Intracellular Oxidation in Murine Stem Cells Due to O2 and Glucose Culture Conditions

Clinical realization of stem cell therapies requires more knowledge at the mechanistic level to control differentiation or maintain pluripotency to yield cellular products with low heterogeneity. Recent characterization of metabolism at different stages of pluripotency suggests that directing embryonic stem cells (ESCs) towards specific phenotypes can be augmented through the use of nutrient and oxygen culture conditions; however, it is unclear how these metabolic conditions synergize with growth factor supplementation and media inhibitors for ESC expansion or differentiation. In particular, a reactive oxygen species (ROS) byproduct of hypoxia and oxidative phosphorylation, H₂O₂, is a known modulator of MAPK and SMAD signal transduction cascades and thus may affect cell differentiation decisions. We hypothesize that hypoxia inducible factor (HIF) activated ROS allows modulation of signaling pathways, thereby allowing one to metabolically tune cellular phenotype using culture conditions in a reproducible manner. In order to test this hypothesis, we are quantifying the relative magnitude and variance of intracellular H₂O₂ levels in mESC and mEpiSC colonies under a range of oxygen and glucose conditions. The epiblast cell line is used as a comparison to hESCs because both use an Activin A/FGF-dependent mechanism for pluripotency maintenance, which is significantly different from the BMP/LIF-dependent mechanism in mESCs. A ratiometric sensor protein, HyPer, that provides quantitative intracellular measurements specific to H₂O₂, was stably transfected in the mESC-D3 cell line to provide unprecedented quantification of H₂O₂. The D3-HyPer cells were cultured in monolayer for three days under oxygen and glucose conditions relevant to diffusion limitations in vivo (20% and 5% O₂; 25 mM and 5.5 mM glucose-supplemented media). Colonies were imaged by live cell confocal microscopy and analyzed for ratiometric (oxidized/reduced) value and variance of cytosolic signal among cells. The mESC HyPer-D3 cell line showed statistically significant (p < 0.0001) increased ratios of oxidized/reduced HyPer for the low glucose conditions when compared to high glucose, regardless of the oxygen environment during culture. The ratio increased by 1.7x for low glucose/high oxygen when compared to high glucose/high oxygen and 1.5x for low glucose/low oxygen when compared to high glucose/low oxygen. Statistically significant differences (p < 0.0001) were observed when glucose supplementation was held constant and oxygen was the variable, but not to the same magnitude. This suggests that the highly glycolytic mESCs are more sensitive to glucose deprivation than hypoxia for eliciting cellular oxidation. Furthermore, despite reports of hypoxia-induced ROS in other cell types, no synergetic effects were noted in the low glucose/low oxygen condition. The mEpiSC HyPer-D3 cell line showed a statistically significant (p < 0.0001) increased ratiometric HyPer value for the low glucose/high oxygen condition when compared to all other conditions. Future studies will investigate how metabolic shifts between oxidative phosphorylation and glycolysis during pluripotent-to-epiblast and epiblast-to-germ layer transitions are reflected in the intracellular H₂O₂ levels and regulation pathways. Increased knowledge of how metabolism regulates signaling and pluripotent transcription factors may result in fundamental shifts in stem cell culturing protocols for expansion at the pluripotent state or directed towards lineage specification in a reproducible and homogeneous manner.