Effect of natural antioxidants on amniotic fluid stem cell cultures

The ex vivo expansion of human amniotic fluid stem cells (AFSCs) is a critical step for their clinical application. In vivo, stem cells reside in specialized microenvironments called niches where the oxygen tension is much lower than ambient oxygen (21% O2) conventionally utilized to expand them in vitro. Hyperoxia can induce an abnormal production of reactive oxygen species (ROS). Besides an emerging role for ROS in the regulation of the stem cell cycle, the unbalance in ROS production triggers oxidative stress that can impair cellular self- renewal. In this context the use of natural antioxidants could be proposed as a strategy to preserve stem cell functionality. Aim of this study was to investigate the effects of sulforaphane (SF) and epigallocatechin-3-gallate (EGCG) treatments in AFSCs focusing on viability and multipotency prior to biobanking. AFSCs were treated with SF 1 μM and/or EGCG 10 μM. Both SF and EGCG were able to reduce intracellular ROS levels as measured by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Interestingly, SF and EGCG co-treatment synergistically increased ROS level reduction in respect to SF or EGCG treatment alone. SF and EGCG co-treatment did not modify population doubling over a cell culture period of 25 days. Moreover, the combined supplementation with SF and EGCG in AFSCs culture increased the expression level of pluripotency markers such as Nanog, Sox2, Oct4, as measured by RT-PCR. In conclusion, our data suggest that the co-treatment with SF and EGCG may reduce the risk of spontaneous unspecific differentiation of AFSCs during the expansion phase, probably mediating a protective response against in vitro hyperoxia.