Natural antioxidants to improve cell culture conditions of amniotic fluid stem cells

Human amniotic fluid stem cells (AFSCs) are a powerful source of cells with a plethora of trophic and immunomodulatory functions. These cells can be appropriately expanded ex-vivo to be differentiated in many cell types, in order to dissect developmental pathways, unravel pathogenic mechanisms and replace defective tissues. Traditionally, in vitro AFSCs are exposed to non-physiological concentrations of O2 increasing the production of reactive oxygen species (ROS). The unbalance in the redox signals may impair the AFSC stemness machinery1. In this context the use of natural antioxidants could be proposed as a strategy to prevent the loss of self-renewal and the extensive cellular damage due to abnormal ROS production. To this aim, AFSCs were exposed to sulphorafane (SF) and epigallo-3-catechingallate (EGCG) to counteract ROS deleterious effects. AFSCs were co-treated with 1 M SF and 10 M EGCG. After 72 h a slight increase in their metabolic activity was observed. However, cell population doubling over a cell culture period of 25 days was not different between untreated and co-treated samples. Interestingly, the co-treatment with SF and EGCG synergistically reduced intracellular ROS level in respect to SF or EGCG treatment alone, as measured by DCFH-DA assay. In addition, co-treated AFSCs displayed higher total GSH level in respect to the untreated samples. Self-renewal capacity was investigated evaluating the expression of pluripotent gene markers such as OCT4, NANOG and SOX2 by RT-PCR. Interestingly, the co-treatment was able to up-regulate the expression of all the three genes. Moreover, the chronic treatment for 21 days with SF and EGCG counteracted the expected decrease of OCT4 and NANOG expression during cell passaging. In conclusion, we propose the co-treatment with SF and EGCG as a strategy to counteract oxidative stress and maintain the functionality of AFSCs.