Strategies for the development and maturation of functional hepatocytes from hES cells

Each year companies that develop new medicines spend millions of pounds on tests that are meant to make sure their new drugs are not only effective, but also safe for us to take. In spite of this, the single biggest problem with new medicines is that sometimes they cause damage to the liver, heart or brain, which results in them being taken off the market. This situation could be greatly improved if the companies involved had ways of testing (at an early stage) the safety of new medicines using cells that more closely match those found in normal human liver, heart and brain. Liver transplantation is an effective treatment for patients suffering from life-threatening liver diseases and disorders but the supply of donor organs is severely limited meaning that many people remain on the waiting list each year. New ways to efficiently make functional liver cells would lead to improvements in both of these areas. Many researchers, including our own groups, have shown that it is possible to turn unspecialised cells called stem cells into liver cells, more specifically cells called hepatocytes. It is the hepatocytes in our livers that are responsible for removing harmful products from our blood throughout our lives. At the moment, the hepatocyte-like cells we can generate from stem cells are not as good as proper liver cells and lack the ability to carry out many liver functions. Also, the methods currently used to turn stem cells into liver cells are very expensive and take approximately 4 weeks. In this proposal we want to develop conditions that allow us to turn stem cells into liver cells more efficiently and cheaply. We also want to test for ways in which we can make hepatocyte-like cells that more closely resemble the normal adult liver cells found in humans. To help us do this we will make stem cells that turn on fluorescent signals (which we call reporters) at particular stages as they progress from stem to liver cells. One of these 'reporters' will switch on part way through the process, after about a week, and will help us to improve the generation of cells formed during the early stages of liver development and we also hope to discover how we can increase the numbers of these precursor cells. We will also make a second reporter stem cell line will switch on only when the cells have developed into mature liver cells. We will use this stem cell line to help us work out those conditions that turn stem cells into hepatocyte-like cells that are as close as possible to proper adult liver cells. We will examine whether the liver cells we generate are like normal adult liver cells by examining their properties. We will determine if they contain proteins only found in liver cells and we will study if they contain the enzymes important for metabolising medicines, since these are very important for proper liver cell function. We hope that our results will define conditions that: (i) improve our ability to turn stem cells into liver cells, (ii) help us to expand the number of liver precursor cells we can make, (iii) enable us to generate hepatocyte-like cells that are more like proper adult liver cells and (iv) improve the efficiency and methods of turning stem cells into liver cells so that they are cheaper to make. The availability of mature hepatocyte-like cells from a renewable source of stem cells will be extremely valuable to companies making new medicines, since it will help them develop drugs that should be safer for patients and the time taken to develop them will be reduced, saving money. Not only would stem cell-derived liver cells useful for testing the safety of new medicines, but in the future they may also be valuable as a source of cells for transplantation. Therefore, the improvements in generating functional hepatocyte-like cells that we discover will also help scientists and doctors who are developing cell transplant strategies and artificial livers as alternative treatments for patients requiring liver transplants.

Human ESCs self-renew and have the capacity to differentiate into a wide range of cell types. Harnessing these properties to generate specific cell types is very attractive and many groups have demonstrated the ability of hESCs to generate hepatocyte-like cells (HLCs). The scarcity of primary human liver material means that a renewable source of fully functional HLCs would be very valuable to the pharmaceutical industry for predictive toxicology, but also accelerate development and improvement in bioartifical liver technology for clinical use. However, HLCs generated from hESCs using current technologies are often not fully functional. In order to deliver functionally mature HLCs, that are scalable and suitable for pharmaceutical and eventual clinical use new approaches are required to improve and enhance (i) in vitro hepatic progenitor development and expansion and (ii) generation of functionally mature hepatocyte-like cells. We propose to address these issues here and our overall aim is to generate HLCs that exhibit a full range of mature hepatocyte functions from hESCs by cost-effective and robust means. To achieve our goals we will focus on the following specific aims:
1: Establishment of tools for the generation and validation of HLCs from hESCs
2: Optimisation of hepatic progenitor generation and expansion
3: Utilise innovative approaches to promote generation of functionally mature HLCs.
In Aim 1 we will generate reporter lines for hepatic progenitors (HNF4A-eGFP) and mature HLCs (ASPGR1-mCherry) using zinc finger nuclease technology. Aim 2 will see us combine recent innovations, with combinatorial approaches (drawing from our understanding of normal development) to improve generation of hepatic progenitors and establish conditions that permit their expansion and maintenance. The focus of Aim 3 will be the generation of HLCs with a fully functional phenotype using approaches informed by hepatocyte physiology and liver maturation.

We anticipate that our research findings will have impact in a number of different areas. Above all, our research will generate new knowledge and it is this knowledge that will be exploited to deliver impacts that benefit the following communities:

1. The pharmaceutical industry: Currently the pharmaceutical industry invests millions of pounds each year in the development of new medicines, only to experience unacceptably high rates of attrition, arising largely as a result of un-predicted toxicity when these medicines are introduced in man. The availability of a renewable resource of functional human hepatocyte-like cells that could be used at both early and later stages of the drug development process to predict drug toxicity would have major impact and benefits across the sector. Our procedures for hESC to HLC differentiation will, in large part, bypass the requirement for multiple and expensive IP licensing agreements (Geron is the major holder of this IP), which in addition to cost effectiveness arising through improved efficiency and HLC functionality, will be beneficial in further reducing costs associated with hESC to HLC differentiation. New chemical entities that exhibit significant toxicity would be identified at an early stage of lead optimisation, allowing alternatives to be generated that avoid this toxicity. Additionally, at later stages of the drug development pipeline, functional hESC-derived HLCs could be used routinely to assess safety and predict toxicity. These approaches would lead to cost savings as companies would only develop drugs that are predicted to be safe, while helping to ensure that once new medicines are launched they will be safe for the patients to which they are prescribed.

2. Regenerative Medicine and Clinicians: The ability to generate functional HLCs from hESCs will also have impact in the area of regenerative medicine. Each year more patients are on the liver transplant list than suitable donors will be available for, for example, in 2009-2010 679 liver transplants were carried out in the UK, but 371 patients remained on the active waiting list (National Transplant Service Activity report). Therefore, alternate sources of functional hepatocytes could benefit such patients by providing cells for use in extracorporeal liver devices, bioartificial livers or for direct cell transplantation. While the latter is some way in the future, owing to a number of safety concerns that need to be addressed (including immunogenicity and the potential for tumour formation), the use of HLCs in support systems could find utility much sooner.

3. The public and patients: The benefits of our research, outlined in points 1 and 2 above, have the potential to improve the health of the general public that in turn would reduce the burden on the NHS. New medicines should become much safer for patients to take, reducing the potential for adverse reactions, which are estimated to account for 0.5-3% of hospital admissions. This will, in turn, mean patients are treated with the best drug the first time, improving their health and wellbeing. In the longer term, the ability to treat patients suffering from liver diseases with therapies that may allow a patient's own liver to regenerate, e.g. using HLC-based transplantation, should reduce the burden on the NHS for supporting these patients.

4. Researchers involved in the project: Our studies will permit the training of research staff in a wide range of areas, allowing them to develop not only research specific skills, but we will also support them to develop a range of transferable skills. This will directly benefit the research staff themselves through the acquisition of a highly desirable set of skills that will be valuable for their career development, within either the academic, clinical or commercial sectors. Skilled staff are key to economic development of stem cell-based commercial ventures, and this will be another benefit arising from our research.