Abstract
The rat is an important model for liver regeneration. However, there is no in vitro culture system that can capture the massive proliferation that can be observed after partial hepatectomy in rats. We here describe the generation of rat liver stem cell lines. Rat liver stem cells, which grow as cystic organoids, were characterized by high expression of the stem cell marker Lgr5, by the expression of liver progenitor and duct markers, and by low expression of hepatocyte markers, oval cell markers, and stellate cell markers. Prolonged cultures of rat liver organoids depended on high levels of WNT-signalling and the inhibition of BMP-signaling. Upon transplantation of clonal lines to a Fah−/− Il2rg−/− rat model of liver failure, the rat liver stem cells engrafted into the host liver where they differentiated into areas with FAH and Albumin positive hepatocytes. Rat liver stem cell lines hold potential as consistent reliable cell sources for pharmacological, toxicological or metabolic studies. In addition, rat liver stem cell lines may contribute to the development of regenerative medicine in liver disease. To our knowledge, the here described liver stem cell lines represent the first organoid culture system in the rat.
Highlights
Institute, Department of Physiology, Development and Neuroscience, University of Cambridge, Tennis Court Rd, Cambridge CB2 1QN, UK. 5Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Rd, Cambridge CB2 1QN, UK. 6Human and Molecular Genetics Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, 53226, WI, USA. *These authors contributed to this work
To establish rat liver stem cell lines, liver tissue was digested with collagenase and differential centrifugation steps were performed to enrich for duct cells
When rat liver cells were subjected to these ‘human’ liver stem cell conditions[17], cystic organoids were lost within 1 week after switching culture conditions, indicating that these conditions fail to support rat liver stem cell self-renewal (Fig. 1B)
Summary
Recent studies using different animal models of liver damage demonstrate that multiple cell types can contribute to liver regeneration, such as hepatocytes[19,20,21,22], duct-derived oval cells[23,24,25] and hepatic stellate cells (a liver-resident mesenchymal cell)[26]. Genetic lineage tracing techniques have recently demonstrated that Lgr[5], which is not expressed in the healthy adult mouse liver or pancreas, becomes activated in a subpopulation of cells upon injury[13,14]. In the presence of the Lgr5-ligand RSPO1, single Lgr5+ mouse liver cells can give rise to cystic organoid cultures that can be expanded indefinitely, while maintaining the potential to differentiate towards hepatocytes and duct cells, demonstrating that liver derived Lgr5+ cells are true bi-potential stem cells in vitro. We chose for the SHR/OlaIpcv (SHR) and BN-Lx/Cub (BN-Lx) rat strains, because these are the two founder strains of the rat HXB/BXH recombinant inbred panel that has been extensively phenotyped at the physiological, behavioral and molecular levels and all genomic variation between both strains has been identified[28,29]
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