Abstract
Clinical studies have proved the therapeutic potential of hepatocyte transplantation as a promising alternative to whole organ liver transplantation in the treatment of hereditary or end-stage liver disease. However, donor shortage seriously restricts cell availability, and the lack of appropriate cell culture protocols for the storage and maintenance of donor cells constitutes a significant obstacle. The aim of this study was to stimulate mature hepatocytes in culture to multiply in vitro and track their fate on transplantation. Rat hepatocytes isolated nonenzymatically were cultured serum free for up to 10 days. They were stimulated into proliferation in the presence of growth factors and conditioned media from nonparenchymal and hepatocyte culture supernatants, as well as 10 mM lithium chloride (LiCl). Cell proliferation was assessed by determining DNA content. Additionally, the extent of cell differentiation was estimated using immunofluorescence staining of hepatic, biliary, progenitor, and mesenchymal markers and gene expression analyses. Transplantation studies were performed on the Fischer CD26-mutant rat following pretreatment with retrorsine and partial hepatectomy. Proliferating hepatocytes increasingly adopted precursor characteristics, expressing progenitor (OV6, CD133), hepatic lineage (CK18), biliary (CD49f, CK7, CK19), and mesenchymal (vimentin) markers. The supplement of LiCl further enhanced the proliferative capacity by 30%. Transplantation studies revealed extensive repopulation by large donor hepatocyte clusters. Furthermore, bile duct-like structures deriving from donor cells proved to be immunoreactive to ductular markers and formed in close proximity to endogenous bile ducts. Mature hepatocytes reveal their potential to "switch" between phenotypes, adopting progenitor characteristics during proliferation in vitro. Following transplantation, these "retrodifferentiated" cells further expanded in vivo, thereby generating bipotentially differentiated progenies (hepatocytes and bile duct-like structures). This apparent plasticity of mature hepatocytes may open new approaches for cell-based strategies to treat liver disease.
Highlights
The unique ability of the liver to regenerate has been common knowledge since as early as the eighth century BC, even featuring in Greek mythology
We previously demonstrated that serum-free-cultured mature hepatocytes are able to repopulate the host liver after transplantation, despite having lost some hepatocyte-specific characteristics in vitro [1]
Since our syngeneic transplantation experiments required the use of Fischer rats, we adjusted and optimized the settings of cell culture to the Fischer 344 (F344) rat strain and tested the proliferative potency and reexpression of progenitor markers in cultured F344 hepatocytes
Summary
The unique ability of the liver to regenerate has been common knowledge since as early as the eighth century BC, even featuring in Greek mythology. Recent translational research in cell transplantation has demonstrated liver regeneration by proliferating and implanting mature hepatocytes into the host liver of animal models. While the majority of studies have concentrated on the differentiation of hepatocytes during proliferation in culture, numerous strategies have attempted to supply cells for transplantation from different sources to overcome the burden caused by limited numbers of donor organs. These studies have focused, for instance, on fetal liver cells and mesenchymal stem cells for therapeutic purposes [24,37]
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