Our ability to grow and manipulate human ES (hES) cells has improved markedly in recent years. A number of procedures have been developed to allow differentiation of human ES cells into specific cell types such as insulin-producing cells and liver cells. Despite these tremendous technical advances, several major technical challenges need to be addressed if the intent of such differentiation is cell replacement therapy. To perform any transplantation assay, whether in animal models or in humans, millions to billions of cells must first be induced to transform into a specific cell type to provide adequate starting material. Therefore, a robust procedure for scaling up the culture procedure is the first requirement. At the same time, the transplantable cells must exist in a pure population, devoid of any unwanted, undifferentiated (and therefore potentially tumorigenic) hES cells. In this proposal, we will solve these problems using a new microfluidic platform that delivers growth factors to the cells in a uniform, efficient manner to decrease the heterogeneity and increase the yield of specialized cells from hES cells. Further, we propose to develop prototype bioreactors to produce specialized cells from hES cells in large-scale. During the course of this experiment, we will focus on generating hepatocytes (liver cells) from human ES cells. Availability of a large number of hepatocytes will significantly impact the field of liver diseases for the following reasons. First, stem cell therapy for severe liver failure is particularly attractive as donor livers are rare and intravenous administration of differentiated hepatocytes usually results in their deposition into the liver. Second, there are significant benefits to the field of hepatitis research from the development of a homogeneous and plentiful source of human hepatocyte. Currently there are no effective animal model systems for studying hepatitis virus infection, as the virus infects only human and primate cells. Thus availability of a reliable source of human hepatocytes will accelerate the rate of progress in the field. Third, hES cell lines with genotypes characteristic of various genetic diseases that affect liver function could provide novel insights into mechanisms of the liver disease process and variations in drug response. In summary, this project attempts to accomplish two goals. One is to develop a widely used technique to grow a large population of homogeneous cells. Second is to produce a large numbers of hepatocytes that will be useful in clinical research.
Statement of Benefit to California:
A primary goal of Proposition 71 is to translate basic stem cell research to clinical applications. The disability and loss of earning power and personal freedom resulting from a disease or disorder are devastating and create a financial burden for California in addition to the suffering caused to patients and their families. Therapies using human embryonic stem cells (hES cells) have the potential to change millions of lives. Using hES cells as models of disease will help us understand the underlying causes of disease and likely aid in the development of drugs to treat those diseases.
In this proposal, we will focus to produce a large number of liver cells to cure various liver diseases. It is astonishing that liver diseases such as cirrhosis and hepatitis affect 25 million people in the United States, and that over 27,000 Americans die from cirrhosis annually, making it the country's third leading cause of death for people between the ages of 25 and 59. The 2006 report submitted to the Center for Health Statistic for the State of California indicated that chronic liver disease and cirrhosis age-adjusted death rate was 10.6 deaths per 100,000 population in California. The number is significantly high and this number did not meet the Healthy People 2010 objective of no more than 3 deaths.
Given the fact that often the only way to cure patients with sever liver disease is by transplanting new healthy livers, and that donor livers are rare, a new approach to obtain liver cells for transplantation purpose is urgently needed. Since liver transplantation will require millions to billions of cells, which must first be induced to transform into a specific cell type to provide adequate starting material, a robust procedure for scaling up the culture procedure is the first requirement. At the same time, the transplantable cells must exist in a pure population, devoid of any unwanted, undifferentiated (and therefore potentially tumorigenic) hES cells. Only when these goals are met, we can consider developing clinical trial methodology that will directly impact human testing of stem cell therapies. Our proposed approach can overcome these major issues.
Additional significant benefits obtaining a reliable source of hepatocytes are the following. It would distinctly increase the pace of development of novel therapies for hepatitis patients. Second, it will serve to develop new improved methods for developing and testing drugs for treating liver diseases. It is also anticipated that, in the long term, the return to the State in terms of revenue, health benefits for its Citizens and job creation will be significant. The State of California will also be viewed as the leader in establishing clinical stem cell research programs.
This application focuses on a significant bottleneck to the application of hESCs for cell-based therapies: the lack of differentiation protocols that produce high quality, pure cultures of specialized cell types. In particular, this proposal is concerned with cell therapy approaches for end-stage liver diseases and the need for large quantities of hepatocytes. The proposed approach depends on a microfluidic platform to deliver growth factors efficaciously, decrease the heterogeneity inherent in culture systems and increase the yield of fully-differentiated cells from hESCs. Hepatocytes differentiated in vitro will be analyzed functionally by transplantation into an appropriate mouse model.
The reviewers agreed that a useful stem cell source of hepatocytes would be of considerable benefit for research, and the potential impact could be extremely high for cell-based transplantation therapies. Transplanted hepatocytes could provide a bridging system in patients with acute liver failure, serving until a suitable liver donor is identified; these patients are most likely to see a significant impact if unlimited quantities of hepatocytes from hESCs were available.
The proposal describes the development of a microfluidic culture system to overcome the differentiation bottleneck, but reviewers had serious concerns about the feasibility of many details of the project’s execution. Reviewers felt that the description of the culture system methodology was imprecise and unclear, and they expressed concerns that the endpoints and measures of mature hepatocyte function were inadequate. Reviewers noted that the proposed procedures were largely derivative and that the research team lacks extensive experience in the analysis of liver function. There was further concern about the feasibility of Aim 2 in that insufficient consideration was given to issues of scale-up, cell removal and reseeding of the bioreactor, and choice of substrates for attachment and differentiation. Overall, reviewers were not convinced that the methods proposed would yield sufficient purity of cell product or sufficient numbers of hepatocytes for clinical application. Since hepatocytes cannot be routinely passaged in culture, some of the required culturing procedures described in the application may be extremely difficult to achieve. The potential complication of rejection of transplanted hepatocytes was not addressed, and this would likely be an important issue during testing of successfully differentiated cells.
The principal investigator is highly qualified, well published, and well supported by the NIH. The team’s strengths are in engineering and stem cell culture, but the addition of personnel experienced in liver biology would have strengthened the application, especially with regard to the proposal’s design. Finally, reviewers felt that the research environment was exceptional and noted that the applicant would be moving into a newer, better, CIRM-funded facility in the near future.
In summary, the experience of the group as well as the novelty of the application is largely in the engineering area and not in cell biology. Although the goals are worthwhile, prospects for the study’s success are severely weakened by lack of essential detail and deficient attention to liver physiology and hepatocyte biology.