The shortage of liver organ donors is increasing and the need for viable alternatives is urgent. Liver cell (hepatocyte) transplantation may be a less invasive treatment compared with liver transplantation. Unfortunately, hepatocytes cannot be expanded in vitro, and allogenic cell transplantation requires long-term immunosuppression. Organoid-derived adult liver stem cells can be cultured indefinitely to create sufficient cell numbers for transplantation, and they are amenable to gene correction. This study provides preclinical proof of concept of the potential of cell transplantation in a large animal model of inherited copper toxicosis, such as Wilson’s disease, a Mendelian disorder that causes toxic copper accumulation in the liver. Hepatic progenitors from five COMMD1-deficient dogs were isolated and cultured using the 3D organoid culture system. After genetic restoration of COMMD1 expression, the organoid-derived hepatocyte-like cells were safely delivered as repeated autologous transplantations via the portal vein. Although engraftment and repopulation percentages were low, the cells survived in the liver for up to two years post-transplantation. The low engraftment was in line with a lack of functional recovery regarding copper excretion. This preclinical study confirms the survival of genetically corrected autologous organoid-derived hepatocyte-like cells in vivo and warrants further optimization of organoid engraftment and functional recovery in a large animal model of human liver disease.

Vasculature performs a critical function in tissue homeostasis, supply of oxygen and nutrients, and the removal of metabolic waste products. Vascular problems are implicated in a large variety of pathologies and accurate in vitro models resembling native vasculature are of great importance. Unfortunately, existing in vitro models do not sufficiently reflect their in vivo counterpart. The complexity of vasculature requires the examination of multiple cell types including endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), as well as vessel location in the body from which they originate. The use of canine blood vessels provides a way to study vasculature with similar vessel size and physiology compared to human vasculature. We report an isolation procedure that provides the possibility to isolate both the endothelial and smooth muscle cells from the same vessels simultaneously, enabling new opportunities in investigating vasculature behavior. Canine primary ECs and VSMCs were isolated from the vena cava, vena porta and aorta. All tissue sources were derived from three donors for accurate comparison and to reduce inter-animal variation. The isolation and purification of the two distinct cell types was confirmed by morphology, gene- and protein-expression and function. As both cell types can be derived from the same vessel, this approach allows accurate modeling of vascular diseases and can also be used more widely, for example, in vascular bioreactors and tissue engineering designs. Additionally, we identified several new genes that were highly expressed in canine ECs, which may become candidate genes for novel EC markers. In addition, we observed transcriptional and functional differences between arterial- and venous-derived endothelium. Further exploration of the transcriptome and physiology of arteriovenous differentiation of primary cells may have important implications for a better understanding of the fundamental behavior of the vasculature and pathogenesis of vascular disease.

Wilson’s disease (WD), an autosomal recessive disorder, results in copper accumulation in the liver as a consequence of mutations in the gene ATPase copper transporting beta (ATP7B). The disease is characterized by chronic hepatitis, eventually resulting in liver cirrhosis. Recent studies have shown that dysregulation of nuclear receptors (NR) by high hepatic copper levels is an important event in the pathogenesis of liver disease in WD. Intracellular trafficking of ATP7B is mediated by COMMD1 and, in Bedlington terriers, a mutation in the COMMD1 gene results in high hepatic copper levels. Here, we demonstrate a reduced Farnesoid X nuclear receptor (FXR)-activity in liver biopsies of COMMD1-deficient dogs with copper toxicosis, a unique large animal model of WD. FXR-induced target genes, small heterodimer partner (SHP), and apolipoprotein E (ApoE) were down-regulated in liver samples from COMMD1-deficient dogs with hepatic copper accumulation. In contrast, the relative mRNA levels of the two CYP-enzymes (reduced by FXR activity) was similar in both groups. These data are in line with the previously observed reduced FXR activity in livers of ATP7B−/− mice and WD patients. Therefore, these data further corroborate on the importance of the COMMD1-deficient dogs as a large animal model for WD.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that...