Redox environment in stem and differentiated cells: A quantitative approach

Abstract

Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa) maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H2DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed.

High doses of synthetic antioxidants induce premature senescence in cultivated mesenchymal stem cells

“…To this end, eMSCs were stained with a fluorescein-containing cell-permeable probe 2′, 7′- dichlorodihydrofluorescein diacetate (H 2 DCFDA). H 2 DCFDA becomes fluorescent in the result of the multi-step intracellular oxidation process and is routinely used as a general indicator of the cellular redox-status 46,47 . Fluorescence signal measured with flow cytometer in the cells treated with the aforementioned AOs, excluding Tempol, was lower than that in the control cells (Fig.…”

Measuring intracellular concentration of hydrogen peroxide with the use of genetically encoded H2O2 biosensor HyPer

“…To monitor the extracellular H 2 O 2 concentration in the course of kinetic measurements, Amplex ® Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen) was used in accordance with the procedure described in Ref. [11]. At H 2 O 2 concentration of 5 μM and cell density of 15 000 cells/ml, the decrease in H 2 O 2 concentration in the extracellular medium did not exceed several percent within 10 min after the peroxide addition to cells and therefore was neglected in the analysis of HyPer oxidation.…”

Loss of heme oxygenase-1 accelerates mesodermal gene expressions during embryoid body development from mouse embryonic stem cells

“…Given that ESCs are characterized by low ROS level [33] , it is not surprising that HO-1 is not required for maintaining pluripotency. As such, we were able to establish HO-1 –/– ESCs displaying pluripotent markers ( Fig.…”

“…Furthermore, it is well recognized that redox homeostasis regulates stem cell renewal and differentiation [32] . ESCs are characterized by low reactive oxygen species (ROS) level and a low capacity to remove extracellular hydrogen peroxide, assessed by a quantitative redox biology approach [33] . During cardiomyocyte differentiation from ESCs, ROS levels are increased [34] .…”