In vitro modelling of biliary development and diseases

Bile is a toxic fluid produced in the liver and transferred through tubular structures known as bile ducts to the gut, where it helps with fat absorption. In bile duct disorders (cholangiopathies), the bile ducts fail to contain and transfer bile effectively. As a result, toxic bile leaks out of the ducts and accumulates in the liver causing significant damage. Unfortunately, our understanding of the exact mechanisms leading to biliary disease is very limited. Research in the field is hindered by technical difficulties in obtaining and maintaining live bile duct samples in the laboratory and the lack of animal models faithfully reproducing the disease. Nevertheless, the need to decipher the disease mechanisms, towards developing new diagnostic tests and treatment options is very pressing. Bile duct disorders account for a major proportion of all liver disease, carrying significant mortality. The only available treatment is liver transplantation, a lifesaving but expensive and challenging operation, requiring lifelong medication and follow-up and complicated by the national shortage of liver donors.

Alagille syndrome (AGS) is an inherited disorder. Although it involves multiple organs, biliary disease caused by poor bile duct development in pregnancy and early life constitutes the most common clinical manifestation. Liver transplantation remains the only treatment option. Compared to other bile duct disorders the study of AGS has two important advantages: Primarily, the disease mechanism is not completely unknown, as the hereditary mutation causing it has already been described. Secondarily, in view of the close association between AGS and bile duct development, elucidating the disease mechanism will provide significant information on how bile ducts normally develop. Duct development is implicated to some extent in all childhood biliary disorders leading to paediatric liver transplantation. Therefore, studying AGS may advance our knowledge on a whole group of biliary diseases.

The aim of this project is to develop a novel method for generating large amounts of fully functional bile duct replicas in the laboratory. The resulting ducts will provide an excellent model to study any biliary disease, test and develop new therapies. I propose applying this method for generating the first proof-of-principle disease model for AGS and elucidating the disease mechanism. I have already made significant progress towards achieving this goal. Using the technology of stem cells, I have successfully created functional bile duct replicas, starting from healthy individuals' skin samples. I intend to use the same approach to generate and study bile ducts from AGS patients. Once the ducts have been created, I will use cutting edge technology to decipher the series of events leading to AGS and attempt to 'rescue' the diseased ducts by intervening and altering these events.

This project will shed light on the mechanisms driving AGS, facilitating the development of new tests for identifying the disease in early stages, new markers for predicting its course and new treatment options alternative to liver transplantation. Ultimately, it will contribute to improving the patients' quality of life, reducing pressure on the transplant waiting list and changing AGS from an untreatable disease to a manageable condition. Furthermore, the platform described has the potential to generate large amounts of bile duct replicas from patients with any biliary disorder. Therefore, it will provide a powerful novel disease modelling tool for the benefit of any research group working on bile ducts, overcoming the difficulties related to lack of biliary tissue and paving the road towards understanding and treating biliary disease in the future.

Cholangiopathies represent a leading cause for liver disease and transplantation. Treatment options are very limited and research in the field is hindered by poor primary tissue access and lack of robust disease models. Alagille Syndrome (AGS) is a monogenic, syndromic infantile cholangiopathy. It is caused by JAG1 or Notch2 gene mutations leading to poor biliary tree development, paucity of bile ducts and hepatic impairment. The pathogenesis of AGS remains elusive and liver transplantation provides the only available therapy.

We propose establishing a novel platform for in vitro modelling of biliary diseases, based on a methodology we developed for generating cholangiocytes from patient derived human Induced Pluripotent Stem Cells (hIPSCs). We will validate this platform by developing the first proof-of-principle in vitro model for AGS, towards exploring the role of JAG-Notch signalling in biliary development and disease. The disease phenotype will be reproduced in vitro using AGS-hIPSCs undergoing biliary differentiation and organoid formation in 3D culture conditions. To demonstrate the association between JAG-Notch signalling and our in vitro phenotype, we will correct the JAG mutation in AGS hIPSCs and silence Notch signalling in generic hIPSCs expecting rescue and reproduction of the AGS phenotype respectively. We will then use CHIP assays, genome wide analysis and pathway inducers/inhibitors to characterise the transcriptional network regulated by Notch in biliary development and identify cross-talk with other key pathways.

Differentiation of hIPSC to cholangiocytes will increase our insight in biliary development, overcome limitations due to lack of primary tissue and provide large amounts of autologous biliary epithelial cells for the study of cholangiopathies. Modelling AGS in vitro will elucidate the disease pathogenesis, promote the generation of novel prognostic, diagnostic and therapeutic modalities and pioneer in vitro biliary disease modelling.

Social impact: Currently, treatments options for biliary diseases are very limited and the only available treatment for liver disease in Alagille Syndrome (AGS) is liver transplantation, a lifesaving but challenging procedure, requiring long-term medication and follow-up and complicated by the national shortage of grafts. This project will generate the first robust in vitro modelling platform for AGS and biliary diseases, providing a novel tool that will promote research in the field of cholangiopathies. Furthermore, it will increase our insight in the pathogenesis of AGS identifying potential diagnostic, prognostic and therapeutic markers. These advances will constitute the first step towards developing new treatments alternative to liver transplantation, improving the patients' quality of life and reducing pressure on the liver transplant waiting list, not only for AGS but ultimately for the whole spectrum of biliary diseases.

Non Academic beneficiaries: The proposal is likely to generate commercially exploitable findings. The platform generated by this project will provide a solution to the challenges of biliary differentiation of stem cells, in vitro biliary disease modelling and drug screening for cholangiopathies. Several companies including GSK, Vertex, DefiniGEN, AstraZeneca, and pFIZER have already shown an interest in cell types generated by the Vallier group, available for disease modelling. HIPSCs-derived cholangiocytes could constitute excellent candidates for such commercial applications.

Economic impact: Liver transplantation remains the only treatment option for the majority of cholangiopathies. The very high cost of the procedure (~ £56,000/ procedure), combined with the cost of the life-long immunosuppression therapy required and multiple diagnostic tests or hospital admissions, associated with procedure complications (~ £81,000/ patient), constitute a significant economic burden for the NHS (Data from the Specialised Commissioning Team report: Organs for Transplant: Costs and Savings for the NHS, October, 2010). The development of a novel modelling platform for biliary disease will promote the identification of novel therapeutic targets, enable screening for new drugs and ultimately provide alternative treatments to liver transplantation, reducing the economic impact of biliary disease on the NHS.

Experiments on animals and animal welfare: The extensive use of animal models in research has given rise to significant ethical and societal concerns. To address these concerns every effort is made to replace and reduce the need for animals in research as well as safeguard their welfare. This initiative is coordinated by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). The development of an in vitro disease modelling platform will contribute significantly towards this cause and limit research costs by reducing the use of animals in the study of biliary disorders or even replacing in vivo models in some cases.

Strategic Impact: This project focuses on advancing the field of regenerative medicine by generating a novel protocol for biliary differentiation of stem cells and translating this basic research in clinical applications by developing a novel disease modelling platform. Both of these areas of focus have been identified as priority targets in the MRC Strategic Plan 2009-2014. Close collaborators involved in the UK PBC (Primary Biliary Cirrhosis) consortium, have already expressed interest in expanding the impact of our research even further, using our platform to facilitate the interpretation of data generated by population wide studies in PBC, a strategic target supported by the recent MRC award for Primary Biliary Cirrhosis Research.