Cell cycle-coupled changes in the level of reactive oxygen species support the proliferation of human pluripotent stem cells

Abstract

The study of proliferation regulation in human pluripotent stem cells is crucial to gain insights into understanding the physiology of these cells. However, redox regulation of the pluripotent cell cycle remains largely unexplored. Here, using human embryonic stem cells (hESCs) as well as human induced pluripotent stem cells (hiPSCs), we demonstrate that the level of reactive oxygen species (ROS) in pluripotent cells oscillates in accordance with the cell cycle progression with the peak occurring at transition from S to G₂ /M phase of the cycle. A decrease of this level by antioxidants leads to hindered S-phase initiation and progression but does not affect the early-G₁ -phase or mitosis. Cells exposed to antioxidants in the early-G₁ -phase accumulate the phosphorylated retinoblastoma protein and overcome the restriction point but are unable to accumulate the main regulators of the S phase-CYCLIN A and GEMININ. Based on the previous findings that CYCLIN A stability is affected by redox homeostasis disturbances in somatic cells, we compared the responses to antioxidant treatments in hESCs and in their differentiated fibroblast-like progeny cells (difESCs). In difESCs, similar to hESCs, a decrease in ROS level results in the disruption of S-phase initiation accompanied by a deficiency of the CYCLIN A level. Moreover, in antioxidant-treated cells, we revealed the accumulation of DNA breaks, which was accompanied by activation of the apoptosis program in pluripotent cells. Thus, we conclude that maintaining the physiological ROS level is essential for promotion of proliferation and accurate DNA synthesis in pluripotent cells and their differentiated descendants.

Keywords: antioxidants; human embryonic stem cells; induced pluripotent stem cells; pluripotent cell cycle; reactive oxygen species; redox homeostasis; redox regulation.