Modelling liver fibrosis using human Induced Pluripotent Stem Cells.

Liver diseases are one of the most common causes of mortality in developing countries, and liver transplantation is the only available treatment. However, this procedure carries considerable risk and also implies indefinite immunosupression treatment associated with heavy side effects and eventual rejection. Furthermore, an increasing number of patients die while on the liver transplant waiting list due to the shortage of suitable organ donor. Therefore, new therapies are urgently needed to solve this growing health care challenge. However, the development of new drug against liver injury is currently impaired by the absence of appropriate in vitro systems for modeling liver pathophysiology. Indeed, current studies are mainly performed on animal models which rarely recapitulate all the aspects of the human disease. In addition, the use of animal is costly, not compatible with high trough put drug screening platforms and also ethically questionable.
Consequently, the aim of this proposal is to address these limitations by developing an in vitro system to model liver disease in a dish using human induced pluripotent stem cells. This system will be compatible with large scale experiments and will only use human cells. Furthermore, its performance will be compared against mouse model to validate its interest for the replacement of model organisms. The resulting in vitro systems will provide an advantageous alternative to animal studies and also novel tools for drug development which will accelerate the delivery of useful therapies.

End-stage liver cirrhosis is one of the most common cause of mortality in modern countries and can only be treated by liver transplantation, which is strongly limited by the lack of organ donors and the side effects of immunosuppressive treatment. Therefore, the development of new therapeutics against liver diseases represents a global health challenge and one of the main objective for the pharmaceutical industry. However, the lack of relevant, physiological and high trough put in vitro system to model liver fibrosis represents a major issue. Current in vitro models of the fibrotic liver consist in cell cultures, precision slice biopsies, and whole organ perfusion systems. 2D culture systems lack functional cell-to-cell interactions and anatomical structures needed to reveal mechanistic details of disease involved in fibrosis, while perfusion and biopsy based models cannot persist long enough to accurately model fibrotic progression. Consequently, most studies are based on animal models which can lack the physiological relevance to human diseases and which are not compatible with high trough put platform necessary for drug screening.
Here, we propose to use of human Induced Pluripotent Stem Cells (hIPSCs) to establish an in vitro model for liver fibrosis. For that, hIPSCs will be differentiated into cell types relevant for the disease including hepatocytes, macrophages, cholangiocytes, and stellate cells. The resulting cells will embedded in a 3-D scaffold-based culture system compatible with high trough put analyses. Then different etiologies will be modeled including liver fibrosis induced by ethanol, tetrachloride and alpha-1 antitrypsin deficiency. The resulting system will be validated by direct comparison with results obtained in mouse model for liver injury. This project will pave the way for the development of new in vitro model for the study of fibrosis in a diversity of organs and will ultimately allow the development of new therapies.

Over the past 30 years mortality from liver disease in young and middle-aged people has increased over six times, with the number of individuals dying from the disease increasing at a rate of 8-10 per cent every year. By 2016, the UK is expected to have the highest liver disease death rates in Europe and, without action to tackle the disease, it could overtake stroke and coronary heart disease as the leading cause of death within the next 10-20 years. Consequently, the development of new therapeutics against liver diseases represents a global health objective. Animal models including mice infected by schistozoma or with induced hepatic injury are currently the preferred approach to study mechanisms involved in liver fibrosis or to screen for new therapeutics. However, they rarely recapitulate the entire clinical manifestations of the human disease as rodent and human differ in their physiology and metabolism. Therefore the possibility to develop a vitro system modelling human liver fibrosis will have a major impact on several areas.

- Replacement of animal models for basic studies of liver cirrhosis: A larger number of reports are published each year based on mice models for liver cirrhosis (Publications with CCL4 mice: 204 in 2014, 153 in 2013, 87 in 2012. Publications with schistosoma infected mice: 165 in 2014, 123 in 2013, and 60 in 2012). Therefore, an in vitro system compatible with molecular analyses and representative of the human physiology will represent an advantageous alternative.

- Replacement of animal models for drug screening: Pharmaceutical industry is using mainly transgenic mice model for liver failure in order to screen drug against liver diseases. The possibility to use a human system in vitro will render possible high trough put drug/toxicology screening not possible with animal models or alternative in vitro systems.

- Reduction in number of animal for toxicology screen: A 3D platform mimicking the architecture of the liver could be also used to reduce animal experiments in the context of toxicology tests. Indeed, pharmaceutical drugs are currently validated through quantitative risk assessment determined by animal experimentation and UK official Government Home Office figures show that safety tests require ~375,000 procedures in 2013. A significant part of these experiments are performed for liver toxicology since Drug-induced liver injury (DILI) is a leading cause for attrition and withdrawal of pharmaceuticals. An in vitro platform encompassing all the cells constituting the liver including major immune cells which are known to be involved in DILI could present novel opportunity to refine toxicology tests and thus to reduce the number of animal used in this context.

- Replacement of Animal model to study fibrosis: A broad number of disorders result in an inflammatory process leading to fibrosis and ultimately organ failure. The current project will provide a proof of principle that these disorders induced by complex interactions between immune cells and specialised cell types can be mimicked in vitro. In addition, the IMLiF approach could easily be expended to a diversity of organs affected by fibrosis such as the lung. The applications of the IMLiF in this context will be extremely vast. Indeed, basic studies and therapeutical development concerning fibrosis are mainly performed on animal models (Publications on fibrosis and animal models: 1079 in 2014, 1504 in 2013, 1422 in 2012). Therefore, this project will not only have a major impact on the use of animal in the context of liver diseases but will also pave the way toward the development of next generation in vitro models for a diversity a disease with a broader remit.