Early Translational I
Liver transplantation offers hope to children born with metabolic diseases, to people suffering from acute liver failure, and to those who have long term liver disease such as that caused by the worldwide epidemic of Hepatitis C. Unfortunately the demand for healthy liver tissue for transplantation far exceeds the available supply. A renewable source of human liver cells would also make the development of safe new medicines faster and more reliable. Many drugs are metabolized in the liver, but this metabolism can result in the conversion of a safe medicine into a toxin, or can convert an active compound into an inactive and ineffective substance. Tests in animals do not always predict how the human liver will metabolize drugs. Thus, during the drug development process, many compounds drop out late because of unpredictable results of human metabolism, adding greatly to the cost of Human embryonic stem cells, or pluripotent stem cells made by reprogramming adult cells, could in principle provide a renewable source of liver cells for transplantation and drug testing. Previous work has shown that it is possible to convert embryonic stem cells into cells resembling liver cells, but the methods described are inefficient and do not yield pure populations of functionally mature cells. While most work to date has attempted to isolate mature liver cells directly, research in several laboratories has recently identified a stem cell population in the fetal and adult liver capable of extensive growth and regeneration. Our preliminary work shows that it is possible to convert embryonic stem cells into cells that resemble these stem cells found in fetal or adult human liver. This project will focus on identifying the most efficient way to convert stem cells in liver progenitors, to grow these progenitors in the laboratory, and to convert them into mature liver cells. We will test their ability to engraft and function in animal models of liver repair. By producing pure, well characterized populations of liver stem cells on a large scale from embryonic stem cells, we will overcome a major bottleneck to the application of stem cell technology to liver disease.
Statement of Benefit to California:
End stage liver disease is one of the leading causes of death in California. Minority populations including Native Americans, Hispanics, and African Americans are particularly affected. There is an acute shortage of liver tissue for transplantation; it is estimated that 15% of patients die on the waiting lists. The development of cell replacement therapy for liver disease could greatly enhance the therapeutic options for treatment of children with metabolic disorders, or adults with acute liver failure in the near term future. In the long run, it may be possible to use such approaches for the treatment of chronic liver disease such as that caused by Hepatitis C, if transplanted cells could be engineered to resist viral infection. Thus, a novel source of healthy tissue could impact strongly on the treatment of liver disease. Production of human liver cells in the laboratory also could greatly accelerate the process of drug development. Since the liver accounts for most drug metabolism, and animal models do not necessarily predict how the human body will handle drugs, many compounds fail in the final stages of testing at a great economic cost when it becomes apparent that the human liver inactivates the drug or worse converts it to a toxic compound. Human cell culture models of liver metabolism have the potential to address this hurdle in the drug development process and would be of great economic value. This proposal addresses both the clinical problems of liver cell transplantation and the practical laboratory use of human liver cells in the pharmaceutical industry. It has the potential to impact on human health and to provide significant economic benefit to the State.
The goal of this effort is to develop an improved methodology for producing mature liver cells for regenerative medicine. Initially, the applicants propose to investigate procedures for efficient conversion of human embryonic stem cells (hESC) to hepatic progenitor cells (HpPC), which would ultimately serve as a source for the generation of mature hepatocytes. These HpPCs will be extensively characterized, and the resulting data will be used to devise procedures that will aid in their enrichment or purification from the general population. Next, the applicants propose to improve methods for in vitro expansion of HpPCs using growth factors identified from the hepatic niche. In addition, a series of parameters will be evaluated for driving HpPCs into the hepatocyte and bile duct lineages, the maturity of which will be assessed by a panel of functional analyses. Finally, the applicants propose to engraft HpPCs into mouse models of liver damage in order to assess their capacity to repair tissue. The reviewers agreed that the proposed research addresses a bottleneck in the production of mature hepatocytes, a critical resource for addressing significant public health issues relating to liver disease and drug toxicity. While indicating that the provided description was somewhat superficial, the reviewers expressed great confidence in the capabilities of the research team and noted the excellent track record of the principal investigator. Despite these assets, however, a number of weaknesses were thought to detract from the overall feasibility of this effort. In general, the applicants failed to provide sufficient background information to distinguish their approach from many ongoing and competing methodologies. For example, although in possession of a specific antibody, the investigators did not delineate how this reagent would be advantageous or distinctive compared to alternatives. Of greater concern, the proposed functional analyses of Aim 4 were thought to be insufficient and naively designed. Many of the described endpoints were not necessarily specific to mature hepatocyte function, and the applicants failed to justify how these parameters would contribute to their overall assessment of maturity. Moreover, this aim largely failed to include experiments that directly compared the properties of the derived cells to those of authentic hepatocytes. Most reviewers felt that these analyses, as well as some of the in vivo validation studies, would be more useful or appropriate if conducted prior to proposed expansion efforts of Aim 3 rather than subsequently. As a result, the timelines and milestones appeared to be inefficient and perhaps unrealistic. Additional concerns were raised about the animal models, which some reviewers felt were not state of the art compared to others that have been published. Based on all of these deficiencies, the reviewers suggested that this effort would strongly benefit from the active participation of an experienced hepatobiologist. Finally, the budget was judged to be highly inflated, and the number of full time employees appeared excessive for a relatively modest set of goals. In summary, the proposed effort addresses a critical unmet need and could offer potential new tools for the treatment and investigation of liver disease. Despite an extremely qualified team and an outstanding research environment, it was not clear that this approach offered advantages over competing methodologies. Furthermore, several weaknesses in the experimental design appeared to limit both the feasibility of this strategy and the efficiency with which it could be achieved. Finally, the budget seemed excessive and beyond the scope of the proposed effort.