Dual Bioelectrical Assessment of Human Mesenchymal Stem Cells Using Plasma and Mitochondrial Membrane Potentiometric Probes

Background: Bioelectrical properties are known to impact stem cell fate, state, and function. However, assays that measure bioelectrical properties are generally limited to the plasma membrane potential. In this study, we propose an assay to simultaneously assess cell plasma membrane and mitochondrial membrane potentials. Materials and Methods: Mesenchymal stem cell (MSC) plasma and mitochondrial membrane potentials were measured using flow cytometry and a combination of tetramethylrhodamine, methyl ester (TMRM), and bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC) dyes. We investigated the shifts in the bioelectrical phenotype of MSCs due to extended culture in vitro, activation with interferon-gamma (IFN-γ), and aggregate conditions. Results: MSCs subjected to extended culture in vitro acquired plasma and mitochondrial membrane potentials consistent with a hyperpolarized bioelectrical phenotype. Activation with IFN-γ shifted MSCs toward a state associated with increased levels of both DiBAC and TMRM. MSCs in aggregate conditions were associated with a decrease in TMRM levels, indicating mitochondrial depolarization. Conclusions: Our proposed assay described distinct MSC bioelectrical transitions due to extended in vitro culture, exposure to an inflammatory cytokine, and culture under aggregate conditions. Overall, our assay enables a more complete characterization of MSC bioelectrical properties within a single experiment, and its relative simplicity enables researchers to apply it in variety of settings.