Tools and Technologies I
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.
Statement of Benefit to California:
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.
The goal of the proposed research is to develop improved technology to generate functional hepatocytes from pluripotent human embryonic stem (hES) cells. Efficient methods to direct differentiation of hES cells to mature endodermal lineages are certainly needed, especially a renewable source of mature human hepatocytes for drug screening, predictive toxicology and clinical transplantation. The basic approach outlined is to utilize growth factors bound to extracellular matrix (ECM) proteins in patterned microarrays to identify an optimal combination of ECM to drive hepatocyte differentiation. Micropatterned co-cultures with cells from mature liver (hepatocytes and non-parenchymal cells) will also be set up in order to obtain potential benefits from factors that would presumably be secreted in the normal hepatic microenvironment. Some preliminary data supports the feasibility of these approaches. The applicants make the assumption that growth factors will be the most critical factors for lineage restriction of ES cells to endoderm, while cellular interactions may be more critical for later stages in differentiation. The main proposed method of analysis for differentiation will be RT-PCR for endodermal and hepatocyte markers. The application has some shortcomings in synthesizing published work on endodermal and hepatic differentiation. It is simplistic to view hepatocyte differentiation as a simple 2-step process. The applicants first plan to generate "endoderm" (not well defined phenotypically and developmentally) and then immediately to jump to generation of hepatocytes. The state of the art in the field already has progressed to somewhat more sophisticated protocols. The relatively efficient generation of definitive endoderm from murine and human ES cells, mainly using Nodal or Activin A, has been reported by multiple groups. The proposed use of patterned microarrays of growth factors to generate endodermal derivatives of hES cells, mainly measured by transcription of Sox17, does not indicate clearly how endoderm differentiation will be improved over that reported in the literature. The absence of a more detailed phenotypic characterization of endodermal derivatives, and of the intermediate steps from mesendoderm and definitive endoderm towards foregut endoderm and hepatocytes is disappointing. It would be important to assess a number of protein markers, not merely Sox17 mRNA. Similarly, isolation of human liver stem cells marked by EpCAM has been reported by more than one group. The use of such a reference standard for authentic differentiation towards hepatic stem cells could be important. Steps in the hepatic lineage from the stem cells to "transient amplifying cells" (marked by turn-on of alpha-fetoprotein, probably bipotential for hepatocytes and biliary epithelium) and then to committed hepatocyte progenitors should be delineated. Similarly, a more complete phenotypic assessment of the terminally differentiated hepatic phenotype would be essential, and should again include more stringent criteria than RT-PCR of (mainly) albumin mRNA. Specific markers of Zone 1, 2, and 3 hepatocytes need to be assessed quantitatively, along with other measurements of hepatic function such as drug metabolism. The use of protein components of ECM to present growth factors is logical. However, there is also strong evidence from published literature dating back almost 2 decades that liver-specific proteoglycans play a crucial role in hepatic differentiation, including data from co-culture experiments. Simply substituting heparin for collagens or laminin will likely not suffice to provide the liver-specific components. Thus the application is weakened by a lack of detail regarding the specific cell types proposed for co-culture experiments, and their possible role along the differentiation pathway. Nonetheless, reviewers felt that overall the work proposed was feasible and likely to generate some useful new information. They were concerned that presentation of the engineering concepts was strong but the biology was relatively weak, and that the collaborators with expertise in this area had not sufficiently contributed to the proposal. The principal investigator (PI) is an Assistant Professor in Biomedical Engineering at an academic medical center. The PI has a strong track record, including good publications and grant support, mainly in the engineering aspects of the project - particularly microarrays and microfabrication technologies. The collaborations with two more senior investigators bring expertise in stem cell biology and growth factors/extracellular matrix, respectively. The other personnel appear well qualified. Overall, this is a team that appears to have been assembled with a clear conception of the need for complementary expertise in engineering and biology.