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.

A ROS-dependent mechanism to drive progression through S phase

“…The concept that the redox environment of cells is coordinated with cell cycle progression dates back to 1931 when Rapkine (Rapkine 1931) postulated a thiol cycle in dividing urchin eggs. Since then, redox oscillations similar to the ROS dynamics we observe here have been inferred by several studies based on indirect experiments with reducing agents such as NAC (Menon et al 2007; Kim et al 2001; Kyaw et al 2004; Havens et al 2006) and directly using the ROS-sensitive fluorescent probe dichlorodihydrofluorescein diacetate DCFH-DA(Kim et al 2001; Kyaw et al 2004; Havens et al 2006; Menon & Goswami 2007; Ivanova et al 2021). However, ROS measurements with DCFH-DA are inherently prone to artifacts and have several limitations (Kalyanaraman et al 2012; Bonini et al 2006).…”

A ROS-dependent mechanism promotes CDK2 phosphorylation to drive progression through S phase

“…The concept that the redox environment of cells is coordinated with cell cycle progression dates back to 1931 when Rapkine ( Rapkine, 1931 ) postulated a thiol cycle in dividing urchin eggs. Since then, redox fluctuations similar to the ROS dynamics we report have been inferred by several studies indirectly by reducing agents such as NAC ( Menon et al., 2007 ; Kim et al., 2001 ; Kyaw et al., 2004 ; Havens et al., 2006 ) or directly by the ROS-sensitive fluorescent probe dichlorodihydrofluorescein diacetate DCFH-DA ( Kim et al., 2001 ; Kyaw et al., 2004 ; Havens et al., 2006 ; Menon and Goswami, 2007 ; Ivanova et al., 2021 ). ROS measurements with DCFH-DA are inherently prone to artifacts and have several limitations ( Kalyanaraman et al., 2012 ; Bonini et al., 2006 ).…”

A chloroplast sulfate transporter modulates glutathione-mediated redox cycling to regulate cell division

“…Reactive oxygen species (ROS) signaling plays an important role in cell cycle regulation in eukaryotic organisms (Burch and Heintz, 2005; Burhans and Heintz, 2009; Tsukagoshi et al, 2010; Chiu and Dawes, 2012; Dolzblasz et al, 2016; Yu et al, 2016; de Simone et al, 2017; Ivanova et al, 2021). Metabolic fluctuation between the oxidative and reductive phases is regulated as a function of the cell cycle with cell division strictly confined to the reductive phase in budding yeast (Tu et al, 2005).…”