Tools and Technologies I
The potential of stem cell biology in regenerative medicine is vast; yet in each organ system, unlocking this potential will require a comprehensive, detailed approach unique to that system or organ. Our lab is dedicated to fully developing all aspects required to bring stem cell therapeutics from the laboratory to clinically useful therapies. We have developed a strategy using embryonic stem (ES) cells differentiated in the laboratory in such a way that that these cells successfully transplant into the liver tissue and function. This process could treat diseases where defective proteins are not made, or made improperly like Hemophilia A and B. Furthermore, if the resident stem cell of the liver can be replaced or augmented, these therapies might be long-term, or even lifelong. However, the biggest roadblock to the clinical testing of these treatments is a proper mouse model, where the liver to be treated responds as a human liver would, when receiving the cell transplants. Only then we can track the human cellular transplant for an adequate duration, and have confidence that the cells are safe and durable. We propose to develop a mouse model, a series of human embryonic stem cells, and strategic genetic changes to achieve the goal of possessing as nearly normal a “humanized” liver as possible. If we are successful in creating a mouse model with the described properties, it is likely that we will learn new information about stem cells in general that could have broader application to stem cell therapeutics in other organs while adding to the understanding of other liver diseases. We believe this model could be produced within the time frame of the award. Our five-year plan would be to specialize in multiple applications where genetically modified stem cells could treat liver based disorders. With development of the model described here, one could follow up with a stem cell based strategy for the treatment of hepatitis C infection. Imagine if viral resistant cells could one day remodel the liver and clear Hepatitis C infection. This could be a one-time treatment for patients with Hepatitis C infection, which is a world wide pandemic. Such lofty goals for liver based stem cell therapeutics begin with a robust humanized liver model.
Statement of Benefit to California:
Liver Disease is the 12th leading cause of death in the United States. However, a strong case can be made that its impact is greater on the State of California than more prevalent diseases. The death rates are skewed such that the liver disease takes life in its prime. The rank is 7th between the ages of 35 to 45, and 4th between the ages of 45 to 55. Among Native Americans between the ages of 35 to 45 it is the second most common cause of death. Currently liver transplantation is the only viable therapy for life threatening liver diseases, but this modality is very expensive and available to only a fraction of those worldwide that need a transplant. However, the greatest need that may one day be addressed by stem cell therapies is viral hepatitis. Outside the U.S, hepatitis B and C are worldwide pandemics, and these are diseases that affect urban and underprivileged populations disproportionately. Through stem cell technologies, the State of California has the opportunity to develop a cure for these slow killers. Now, there is a benefit California! Since these are chronic viral infections they are very difficult and extremely expensive to treat, even partially. The vast majority of the 350 million with hepatitis B and 170 million people with hepatitis C are in developing regions, and are not likely to benefit from future antiviral therapies and certainly cannot avail themselves to current medical regimens. Eighty percent of these infected individuals will eventually develop life threatening complication of the chronic infection such as cirrhosis, and liver cancer. We are proposing that a stem cell, genetically engineered to be resistant to these viral particles, could become a one-time cure. For this promise to come to fruition, we must master the technologies to safely engraft Human Embryonic Stem Cell derivates into the liver for the diseases that can most directly be treated in this manner.
This proposal focuses on the development of a stable, humanized liver mouse model. The Principal Investigator (PI) proposes to genetically modify both the human embryonic stem cells (hESCs) and the host mouse to generate the model. In the first aim, using hESC, the PI plans to genetically disrupt the function of a gene involved in the innate immune response, to prevent the destruction of xenogeneic cells after transplanted hESC differentiate into hepatocytes. In the second aim, s/he proposes to generate a transgenic mouse line modified so as to allow the creation of a hepatic niche for transplanted hepatocytes of human origin. The PI will engineer a mouse that will have reduced proliferation of endogenous hepatocyte precursor cells because of an altered cell cycle regulator, allowing a competitive advantage to proliferation of transplanted hESC-derived hepatic precursor cells, and repopulation of the human cells in the niche where the endogenous precursors are cell cycle disabled.. The reviewers agreed that the primary goal of this proposal is an important one, as humanized livers in mouse models are a missing tool for predictive toxicology and drug development, but felt that the PI’s approach was based on faulty hypotheses. They also questioned the feasibility of the research plan. The reviewers found the PI and the research team qualified to conduct the work described in the proposal. While the reviewers recognized that the ability to humanize the mouse liver using hESCs as a source of hepatocytes would have significant utility for preclinical research, they raised questions about the applicants’ approach. Specifically, they criticized the proposal’s guiding hypothesis. The hypothesis is that an innate immune response is a critical limiting factor in the repopulation of the mouse liver by human cells. The single paper cited to support this hypothesis has been strongly put into doubt by subsequent reports by other leaders in the field. Thus the immune damage noted in the cited model may be unique to only one mouse strain, or to that strain following the transplantation of human cells. In the absence of independent evidence to the contrary, the proposal's major focus on the technically difficult task of disrupting a gene in hESCs seems misguided. The reviewers also raised significant questions about the project’s feasibility. One reviewer wondered how teratoma formation would be monitored and prevented. Other reviewers believed that the overall strategy of the second aim, using over-expression of a cell cycle regulator gene in the mouse liver to give an advantage to proliferation of transplanted hESCs was problematic. They noted several potential issues with this approach. Similar transgenic mice have been created and the data from these animals suggest that the level of expression of the chosen transgene must be carefully regulated, as animals with high transgene copy numbers are not viable. The selection of the genetic locus for transgene expression was also questioned, given that expression at this locus is normally massively downregulated in adult liver, where hESC derivatives will be transplanted to populate the liver stem cell niche. In addition, the reviewers expressed concern that this approach might lead to lethality in the fetal period due to poor liver development. Although the applicants propose investigating inducible expression to get around some of these potential pitfall, an inducible expression strategy would add additional work to a project the reviewers felt was already ambitious for a two-year timeframe. The reviewers found the PI and assembled research team qualified to carry out the proposed research. The PI is an accomplished transplant surgeon and has a good track record with ESC research. Overall, the reviewers did not believe that this proposal addresses significant roadblocks in stem cell research and questioned its feasibility as written.