Despite the availability of an effective anti‐hepatitis B virus (HBV) vaccine, ±400 million people world‐wide are chronically infected with HBV, and are at high risk of developing cirrhosis and liver cancer. Potent drugs are available to treat HBV infections. However, as HBV establishes a chronic infection by delivering a very stable form of viral DNA in the hepatocyte nucleus, life‐long therapy is needed in most patients. Therefore the pharmaceutical industry is now aiming to develop strategies to eradicate the virus from the infected liver. Such studies are hampered by the lack of appropriate in vitro or animal models due to the narrow tropism of HBV for human hepatocytes. In addition, good culture systems are lacking, as human liver cells are in short supply, culture of these cells causes to loose their phenotype and function very quickly, We hypothesized that it would be best to create a culture system that contained hepatocytes (the functional liver cells), but also liver specific endothelium (cell lining blood vessels), through which the virus needs to pass from the bloodstream to the hepatocytes and that support the function of hepatocytes, and stellate cells, responsible for scaring of the liver following liver damage, and that also support the normal function of hepatocytes. To create such combined cultures, we also hypothesized that matrix that mimics the biophysical and biochemical features of the liver would be needed to create an optimal culture system. Finally, we hypothesized that all three cell types should be generated from stem cells as these can be expanded indefinitely and differentiate in all three desired cell types.
The overall objective of the ‘LiVirIn’ was to develop superior in vitro three dimensional culture systems of three different liver cells derived from human stem cells that would allow the study of hepatitis B virus infection (acute and ‘chronic’) and test of anti-HBV drugs. The hypothesis of the project was that (1) co-culture of three cells next to one another in the liver lobule, and this in (2) in a matrix that mimics the biophysical and biochemical features of the liver would improve maturation and function of hepatocytes, stellate and endothelial cells, mimicking liver tissue in vivo.
Conclusion
To conclude these studies, we created unique 3D microenvironmental niches that allow further maturation of stem cell derived hepatocytes, and can also maintain the phenotype of liver stellate cells and liver endothelial cells. These unique microenvironments are completely defined as opposed to other naturally used 3D culture system. Currently, we are testing the effect of the top 3 microenvironments on the co-maturation of hepatocytes, liver stellate cells and liver endothelial cells, all derived from pluripotent stem cells (PSCs). Once this has been optimized we will test the improvement in infection of hepatitis B and test the effect of drugs on the infection rate.
Furthermore, we adapted the stem cell-hepatocyte differentiation assay into a high throughput screening platform to identify molecules that can improve hepatocyte maturation. The top hits from this studies will be used along with 3D coculture system, will further improve hepatocyte maturation and thus HBV infection. In addition, the high throughput screening platform will also be extremely useful for identifying anti-hepatitis B drugs using a high throughput screen approach.
