Reactive oxygen species (ROS) and antioxidants are essential to maintain a redox balance within tissues and cells. Intracellular ROS regulate key cellular functions such as proliferation, differentiation and apoptosis through cellular signaling, and response to injury. The redox environment is particularly important for stem/progenitor cells, as their self-renewal and differentiation has been shown to be redox sensitive. However, not much is known about ROS and antioxidant protein function in freshly isolated keratinocytes, notably the different keratinocyte subpopulations. Immunostaining of neonatal cutaneous sections revealed that antioxidant enzymes [catalase, SOD2, gluthatione peroxidase-1 (GPx)] and ROS are localized predominantly to the epidermis. We isolated keratinocyte subpopulations and found lower levels of SOD2, catalase and GPx, as well as decreased SOD and catalase activity in an epidermal side population with stem cell-like characteristics (EpSPs) compared to more differentiated (Non-SP) keratinocytes. EpSPs also exhibited less mitochondrial area, fewer peroxisomes and produced lower levels of ROS than Non-SPs. Finally, EpSPs were more resistant to UV radiation than their progeny. Together, our data indicate ROS and antioxidant levels are decreased in stem-like EpSPs.

The epidermis is a continuously renewing epithelium that is maintained by the presence of stem/progenitor cells. Besides the well known pathophysiological impact of UV‐induced oxidative stress in cutaneous malignancy, redox state may play a role in basal homeostasis of the epidermis, and particularly in stem/progenitor cell differentiation. However, no data existed until now, because isolating epidermal stem/progenitor cells was problematic. We used our previously reported method to separate stem/progenitor cells from their progeny (transient amplifying cells, or TA), and measured the levels of several antioxidant enzymes. Using Western blot analysis, copper‐zinc superoxide dismutase (SOD) was not detected in any murine extracts. Furthermore, glutathione peroxidase was equally present in all groups. However, the levels of manganese SOD and catalase were decreased in the progenitor cell population, compared to the total epidermal population and the TA cells. These data were confirmed by enzymatic activity assays, showing that catalase activity was decreased in stem/progenitor cells compared to the TA cells. These results suggest that different levels of reactive oxygen species are present in these cells. These data support the view that the antioxidant defense of stem/progenitor cells was decreased compared to more differentiated cells, and is consistent with their slow cycling property.

The epidermis is a stratified cornified epithelium that protects the organism against physical and microbial insults. These environmental exposures can result in oxidative stress and affect cellular proliferation and differentiation. However, very few studies have evaluated the base line levels of reactive oxygen species (ROS) and antioxidant proteins in the epidermis or in the context of stem/progenitor cells, cellular types critical to the homeostasis of the epidermis. Here, we show that ROS are mainly produced by epidermal cells and that antioxidant proteins (MnSOD, GPx1, catalase) are differentially expressed in the epidermis. Using our previously sorted epidermal progenitor cells (side population, EpSPs) and more differentiated cells (non‐SPs), we show that catalase expression and activity are significantly decreased in EpSPs compared to non‐SPs. Furthermore, we found fewer peroxisomes per cell and decreased mitochondrial surface area in the EpSPs compared to non‐SPs. Using dihydroethidium (DHE), we show that EpSPs have less DHE signal compared to more differentiated cells. In summary, our data suggest that EpSPs have decreased levels of antioxidant proteins and ROS. Together, these studies indicate that the intracellular redox environment of EpSPs is reduced relative to their more differentiated progeny.Grant Funding SourceNational Institute of Health

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