Oxidative stress promotes exit from the stem cell state and spontaneous neuronal differentiation

• DOI: 10.18632/oncotarget.23786
• Publication Date: 2017-12-30T14:57:22Z
• Authors: Qidong Hu; Puja Khanna; Belinda Shu Ee Wong; Zealyn Shi Lin Heng; Charannya Sozhees...; Lal Zo Thanga; Sharon Wui Sing Tan; Gyeong Hun Baeg
• Keywords: 570; antioxidant; NCAM gene; paraquat; polymerase chain reaction; TDGF1 gene; embryo; 610; reactive oxygen metabolite; NEUROD1 gene; GFRA1 gene; transcription factor Nrf2; Article; nerve cell differentiation; Western blotting; 03 medical and health sciences; immunocytochemistry; oxidative stress; controlled study; human; gene; 0303 health sciences; neurite; PAX6 gene; OCT4 gene; quantitative analysis; mitogen activated protein kinase; human cell; embryonic stem cell; TUJ1 gene; alpha [amino(4 aminophenylthio)methylene] 2 (trifluoromethyl)phenylacetonitrile; mitogen activated protein kinase 3; cell level; mitogen activated protein kinase 1; HOXA1 gene; NANOG gene; gene expression; signal transduction; cell structure; Research Paper

Reactive oxygen species (ROS) play important roles in fundamental cellular processes such as proliferation and survival. Here we investigated the effect of oxidative stress on stem cell maintenance and neuronal differentiation in a human embryonic stem cell (hESC) model, Ntera2 (NT2). CM-H2DCFDA and DHE assays confirmed that the oxidizing agent paraquat could induce a high level of ROS in NT2 cells. Quantitative PCR, Western blotting and immunocytochemistry showed that paraquat-induced oxidative stress suppressed the expression of stemness markers, including NANOG, OCT4 and TDGF1, whereas it enhanced the spontaneous expression of neuronal differentiation markers such as PAX6, NEUROD1, HOXA1, NCAM, GFRA1 and TUJ1. The treated cells even exhibited a strikingly different morphology from control cells, extending out long neurite-like processes. The neurogenic effect of ROS on stem cell behaviour was confirmed by the observations that the expression of neuronal markers in the paraquat-treated cells was suppressed by an antioxidant while further enhanced by knocking down Nrf2, a key transcription factor associated with antioxidant signaling. Lastly, paraquat dose-dependently activated the neurogenic MAPK-ERK1/2, which can be reversed by the MEK1/2 inhibitor SL327. Our study suggests that excessive intracellular ROS can trigger the exit from stem cell state and promote the neuronal differentiation of hESCs, and that MAPK-ERK1/2 signaling may play a proactive role in the ROS-induced neuronal differentiation of hESCs.