Periodic Reporting for period 1 - LiVirIn (3D Liver Organoids: Modelling Host Hepatitis B Virus Interaction)
Reporting period: 2015-03-01 to 2017-02-28
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.
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 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 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.