The liver is the largest internal organ in the body and is essential for bodily function. It metabolizes the food we eat and the drugs we take and detoxifies foreign compounds that we are exposed to. It produces proteins that circulate in the blood that carry lipids, vitamins and hormones, as well as enzymes that help our blood clot. The liver has a legendary capacity to regenerate and repair itself but many times it cannot. Excess alcohol, exposure to chemicals and viral infections can all cause the liver to become permanently diseased and fail to carry out its essential functions.
Liver failure results in more than 40,000 deaths annually in the United States. Currently, the only viable treatment for liver failure is liver transplantation. However, liver transplantation as a treatment can be problematic due to a shortage of available donor organs (particularly in the U.S.), risk of rejection and complications due to chronic treatment with immunosuppressants. An alternative approach for treating acute and chronic liver failure is hepatocyte transplantation therapy. Hepatocytes are cells in the liver that carry out liver functions. Hepatocyte transplantation has several advantages over whole organ transplantation but is still limited by a reliable source of hepatocytes.
Hepatocytes are also important for drug testing. Before a drug can be tested in humans it must undergo pharmacokinetic and toxicological tests in the liver. In the past, these tests were done in animals (usually rats, mice or dogs) but we now know that there are many important differences between humans and other mammals so it is preferred to test drugs in human systems in vitro (i.e., in a petri dish) before they are given to humans. These in vitro or cell-based assays also save the lives of millions of animals that in the past had to be used for this testing. To do perform the in vitro testing, pharmaceutical companies typically buy freshly isolated human hepatocytes from organ donors who die in accidents. However, this supply of hepatocytes, like that for treating liver failure, is erratic, not well controlled and extremely expensive.
Both of these problems – a shortage of hepatocytes to treat liver disease as well as to use in pharmaco-tox testing – can be solved using human embryonic stem (ES) cell technology. ES cells are cells that have the capacity to regenerate themselves (i.e., “proliferate”) as well as to turn into any other cell type in the body under the proper conditions (i.e., “differentiate”). Hepatocytes are among the cell types that researchers have been able to derive from human ES cells, however much more work needs to be done in this area. In this proposal, we will establish an optimized protocol to differentiate human ES cells into hepatocytes. These hepatocytes can be used as a reliable, controlled source of material to treat liver disease as well as to test new drugs for treating other diseases.
Statement of Benefit to California:
California will benefit in many ways from this research. It has been estimated that it requires hundreds of millions of dollars and at least 10 years for a single new compound to reach the marketplace as a new drug. There are many hurdles that contribute to this long lag time and costly process. One of the main hurdles is toxicological testing. In order for pharmaceutical companies to develop new drugs to treat diseases they must first test them to determine whether the drug is toxic to human cells and how it will be metabolized in the human body. The current gold standard for toxicological testing are primary human hepatocytes. Pharmaceutical companies spend over $4 billion per year on drug testing using human hepatocytes. This need for human hepatocytes is greatly increasing as companies are automating the drug discovery process using high throughput screens done by robots. Between that automation and the human genome sequence that is now available, the number of new drug candidates is increasingly exponentially. That means that the need for human hepatocytes to test these drugs on before they can be tried in humans is also increasing greatly.
The problem is that the supply of human hepatocytes is very limited and is not well controlled since it must rely on organ donors who typically die in accidents. There are several companies that sell human hepatocytes for drug testing, and research, but none of these major companies are based in California. This proposal aims to develop a viable, renewable source of human hepatocytes using embryonic stem cells. Companies will then buy their hepatocytes from California, bringing large amounts of revenue into the state.
SYNOPSIS: The long-term goal of the proposed research is to establish human embryonic stem cell (hESC)-derived human hepatocytes as a research tool and a therapeutic option for treatment of liver failure. The first Aim is to develop an optimized protocol for the differentiation in vitro of hESCs to hepatocytes by monitoring the appearance and persistence of liver-enriched transcription factors known to control the metabolic functions of mature hepatocytes. The efficacy of a published differentiation protocol will be more thoroughly examined by microarray expression analyses, cellular morphology, detection of multiple hepatic markers, and analysis of spliced isoforms for HNF4alpha and other HNFs. The second Aim will generate derivative hESC lines bearing transgenic reporters to mark and thereby track the effectiveness of hepatocyte development and differentiated functions in mouse transplant models.
INNOVATION & SIGNIFICANCE: Others have presented evidence that hESCs can be encouraged toward an hepatic phenotype in vitro. A concerted effort to optimize the production of differentiated hepatocytes using rigorous criteria for differentiated functions is timely. The innovation in this proposal is the insight into hepatic transcriptional regulators to define precise criteria to monitor the course of in vitro differentiation and to set specific molecular goals for in vitro differentiation protocols. The demand for liver-donors for transplantation hepatocytes far exceeds availability. An effective strategy for large-scale production of functional hepatocytes from hESCs would be an exceedingly valuable therapeutic resource for a wide variety of liver diseases. A replenishable source of homogeneous human hepatocytes also would be extremely useful to test drugs for toxicity and pharmacokinetics, prerequisites for clinical trials. One of the reviewers thought that generation and analysis of hepatocytes from hESC is a therapeutically important goal but otherwise, the application lacks innovation.
STRENGTHS: Both reviewers agreed that a strength is the significant prior experience of the Principal Investigator (PI) in analyzing transcription factors regulating hepatocyte differentiation. The PI is an established investigator with extensive experience with transcription factors controlling liver function, which will help guide the development of more effective differentiation protocols. The PI has become well-trained in embryonic stem cell technology at the Pasteur Institute, a WiCell workshop and the 2005 ISSCR meeting, and has recruited the collaboration of a new Assistant Professor with training and a publication record documenting extensive expertise with hESCs.
WEAKNESSES: Although organ-specific spliced isoforms of hepatic transcription factors likely have important distinctive biological functions, it is as yet unclear that they play central roles for ESC differentiation. The goals emphasize characterization of two ESC lines and data mining. The more specific aspects of the experimental plan are narrowly focused on a single spliced isoform of one liver-enriched factor rather than on a more general strategy to coax the differentiation of hESCs toward an hepatic phenotype. This implies a greater interest in the biology of HNF4alpha than developing the technology to create hepatocytes from hESCs. HNF4alpha1/4 as well as other endoderm and liver markers appear to be expressed in primitive endoderm as well, and may confuse the use of some markers to follow the formation of definitive endoderm and hepatocytes. A plan is needed that considers this complication and includes markers for visceral/primitive endoderm. Although this could be a useful project, it is not clear what particular insights will make this attempt different or more effective than previous ones.
A number of concerns about the transgenic lines of Aim 2 remain. Targeted integration in hESCs has been technologically difficult, and specific insights into this technical problem are not presented. Because precise recapitulation of correct developmental expression by the transgenes may be crucial, it is important to know beforehand the gene regions that contain all critical developmental information for the albumin, P1, P2, HNF4a1, HNF4a7 promoters and, if possible, a plan to confirm their regulatory potential in vivo. Inserting liver-specific transgenes in a locus (ENVY) expressed ubiquitously may eliminate the desired hepatocyte specificity of these engineered markers. Because transgenes of differentiation markers would only track differentiated hepatic ESCs in mice models, a second ubiquitously expressed transgene would be needed to test the efficiency and overall developmental potential of lines.
Some specific concerns are: (1) Optimization of existing protocols for differentiation of ESC into hepatocytes are poorly articulated. Transgenic constructs generated in aim 2 should be used as convenient reporters in testing optimization parameters. (2) Experiments in aim 2 are largely descriptive and it remains unclear if they will yield clear and important insight into molecular pathways regulating human hepatocyte differentiation.
The D’Amour protocol for generating definitive endoderm might be an excellent starting point.
DISCUSSION: There was no further discussion following the reviewers' comments