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Monitoring Glutathione Dynamics and Heterogeneity in Living Stem Cells.

ABSTRACT: Glutathione (GSH) is a major antioxidant in cells, and plays vital roles in the cellular defense against oxidants and in the regulation of redox signals. In a previous report, we demonstrated that stem cell function is critically affected by heterogeneity and dynamic changes in cellular GSH concentration. Here, we present a detailed protocol for the monitoring of GSH concentration in living stem cells using FreSHtracer, a real-time GSH probe. We describe the steps involved in monitoring GSH concentration in single living stem cells using confocal microscopy and flow cytometry. These methods are simple, rapid, and quantitative, and able to demonstrate intracellular GSH concentration changes in real time. We also describe the application of FreSHtracer to the sorting of stem cells according to their GSH content using flow cytometry. Typically, microscopic or flow cytometric analyses of FreSHtracer and MitoFreSHtracer signals in living stem cells take ~2~3 h, and the fractionation of stem cells into subpopulations on the basis of cellular GSH levels takes 3~4.5 h. This method could be applied to almost every kind of mammalian cell with minor modifications to the protocol described here.

SUBMITTER: Jeong EM

PROVIDER: S-EPMC6657947 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A remarkable feature of the self-renewing population of embryonic stem cells (ESCs) is their phenotypic heterogeneity: Nanog and other marker proteins of ESCs show large cell-to-cell variation in their expression level, which should significantly influence the differentiation process of individual cells. The molecular mechanism and biological implication of this heterogeneity, however, still remain elusive. We address this problem by constructing a model of the core gene-network of mouse ESCs. The model takes account of processes of binding/unbinding of transcription factors, formation/dissolution of transcription apparatus, and modification of histone code at each locus of genes in the network. These processes are hierarchically interrelated to each other forming the dynamical feedback loops. By simulating stochastic dynamics of this model, we show that the phenotypic heterogeneity of ESCs can be explained when the chromatin at the Nanog locus undergoes the large scale reorganization in formation/dissolution of transcription apparatus, which should have the timescale similar to the cell cycle period. With this slow transcriptional switching of Nanog, the simulated ESCs fluctuate among multiple transient states, which can trigger the differentiation into the lineage-specific cell states. From the simulated transitions among cell states, the epigenetic landscape underlying transitions is calculated. The slow Nanog switching gives rise to the wide basin of ESC states in the landscape. The bimodal Nanog distribution arising from the kinetic flow running through this ESC basin prevents transdifferentiation and promotes the definite decision of the cell fate. These results show that the distribution of timescales of the regulatory processes is decisively important to characterize the fluctuation of cells and their differentiation process. The analyses through the epigenetic landscape and the kinetic flow on the landscape should provide a guideline to engineer cell differentiation.

Dataset Information

Monitoring Glutathione Dynamics and Heterogeneity in Living Stem Cells.

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