Redox status in mammalian cells and stem cells during culture in vitro: Critical roles of Nrf2 and cystine transporter activity in the maintenance of redox balance

• Rapid air-oxidation of cysteine to cystine occurs in culture media.
• Cystine transporter activity maintains cellular GSH and cysteine pools.
• Nrf2 and ATF4 upregulate expression of the cystine transporter xCT.
• Many lymphoid and stem cells lack cystine transporter activity.
• Fibroblast feeder cells can supply cysteine to cystine transporter deficient cells.

Culturing cells and tissues in vitro has provided valuable insights into the molecular mechanisms regulating redox signaling in cells with implications for medicine. However, standard culture techniques maintain mammalian cells in vitro under an artificial physicochemical environment such as ambient air and 5% CO2. Oxidative stress is caused by the rapid oxidation of cysteine to cystine in culture media catalyzed by transition metals, leading to diminished intracellular cysteine and glutathione (GSH) pools. Some cells, such as fibroblasts and macrophages, express cystine transport activity, designated as system xc−xc−, which enables cells to maintain these pools to counteract oxidative stress. Additionally, many cells have the ability to activate the redox sensitive transcription factor Nrf2, a master regulator of cellular defenses against oxidative stress, and to upregulate xCT, the subunit of the xc−xc− transport system leading to increases in cellular GSH. In contrast, some cells, including lymphoid cells, embryonic stem cells and iPS cells, express relatively low levels of xCT and cannot maintain cellular cysteine and GSH pools. Thus, fibroblasts have been used as feeder cells for the latter cell types based on their ability to supply cysteine. Other key Nrf2 regulated gene products include heme oxygenase 1, peroxiredoxin 1 and sequestosome1. In macrophages, oxidized LDL activates Nrf2 and upregulates the scavenger receptor CD36 forming a positive feedback loop to facilitate removal of the oxidant from the vascular microenvironment. This review describes cell type specific responses to oxygen derived stress, and the key roles that activation of Nrf2 and membrane transport of cystine and cysteine play in the maintenance and proliferation of mammalian cells in culture.

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