Development of an hES Cell-Based Assay System for Hepatocyte Differentiation Studies and Predictive Toxicology Drug Screening
Drug-induced liver toxicity, including that from FDA-approved drugs, is the leading cause of liver failure in the US. One of the biggest road blocks to testing drug-induced liver toxicity prior to clinical studies or release of the drug into the market is the absence of a good model of human drug metabolism in the liver. Development of a clinically predictive drug screening system would allow earlier detection of drug-induced liver toxicity, thus decreasing drug costs, decreasing the scale of pre-clinical animal testing, and increasing drug safety. Unfortunately, use of primary human liver cells for drug screening is hampered by their limited availability and poor viability in culture. Human embryonic stem (hES) cells, however, could provide a renewable, scalable, relevant source of liver cells since they can be induced to turn into these types of cells. Unfortunately, though, current hES protocols yield primarily immature liver cells, even though mature adult-like liver cells would be needed for drug screening. Here we propose development of a new hES cell line tool that attaches a fluorescent molecule to a protein found in mature liver cells. This would be a very powerful tool for two key avenues of study and development. First, it will facilitate testing of new methods to enhance the maturation of hES-derived liver cells, ultimately leading to better protocols for transplantation and regenerative medicine purposes. Second, it will also be instrumental in screening for drug-induced liver toxicity effects. While [REDACTED] interests lie more with the usage of this tool for drug screening purposes, we plan to openly share this tool with the scientific community under standard licensing agreements so that rapid progress can be made in both these areas.
This proposal has been submitted by a California company whose mission is to develop and commercialize ES cell-based assays to aid in drug discovery and development. Using stem cells to model how the liver metabolizes drugs would lead to earlier detection of drug-induced liver toxicity, keeping many of the more dangerous clinical drugs from ever reaching the market. This would increase drug safety, decrease drug costs, and decrease the scale of pre-clinical animal testing that is currently used in drug development benefiting all Californians.
The work outlined in this proposal will also bring significant revenue into California, in various different forms. Whenever possible, we will continue to order supplies and/or use services through our standing relationships with California vendors, such as E&K Scientific (Santa Clara, CA), Invitrogen (Carlsbad, CA), and Stanford University Core Research facilities in order to support California businesses and universities. Partnering and licensing of the technology developed in this proposal, as well as commercialization of new and safer drugs, would bring revenue and additional jobs into California. In addition, patents arising from these technologies are also potentially significant for California, due to the licensing revenue fees that would go back to the state.
The ES cell line tool generated as a result of this proposal, along with the reagents used to make it, will be shared openly with the scientific community under transfer and/or licensing agreements that are standard to both academic and industrial scientific entities. This will facilitate rapid progress toward two key avenues of research and development: 1) screening drugs for potential toxic effects on the liver and 2) for better understanding liver development, ultimately leading to better protocols for transplanting ES-derived liver cells into patients with liver disease or drug-induced liver toxicity. This would also result in more jobs in California to carry out this work and would form the basis of additional academic and corporate collaborations that would increase California's leadership role in stem cell applications.
The leading cause of liver failure in the US is drug-induced liver toxicity. Currently there is an absence of a good model of human drug metabolism in the liver, which poses one of the biggest road blocks to testing drug-induced liver toxicity prior to clinical studies or release of the drug into the market. We are using human embryonic stem (hES) cells to develop a clinically predictive drug screening system that should allow earlier detection of drug-induced liver toxicity, thus decreasing drug costs, decreasing the scale of pre-clinical animal testing, and increasing drug safety. There are two arms to this work. The first is to engineer a new hES cell line that attaches a fluorescent molecule to a protein found in mature liver cells. To date we have completed the genetic molecules necessary for development of this cell line, and we are currently using these molecules to generate the engineered hES cell line. The second arm is to test new methods to enhance the maturation of hES-derived liver cells, since current hES protocols only yield immature liver cells. As part of this approach, we are testing a novel 3D culture system that has already been shown to improve maturation of other cell types, such as heart cells and fresh liver cells from humans. By combining our new hES cell line with improved protocols for generating mature hES-derived liver cells, we will have a powerful system not only for screening drugs for potential liver toxicity effects but also for improving protocols for transplantation and regenerative medicine purposes. We plan to openly share this new cell line with the scientific community under standard licensing agreements so that rapid progress can be made in both these areas.
We have developed and validated two human ES cell clones that have the BLA reporter correctly targeted into the CYP3A4 gene. In addition, we have made major improvements to the hepatocyte differentiation protocols, resulting in cultures with greater than 85% hepatocytes, which express significant levels of mature hepatocyte proteins and drug metabolizing enzymes, including albumin, CYP1A2 and CYP3A4. Furthermore, these cultures demonstrate CYP1A2-dependent metabolism of acetaminophen, which is approximately 20% of the activity, on a per cell basis, seen from primary human hepatocytes. This is significantly more than we have seen with any other protocols. We expect to use these cells in our drug development programs as screening assays for liver metabolism and toxicity studies.
We have successfully achieved the major aim of this grant, which was to develop a new human embryonic stem (hES) cell line in which a fluorescent molecule is attached to a protein found in mature liver cells. This protein is responsible for metabolizing the majority of drugs currently in the market, so it is critical to understand the effect that different drugs have on the function of this protein so that drug-induced liver toxicity, which is the leading cause of liver failure in the US, can be reduced. We have done extensive validation of this cell line tool, and we are currently in the process of developing it into an assay system for screening compounds for drug-induced liver toxicity effects. There is a great need for this type of assay since there is currently an absence of a robust model of human drug metabolism in the liver. As part of this development, we have also done extensive work optimizing the generation of liver cells from hES cells. We now can differentiate hES cells into nearly pure populations of cells that are precursors to liver cells and we are currently using our new cell line to facilitate testing new methods to enhance the maturation of these precursors into hES-derived liver cells. The combination of using our new cell line tool in an optimized protocol for generating hES-derived liver cells will hopefully result in a clinically predictive drug screening system that will allow earlier detection of drug-induced liver toxicity, decreased pre-clinical animal testing, and increased drug safety. We are committed to aggressively continuing this work even beyond the close of this grant funding to achieve the goal of a human cell-based, clinically predictive liver toxicity drug screening system.