The liver is an essential part of the body because it removes wastes and plays a central role in metabolism. Therefore, inherited or acquired liver disorders represent a major health problem. Currently, the only established successful treatment for end-stage liver disease is liver transplantation. However, the number of donors available is inadequate and a large fraction of patients with liver failure do not have an opportunity to receive a liver transplant. The shortage of donor organs strongly supports the need for alternative treatments for liver diseases. Liver-related cell therapies, where cells are transplanted instead of the whole organ, is one very promising alternative to liver transplantation. However, human liver cells (hepatocytes) are also in very short supply which further complicates the development of new cellular therapies. Human embryonic stem cells (hESCs) represent an ideal source of liver cells for transplantation. A small number of these cells can be expanded into a much larger population of the necessary cells, eliminating the problem of cell availability. In addition, hESCs can be differentiated into many cell types including liver cells. However, success in driving hESC toward liver-like cells has been limited with only a very small percentage of cells acquiring hepatocyte-like functions. We believe that the lack of success in stem cell-to-hepatocyte conversion is partly due to the limitations of traditional cell culture approaches which only allow investigators to study one individual biological change in the culture conditions at a time, the need to use a large number of cells to test these changes, and the considerable time and cost investment. These limitations make it very difficult to thoroughly analyze a variety of different culture conditions that may be successful in helping the stem cells to differentiate into liver cells. The overall goal of this project is to develop novel cell culture technologies that will enhance the ability to precisely control the factors that will induce hESCs to become functional hepatocytes. In this project, technologies commonly employed in the semiconductor industry and in study of the human genome will be used to create miniature stem cell culture platforms where multiple experiments can be performed in parallel and in a shorter period of time then current standard cell culture conditions. This novel "combinatorial" culture platform will allow the rapid discovery of the biological stimuli required for stem cell-to-hepatocyte conversion and significantly advance the field, allowing others to adapt these conditions to their cells of interest. A reliable method for differentiation of human hepatocytes from hESCs will provide a means to obtain a stable source of transplantable liver cells in sufficient quantity and will hasten the development of liver-related cellular therapies.
In 2003, chronic liver disease or cirrhosis was the cause of 513,000 patient discharges and 26,549 deaths in the United States alone (National Center for Health Statistics). Similarly, a total of 6,808 persons died in California during 2003 as a result of end stage liver disease (ESLD) at a rate of 20.1 per 100,000 population, an increase from the the 5,574 deaths and a rate of 18.2 per 100,000 reported during 1999 (California Center for Health Statistics). As a consequence of the limited supply of donor livers, more than 17,000 patients are currently on the liver transplant waiting list and more than 1,500 patients will die this year while waiting for a liver transplant (National Institutes of Health). Therefore, there is a great need to improve strategies for overcoming liver disease-related mortality and morbidity at both the national and state level. As an alternative to transplanting a whole organ, liver cells may be transplanted to achieve a therapeutic effect. However, one barrier to the use of liver-related cell therapies is the limited supply of human liver cells (hepatocytes). Our long-term goal is to employ embryonic stem cells for developing liver-related cell therapies. The unique features of embryonic stem cells, their ability to become many cell types, and their capacity for extensive expansion to a large quantity of cells make embryonic stem cells a very promising source of hepatocytes. However, it is currently very difficult to convert embryonic stem cells to liver cells with high efficiency and yield. This project is focused on overcoming these existing difficulties by developing a "smart" culture dish capable of uncovering the biological stimuli required to drive embryonic stem cells towards a liver cell type. The technologies used in the semiconductor industry will be adapted to create a miniature culture factory that allows scientists to expose the cells to an array of stimuli at the same time, and requires fewer cells as well as other costly cell culture additives. The proposed studies are expected to greatly improve our ability to differentiate embryonic stem cells into liver cells. Creating a method to provide an unlimited supply of human liver cells will bring liver-related cell therapies closer to reality, and will help alleviate the morbidity and mortality currently associated with liver diseases. In addition, human hepatocytes obtained from embryonic stem cells will have immediate applications for use by the pharmaceutical industry and biotechnology companies for drug discovery and development where the potential effects of new drugs on liver cells commonly need to be tested. Overall, we expect the outcome of the proposed project to be of significant benefit to the citizens of the State of California.