EXECUTIVE SUMMARY

S-Nitrosoacetylpenicillamine (SNAP) Induced Maturation of

Embryonic Stem Cell Derived Liver Lineage

SUMMARY:  Dr. Martin Yarmush and Dr. Eric Novik in the Department of Biomedical Engineering at Rutgers University have utilized previously reported primary stem cell differentiation systems to generate liver lineage cells.  These cells are then plated into a collagen sandwich configuration and supplemented with a morphogen, S-nitrosoacetylpenicillamine (SNAP) to induce maturation and maintenance of liver function in embryonic stem cell derived liver lineage cells.  This technique confers the following benefits: 1) the system can maintain liver function of differentiated stem cells for extended periods of time; and 2) the cells have more function than previously reported results.

BACKGROUND:  The liver is the metabolic hub of the human body and performs a variety of functions such as exocrine, endocrine, clotting, organic metabolism and cholesterol metabolism.  Hepatocytes are the primary cell type of the liver and are responsible for most of the liver’s metabolic functions.  Isolated human hepatocytes, which carry most of the livers detoxification work load, are generally recognized as one of the most relevant first line screens in pre-clinical candidate drug assays.  In particular, the cytochrome P450 enzymes in hepatocytes are responsible for most Phase I and II drug metabolism and their basal expression and function is of great interest to drug metabolism and pharmacokinetic researchers.  According to the PhRMA (Pharmaceutical Research and Manufacturers of America) U.S. drug companies spent $39.4 billion on research and development in 2005.  Studies indicate that it can cost from $802 to $868 million dollars, of which 80% is spent on clinical trials and development, and 8-10 years of development to bring a novel drug to market.  Experiments can include basic in-vivo/in-vitro drug clearance correlations, CYP450 induction studies as well as genetic screening.  Limitations to using isolated hepatocytes include the rapid loss of liver-specific function when maintained in-vitro as well as the procurement of healthy cells.  In general, healthy livers are used for transplantation and only livers which are not fit for transplantation are used for hepatocyte isolation.  A sustainable source of cells could alleviate both cost and quality pressures.

In addition, in 2001 in the U.S., 97 patients with ALF died while waiting for an OLT.  Development of a bio-artificial liver (BAL) device can bridge the gap between supply and demand and give people the time to wait for a donor liver to become available or allow enough time for the liver to regenerate.  A BAL requires approximately 10¹¹ cells to sustain liver function.  A major bottleneck to such therapies is finding a cell source that can readily sustain such a large quantity of cells.  To make BAL therapy a viable treatment option would require a renewable source of functional hepatocyte cells.  Adult differentiated hepatocytes have been studied as the source for these cells however; the shortage of donor livers and decrease of function with time necessitates the investigation of alternative cell sources for use in a BAL.  Acute liver failure affects hundreds of thousands of people per year around the globe and in many cases is resolved with a liver transplant.  Due to a shortage of donor organs, many patients will die while waiting for a donor organ to become available.  Extracorporeal liver assist devices could help to bridge patients to transplant.  However, this technology is limited by a lack of an adequate hepatocyte cell source.  Pluripotent embryonic stem cells represent a promising renewable cell source to generate hepatocyte lineage cells, which have been incorporated into implantable engineered tissue constructs and ex vivo cell based therapeutic devices.  However, current differentiation techniques have not yet generated the large and functionally sustainable cell masses which would be required to make such therapies clinically available.

TECHNOLOGY:  Drs. Yarmush and Novik have been able to obtain cells which not only have secreted function in the form of albumin secretion, but also detoxification via CYP450 metabolism.  Using 3-MC for 48 hours to induce CYP450 activities, both BROD (CYP 2B2) and MROD (CYP 1A2) detoxification was observed at a level similar to the Hepa1-6 mouse hepatocyte carcinoma cell line.  Although, there have been reports of induction of CYP450 mRNA in ES derived hepatocyte-like cells, there have been few reports of detoxification, a function which would be critical for use of these cells in drug discovery studies as well in a bioartificial liver device.  In addition, the population of hepatocyte lineage cells has been amplified and they maintain their function over extended periods of time.  Together, an induction of detoxification function and an increase in cell mass, greatly improve the prospect of using these cells in drug screening assays as well as incorporating them into bio-artificial liver devices and other tissue engineered constructs.

PATENT STATUS:  A patent application is being filed.

PUBLICATIONS:  E. I. Novik, J. Barminko, T. J. Maguire, N. Sharma, E. J. Wallenstein, R. S. Schloss and M. L. Yarmush, Augmentation of EB Directed Hepatocyte-Specific Function Via Collagen Sandwich and SNAP, has recently been accepted for publication in Biotechnology Progress.