Differential Response of Human Embryonic Stem and Somatic Cells to Non-Cytotoxic Hydrogen Peroxide Exposure: An Attempt to Model In Vitro the Effects of Oxidative Stress on the Early Embryo

Abstract

Human Embryonic Stem Cells (hESCs) potentially offer a unique in vitro model to study how an adverse environment during the early developmental stages post-fertilization can affect the physiology of the undifferentiated embryonic stem cells existing in the early embryo and predispose to long term effects on the offspring, according to the Developmental Origins of Health and Disease (DOHaD) concept. A number of unfavourable conditions can affect the development of the early embryo inducing oxidative stress both in vivo, for instance in gestational diabetes and in vitro, when embryos are derived from Assisted Reproductive Technologies (ART). Therefore, the aim of this study was the development of a novel in vitro model to analyse the effects of oxidative stress and the antioxidant response against Reactive Oxygen Species (ROS) in embryonic stem cells in comparison with somatic cells, fibroblasts and endothelial cells. To this purpose we designed an in vitro protocol based on hydrogen peroxide (H2O2) treatment of 72 h, in order to better resemble the period of embryonic development from the early cleavages to the blastocyst stage. We demonstrate that H2O2 treatment induces the modification of crucial oxidative stress biomarkers like ROS and lipid peroxidation levels, and mobilizes several antioxidant enzymes through NFkβ translocation. Moreover we show differences between somatic and embryonic cells in their antioxidant response towards H2O2 induced damage. Therefore this study presents a promising in vitro model to investigate the effects of oxidative stress conditions on early human embryonic cells.

Comparative Analysis of AGE and RAGE Levels in Human Somatic and Embryonic Stem Cells under H2O2-Induced Noncytotoxic Oxidative Stress Conditions

“…In a previous work [ 12 ], we developed a novel in vitro model to analyse the effects of oxidative stress and the antioxidant response against reactive oxygen species (ROS) on embryonic stem cells in comparison with somatic cells, demonstrating that the nonlethal doses of H 2 O 2 resulted in an increase in oxidative stress in treated cells. To evaluate the nominal concentration-effect relationship for the cytotoxic action of H 2 O 2 , human somatic cells (Hs27 and HUVEC) and embryonic stem (HUES3 and HUES7) cell lines were exposed to rising concentrations of H 2 O 2 between 4 and 768 μ M during 72 h and cell viability was analysed by AlamarBlue® reduction and normalized to the nontreated control samples of each cell line ( Figure 1(a) ).…”

“…Although relevance of AGEs in the pluripotent embryonic stages remains to be clarified, since the accumulation in the embryo is not a consequence of adverse environment, it can be assumed that the low RAGE expression in oocytes and preimplantation embryos prior to blastocyst stage [ 14 ] avoid the detrimental effects of AGE-RAGE interaction. Therefore, in our oxidative stress treatment, we incubated both embryonic and somatic cells with different H 2 O 2 concentrations for 72 h where no effect on cellular viability or proliferation was observed, meanwhile, on the contrary, significant increase in terms of intracellular ROS accumulation and lipid peroxidation has been demonstrated [ 12 ].…”

“…At the end of the protocol, 3 days after the initiation of chemical exposure, the viability, gene expression, and protein levels were evaluated in exposed versus not exposed control (CTR) cells. (b) Dose-response curves, modified from Barandalla et al [ 12 ]. HUES3, HUES7, Hs27, and HUVEC were exposed to increasing concentrations of hydrogen peroxide for 72 hours, and cell viability was determined by AlamarBlue reagent.…”

“…Human embryonic stem cells (hESCs) have attracted huge attention for their potential applications in cell therapy, but much still remains to be explained about how their unique properties are altered by oxidative stress [ 11 ]. In preliminary studies [ 12 ], we have identified the nonscytotoxic window of exposure to H 2 O 2 of hESCs (HUES3 and HUES7) and somatic cells (HS27 and HUVEC), being between 4 and 16 μ M H 2 O 2 for Hs27 and HUES cells, and up to 32 μ M for HUVEC cells. In this range, we demonstrated that while no modifications of cell number and morphology occurred, a significantly increased level of ROS and lipid peroxidation was observed together with the expression of antioxidant enzymes response.…”

“…Therefore, the objective of this study is to analyse and compare the response against oxidative stress in terms of AGE and RAGE levels between embryonic and differentiated somatic cells. We have applied a previously described noncytotoxic H 2 O 2 treatment to generate an oxidative stress status identified by an increase of ROS [ 12 ]. Then, we have analysed the levels of AGEs and RAGE in two treated hES cells and in two treated somatic cell lines, revealing that oxidative stress affects the AGE accumulation and RAGE expression in a different way in embryonic versus differentiated cell lines.…”