Human liver stem cells and derived products in experimental models of liver ischemia reperfusion injury and of liver isolated normothermic perfusion.

Ischemia reperfusion injury (IRI) is an antigen-independent inflammatory event that affects several clinical settings, including surgical procedures such as liver resection and liver transplantation. IRI is still a major concern in the transplantation setting, causing up to 10% of early graft failure and leading to a higher incidence of acute and chronic rejection. IRI is initiated by Kuppfer cells and hepatocytes through a massive production of reactive oxygen species (ROS) during the ischemic phase and, in a major degree, during the post-reperfusion phase. ROS directly damage hepatocytes and endothelial cells and promote the recruiment of neutrophils and T-cells, starting an inflammatory cascade that triggers apoptosis and necrosis. Human Liver Stem Cells (HLSC) have been identified as a population of pluripotent resident liver cells able to express markers characteristic of the mesenchymal lineage (CD73, CD90, CD29, CD44) together with hepatic markers (alpha-fetoprotein, cytokeratin 18, cytokerain 8 and albumin), suggesting a partial hepatic commitment. HLSC share self-renewal properties with mesenchymal stem cells and can actively take part to tissue remodeling and liver regeneration. We found that HLSC are able to localize within the injured tissue and promote liver regeneration in a murine model of fulminant liver failure and to generate a functional “humanized liver-like tissue” when injected in rat acellular liver scaffolds. Growing evidence suggests that the biological effects of stem cells on neighboring cells are mediated by paracrine mechanisms including the release of extracellular vesicles (EV). EV are an heterogeneous population of cell-secreted vesicles originating from the endosomal compartment or from direct budding of plasma membrane that are able to modify the phenotype and function of neighboring cells. The regenerative effects of EV are well documented and ascribed to a horizontal transfer of proteins, lipids and, above all, specific subsets of mRNA and miRNA. In a rat model of 70% hepatectomy, animals treated with Human Liver Stem Cells-derived Extracellular Vesicles (HLSC-EV) revealed a significant reduction of liver injury and higher regeneration rate of the remnant liver after surgery . The effect of HLSC-EV was investigated on two different experimental models: First Project We set up a short-duration model of ex vivo isolated rat liver Normothermic Machine Perfusion (NMP) in which oxygen delivery was kept suboptimal through a low hemoglobin content in the perfusate. In this model, we investigated whether adding HLSC-EV to the circuit could result in i) their rapid uptake by the liver, and ii) an appreciable reduction of the hypoxic tissue injury. Second Project An in-vivo model of IRI was set up in mice. The aim of this study was to investigate the effects of systemic administration of HLSC-EV on tissue injury after partial clamping of the hepatic hilum (70%) .