Periodic Reporting for period 1 - LiVirIn (3D Liver Organoids: Modelling Host Hepatitis B Virus Interaction)
Reporting period: 2015-03-01 to 2017-02-28
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.
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.
To achieve its objectives ‘LiVirIn’ has produced research results of high quality and subsequently published them in international conferences and will publish in high impact journals:
The major objective of the project was to improve the functional maturation of hepatocytes from human pluripotent stem cells (hPSCs), which then could serve as in vitro model to study HBV infection. For this, a 3D micro-environmental niche was developed that supports hepatocyte maturation and this in a stable manner for 10-20 days. This was accomplished in fabricating 3D bioactive matrices (hydrogels) derived from natural as well as synthetic polymers (Polyethylene glycol (PEG)), mimicking the biophysical and biochemical characteristics of the liver. As initial simple tests of different matrices did not improve differentiation and maturation, we developed a complex “design of experiment” (DOE) approach to screen matrices at four levels: (1) mechanical properties of the matrix, (2) proteolytic degradability, (3) linking the matrices to peptides representative of extracellular matrix proteins and (4) cell-cell interaction proteins (24 different peptides). We tested the effect of hundreds of unique microenvironments on the maturation of hepatocytes derived from stem cells and identified multiple conditions that significantly improved the hepatocyte phenotypes. A system level analysis allowed us to select 3 unique microenvironments that improved hepatocyte phenotype and function by 50 fold. We also tested the top 3 hydrogels for their ability to support the mature fate of human stellate cells (primary as well as hPSCs derived) and endothelial cells (derived from hPSCs), and are currently performing co-cultures of the three cell type in these top 3 microenvironments.
In another approach to improve hepatocyte differentiation, we also performed a small molecule screen, as small molecules are known to affect signalling pathways and can hence also activate maturation signalling pathways. We first adapted the stem cell-hepatocyte differentiation protocol to a robust 384-well format, performed successfully a pilot screen of ±160 molecules, which was followed by a screen ±3000 molecules. A number of hits were found which we are currently validating.
The major objective of the project was to improve the functional maturation of hepatocytes from human pluripotent stem cells (hPSCs), which then could serve as in vitro model to study HBV infection. For this, a 3D micro-environmental niche was developed that supports hepatocyte maturation and this in a stable manner for 10-20 days. This was accomplished in fabricating 3D bioactive matrices (hydrogels) derived from natural as well as synthetic polymers (Polyethylene glycol (PEG)), mimicking the biophysical and biochemical characteristics of the liver. As initial simple tests of different matrices did not improve differentiation and maturation, we developed a complex “design of experiment” (DOE) approach to screen matrices at four levels: (1) mechanical properties of the matrix, (2) proteolytic degradability, (3) linking the matrices to peptides representative of extracellular matrix proteins and (4) cell-cell interaction proteins (24 different peptides). We tested the effect of hundreds of unique microenvironments on the maturation of hepatocytes derived from stem cells and identified multiple conditions that significantly improved the hepatocyte phenotypes. A system level analysis allowed us to select 3 unique microenvironments that improved hepatocyte phenotype and function by 50 fold. We also tested the top 3 hydrogels for their ability to support the mature fate of human stellate cells (primary as well as hPSCs derived) and endothelial cells (derived from hPSCs), and are currently performing co-cultures of the three cell type in these top 3 microenvironments.
In another approach to improve hepatocyte differentiation, we also performed a small molecule screen, as small molecules are known to affect signalling pathways and can hence also activate maturation signalling pathways. We first adapted the stem cell-hepatocyte differentiation protocol to a robust 384-well format, performed successfully a pilot screen of ±160 molecules, which was followed by a screen ±3000 molecules. A number of hits were found which we are currently validating.
Current Pluripotent stem cells (PSC) derived hepatocytes have a maturity akin to fetal liver hepatocytes, and no studies have been published describing PSC derived sinusoidal endothelial or stellate cells. Furthermore, environmental engineering studies, provided novel insights in progressive increased maturation of the hepatocytes from PSCs which will be further extended to stellate and endothelial cells, knowledge that is currently incompletely (hepatocytes) or not (stellate and endothelial cells) known. In LiVirIn, we identified unique microenvironment using a DOE approach by matrix screen allowing improved PSC-HLCs (hepatocytes like cells) maturation. These 3D culture enable not only in the improvement of hepatocytes phenotype but also allowing human stellate cells and endothelial cells differentiation. Currently, we are testing three cell populations as co-culture in 3D microenvironment, this enable us not only to determine how they might maintain/enhance each other’s maturity, but will also have cells and models that make it possible to determine the role of these non-parenchymal cells in HBV infection studies, test anti-viral drugs, as well as for the study drug metabolisation /toxicity studies. Having good functional liver models will also decrease the need for animal studies, in line with the 3R principle
The high throughput screen for small molecules that improve hepatocyte maturation 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.
The high throughput screen for small molecules that improve hepatocyte maturation 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.