Changes of Reactive Oxygen Species Levels and DNA Oxidative Damage during The Differentiation of Human Umbilical Cord Msenchymal Stem Cells into Hepatocyte-Like Cells
Objective: Umbilical cord mesenchymal stem cells (UCMSCs) are multipotent stem cells that their ability to differentiate into hepatocyte-like cells is demonstrated. The hepatic differentiation is a physiological event that is associated with various molecular and biochemical factors. One of the most important factors affecting on stem cells maintenance and differentiation are reactive oxygen species (ROS) that can be produced during liver regeneration as a result of normal cellular metabolism or generated under pathologic processes. ROS play an important role in carcinogenesis, by generating oxidative DNA damage. Among the markers of DNA oxidative damage, 8-OH dG is the most commonly produced base lesion, and measured as an index of oxidative DNA damage. The current study aims to determine the effect of hepatic differentiation on the levels of ROS production and DNA oxidative damage in hepatocyte-like cells derived from human UC-MSCs. Materials and Methods: Human UC-MSCs were isolated, characterized morphologically, cytofluorometrically, and by their differentiation potential. The hepatic differentiation was performed with DMEM supplemented with 5% FBS, 20 ng/ml HGF, 10 ng/ml FGF-4, 20 ng/ ml OSM, and 10-6 M dexamethasone. The expression of markers specific for hepatic was assessed to demonstrate the hepatic differentiation. Then the levels of ROS generation during the differentiation assessed using 2', 7’-dichloroflurescein diacetate (DCF-DA) assay. To analyze DNA oxidative damage, 8-oxo-dG was measured by 8-OH dG ELISA Kit. Results: Following hepatic differentiation, the MSCs began to show a transition from a fibroblast-like morphology, to a round shape and a cubical morphology. The differentiated cells expressed hepatic-related genes and displayed antibody detectable expression of markers specific for hepatic maturation. UC-MSCs produced ROS at high levels compared with the differentiated cells, but ROS generation was decreased upon the hepatic differentiation. The 8-OH dG levels were not statistically changed during the differentiation; but, its levels were decreased on day 30 of differentiation. Conclusion: Using this protocol of the hepatic differentiation, ROS were generated at low levels, suggesting there was very little DNA oxidative damage during hepatic differentiation.