| dc.description.abstract | A new culture method for culturing hepatic stem cells in three dimensional cultures is called liver organoids. In this culture method the genomic integrity of the stem cells is maintained and also the differentiation potential of these cells is retained. Liver organoids can be used in disease modeling or cell transplantation. This research is focused on copper toxicosis, a liver disease often seen in Bedlington terriers. This disease has many similarities with Wilson’s disease in humans. In the case of copper toxicosis, a mutation in het COMMD1 gene causes copper accumulation in the hepatocyte, which eventually leads to chronic hepatitis and cirrhosis. Current treatment with D-penicillamin has several limitations. The development of a disease model could give more understanding of the underlying mechanisms leading to the copper accumulations, but also new drugs could be tested on this model. The first part of this research was aimed at the development of a model for canine copper toxicosis. Wild type liver organoids, COMMD1 deficient liver organoids and COMMD1 deficient organoids transduced with a vector containing the correct COMMD1 gene were cultured and tested for their ability to take up and accumulate copper. Intracellular copper was measured using a copper sensor (CS1) that becomes red fluorescent when binding intracellular copper. After lysis of the cells the fluorescence intensity was measured and an estimation of the intracellular copper concentration was made. Even though, lots of variations of the disease model have been tested, we didn’t succeed to find a disease model that is repeatable and mimics the disease in vitro.
The second part of this project focused on using liver organoids for transplantation. Several liver diseases in humans eventually lead to chronic hepatitis and liver cirrhosis, which can only be treated with liver transplantation. However, shortage in liver donors restricts this treatment option. A lot of research has been done on the possibility to transplant cells instead of a complete new liver. Liver organoids are very promising for this use as well. In this study, hepatic progenitor cells were isolated from liver biopsies of dogs with COMMD1 deficiency. They were cultured as liver organoids and genetically corrected by a viral transduction. These transduced cells were then expanded and differentiated towards hepatocyte-like cells. These differentiated organoids were then injected into the portal vein using a port-a-cath. Our hypothesis was that these organoids will engraft in the liver tissue and have a growth advantage in comparison to the dog’s own hepatocytes. The liver tissue will eventually be (partly) repopulated with the new cells and a new functional liver will form. When this alternative to liver transplantation is successful in dogs, the next step could be to test this in humans with for example Wilson’s disease. We succeeded to transplant two COMMD1 deficient dogs with these patient specific liver organoids. For now, the results of the COMMD1 staining on liver biopsies taken after transplantation look promising. However, more dogs remain to be transplanted and more biopsies from several time-points need to confirm the presence of COMMD1 positive cells to confirm our hypothesis. | |
