There is currently no drug available to prevent or reverse the accumulation of fat in the liver caused by non-alcoholic fatty liver disease (NAFLD), or its acute form non-alcoholic steatohepatitis (NASH). Therefore, developing new therapeutics against liver disease is a priority for the pharmaceutical industry. Research typically uses a wide choice of dietary murine models that are fed high fat foods to induce NASH/NAFLD or genetic murine models to validate new target genes for therapeutic development against NAFLD/NASH. However, these models only partially recapitulate NAFLD pathogenesis and the complexity of human genetics induces large variance in individual metabolism thereby hampering the clinical translation of results. Animal models are also unsuitable for large scale analysis. Finally, conventional in vitro models typically use transformed cell lines or primary hepatocytes, which can provide valuable information but do not accurately predict in vivo toxicity.
With previous NC3Rs funding, Professor Vallier developed an in vitro model for NAFLD/NASH using human induced pluripotent stem cell (hiPSC)-derived hepatocytes grown in a 3D scaffold-based culture system. This model enables the assessment of lipid accumulation/ toxicity in hepatocytes and the function of genes associated with disease progression.
Why we funded it
This Technologies to Tools grant aims to replace animals used in NAFLD/NASH drug development, by developing high throughput methods for lipidomic and proteomic analyses enabling rapid phenotyping of hiPSC derived hepatocytes.
Working with the Medicines Discovery Catapult (MDC), Professor Vallier aims to develop methods to perform lipidomic analyse on the 3D hepatocyte cultures using only a small volume of supernatant, followed by validation of the technology for drug screening and therapeutic target identification. This will make the system compatible with high-throughput lipidomic and proteomic screening, increasing the suitability of this platform for end users.
Novel compounds are typically screened for the potential to cause liver damage in murine models. In the past five years, 486 publications have used a methioninecholine deficient diet or a high fat high fructose diet model of NAFLD and 1733 studies were performed using genetic NAFLD models (specifically ob/ob and db/db obese mouse models). If on average 20 animals are used per study, approximately 10,000 mice are required per year. High throughput methods for lipidomic and proteomic analyses, combined with a hiPSC-derived hepatocyte model could replace a large number of these animals for screening purposes if taken up widely. The pharmaceutical industry also uses transgenic mice to validate new target genes for therapeutic development against NAFLD/NASH. The possibility to replace the animal models for a human system in vitro will provide a faster and more physiological platform to study the importance of these genes in disease and validate their therapeutic potential. Animal models for studies of liver cirrhosis or fibrosis could also be replaced using this technology, further increasing the 3Rs impacts.
The culture conditions (2D, 3D or 3D co-culture with macrophages, hepatic stellate cells and cholangiocytes) for the hiPSC derived hepatocytes will be compared. The cells will be cultured for 1 week either without fatty acids, or in the presence of oleic acid to model lipid accumulation associated with NAFLD/NASH, or palmitic acid to model lipotoxicity. Cell survival will be characterised using standard methods and a small volume of supernatant or whole cell lysate will be analysed in the prototype acoustic mist ionisation mass spectrometer at the MDC to provide a lipidomic profile. Lipidomic data will be compared between the different culture systems (2D, 3D or 3D co-culture) and the most informative (i.e. the culture system providing high lipid variation between the presence and absence of fatty acids) will be selected for further experiments. The cells will be grown for 24 hrs, 1 week and 4 weeks, the cell survival analysed as described above and the most informative time of incubation will be selected for continued studies.
hiPSC-derived hepatocytes carrying a PNLPA3 genetic variant (increases susceptibility to NAFLD/NASH) and the corrected isogenic counterpart, will be cultured using the optimised method. Lipidomic analysis will be performed and the resulting lipidomic profiles compared to those of patients already publicly available. Similar experiments will be performed using wild type cells grown with different doses of FGF19 and Obeticholic acid to determine the efficacy of each drug.
Coll A et al. (2019). GDF15 mediates the effects of metformin on body weight and energy balance. Nature 578:444-448. doi: 10.1038/s41586-019-1911-y
Fourrier A et al. (2019). Regenerative cell therapy for the treatment of hyperbilirubinemic Gunn rats with fresh and frozen human induced pluripotent stem cells derived hepatic stem cells. Xenotransplantation 27(1):e12544. doi: 10.1111/xen.12544
Sampaziotis F et al. (2019). Use of Biliary Organoids in Cholestasis Research. Experimental Cholestasis Research 1981:373-382. doi: 10.1007/978-1-4939-9420-5_25
Tysoe O et al. (2019). Isolation and propagation of primary human cholangiocyte organoids for the generation of bioengineered biliary tissue. Nature Protocols 14:1884-1925. doi: 10.1038/s41596-019-0168-0