Co-culture of hepatocytes with mesenchymal stem cells for cellular therapy in liver disease
A major hurdle facing current hepatocyte transplantation practice is the marginal quality of isolated hepatocytes. Previous studies showed that mesenchymal stem cells (MSCs) could maintain morphology and improve liver-specific metabolism of co-cultured hepatocytes. The present work aimed to optimise the MSCs co-culture system by testing adipose tissue (AT), bone marrow, and umbilical cord-derived MSCs at predefined seeding ratios. Liver-specific metabolism and apoptosis assays were performed to investigate hepatotrophic and antiapoptotic effects of MSCs co-culture. Indirect co-culture was established to investigate the role of paracrine factors in hepatotrophic effect of MSCs co-culture. Hypoxia-preconditioned (HPc) MSCs were co-cultured with hepatocytes to investigate potentiative effect of HPc induction. Intracellular reactive oxygen species (ROS) activity quantitation and antagonisation experiments were performed to investigate whether HPc potentiated MSCs co-culture by an intracellular ROS-dependent mechanism. Tumour necrosis factor alpha (TNF-α), transforming growth factor beta1 (TGF-β1), extracellular collagen, and apoptosis- associated caspase and BAX/BCL-2 signalling pathways were analysed to investigate the contribution of soluble factors, extracellular collagen, and gene signalling to the hepatotrophic effects of MSCs co-culture and potentiative effect of HPc induction. All the three types of MSCs exhibited a similar hepatotrophic effect, with a comparable effect even in low-density AT-MSCs co- culture. Hepatotrophic and antiapoptotic effects of MSCs showed a cell contact dependent manner, and HPc potentiated MSCs co-culture by a cell-contact intracellular ROS-dependent mechanism. Decreased hepatocyte autocrine TNF-α, increased MSC autocrine TGF-β1, and enhanced MSCs deposition of extracellular collagen contributed to the hepatotrophic effects of MSCs co-culture and potentiative effect of HPc induction, with downregulated expression of proapoptotic CASP9, BAX, and BID and upregulated expression of antiapoptotic BCL-2. It is concluded that synergistic effects of cell contact, intracellular ROS-dependent soluble factors, extracellular matrix, and apoptosis- associated signalling in MSCs co-culture contribute to hepatotrophic effect and HPc-induced potentiative effect. Co-transplantation with MSCs should improve therapeutic effects of HCT by enhancing survival and metabolism of co-transplanted hepatocytes.
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