Hypoxic preconditioning potentiates the trophic effects of mesenchymal stem cells on co-cultured human primary hepatocytes

Abstract

Introduction: Mesenchymal stem/stromal cells (MSCs) improve the metabolic function of co-cultured hepatocytes The present study aimed to further enhance the trophic effects of co-culture with hepatocytes using hypoxi preconditioning (HPc) of the MSCs and also to investigate the underlying molecular mechanisms involved Methods: Human adipose tissue-derived MSCs were subjected to hypoxia (2 % O2; HPc) or normoxia (20 % O2) for 2 h and then co-cultured with isolated human hepatocytes. Assays of metabolic function and apoptosis were performe to investigate the hepatotrophic and anti-Apoptotic effects of co-culture. Indirect co-cultures and co-culture wit MSC-conditioned medium investigated the role of paracrine factors in the hepatotrophic effects of co-culture Reactive oxygen species (ROS) activity was antagonised with N-Acetylcysteine to investigate whether HPc potentiate the effects of MSCs by intracellular ROS-dependent mechanisms. Tumour necrosis factor (TNF)-α, transforming growt factor (TGF)-β1, and extracellular collagen production was determined and CASP9 and BAX/BCL-2 signalling pathway analysed to investigate the role of soluble factors, extracellular matrix deposition, and apoptosis-Associated gen signalling in the effects of co-culture Results: HPc potentiated the hepatotrophic and anti-Apoptotic effects of co-culture by ROS-dependent mechanisms There was increased MSC TGF-β1 production, and enhanced MSC deposition of extracellular collagen, with reduce synthesis of TNF-α, as well as a downregulation of the expression of pro-Apoptotic CASP9, BAX, BID and BLK genes an upregulated expression of anti-Apoptotic BCL-2 in hepatocytes Conclusions: HPc potentiated the trophic and anti-Apoptotic effects of MSCs on hepatocytes via mechanisms includin intracellular ROS, autocrine TGF-β, extracellular collagen and caspase and BAX/BCL-2 signalling pathways.