and the oxidized/reduced HyPer (Oxi/Red) ratio can serve as a sensitive indicator of the intracellular H₂O₂. We differentiated ESCs toward neural lineages via embryoid body formation and treatment with retinoic acid for 3 days followed by a neural differentiation regimen for another 5 days. Our results show that ~3-fold dynamic range in the Oxi/Red ratio for fully oxidized (1.31±0.47, hydrogen peroxide treated) vs. fully reduced HyPer signal (0.51±0.18, dithiothreitol treated, p < 0.0001). The baseline H₂O₂ level in ESCs, with an Oxi/Red ratio of 0.46(±0.14), is lower than reported in the literature for any other cell type, reflecting a highly reduced intracellular environment. In contrast, neural lineages derived from ESCs show stronger oxidized HyPer signal and weaker reduced signal, resulting in a statistically significant increase in the intracellular H₂O₂ level (Oxi/Red ratio = 0.79±0.17, p < 0.0001). The increase in H₂O₂ is consistent with known metabolic shifts to oxidative phosphorylation upon loss of pluripotency. ESCs also display a significant heterogeneity in intracellular H₂O₂, which may correlate with the differentiation potential of individual cells, as the oxidative response of the neural-derived cells demonstrated a smaller variance within the population. This unique imaging tool provides the opportunity to study spatiotemporal redox patterning within 3D cell aggregates and allows profiling the redox statuses in various cellular systems ranging from ESCs to differentiated cells. Future studies will provide significant insights into the lineage-specific redox regulation of stem cell differentiation.