Executive Summary The aim of this proposal is to establish new therapies for chronic liver disease through the use of differentiated human embryonic stem cells (hESC) or human fetal hepatocytes immortalized with telomerase. The first deliverable will be consistent methods to differentiate and isolate pure hepatocyte-like cells from hESC. A second deliverable will be preclinical studies to investigate efficacy and safety. The final component will be development of clinical scale-up and good manufacturing practices (GMP)-level testing of cells. The goal is to develop a Phase I trial. Ultimately the hope is to fill the unmet need for successful treatment of end-stage liver disease. End-stage liver disease is devastating and calls for new therapies. It is currently best treated by transplantation but there is a dearth of available organs and the transplant procedure is daunting. Thus, some reviewers agreed that a cell-based therapy would be advantageous if fully differentiated hepatocytes were available. However, one reviewer expressed strong doubt that chronic liver disease is an appropriate target for cell-based therapy, based on the pathophysiology of the disease, and suggested that such therapy is more likely to be effective for patients with acute liver failure or for patients with inborn errors of metabolism. Reviewers differed in their opinions of the maturity of the proposed concept, and acknowledged that the field of liver stem cells and hepatocyte differentiation from hESCs is young but maturing. The PI has evidence that telomerase-immortalized fetal hepatocytes may be suitable cells for liver reconstitution. However, one reviewer questioned the maturity of the science as the proposal still placed much emphasis on optimizing hepatocyte differentiation and even choosing which cells to use. Furthermore, there is no mention as to how cell delivery will be accomplished, how the immune response will be managed, and which animal models will be used, yet these would seem to be critical components leading up to clinical trials. The PI addresses the need to eliminate teratoma potential using several methods, including genetic engineering of the cells, yet reviewers pointed out that acceptability of such maneuvers by the FDA must be considered early on. In light of this, reviewers also emphasized that the combination of telomerase-mediated immortalization of therapeutic cells and the anticipated immune suppression will predispose the transplanted cells to become cancerous. Concern was also expressed, as was acknowledged by the PI, as to whether the cells that will be developed by this strategy will truly be able to exhibit all differentiated cell functions usually expressed by hepatocytes. The PI is a well-respected expert on liver and the use of stem cells for replacement of liver. S/he is a Professor of Medicine and the Director of a Transplant Research Program at the home institution, working collaboratively with many scientists on campus and in a nearby institution. The PI has considerable experience and accomplishments in the field of gastroenterology, has had continuous NIH funding since 1979, and has helped design a clinical trial of autologous adult cell transplantation in patients with end stage liver disease. S/he has assembled an impressive number of collaborators with extensive expertise in the fields of stem cell biology, molecular imaging for follow-up, development of scale-up and GMP testing, and in clinical trial design and execution. However, it is not clear as to what degree the individuals listed in this application have experience with clinical trials as the principal investigator rather than performing supporting roles. Additionally, one reviewer pointed out that the home institution is not recognized to be a particularly active liver disease center, especially in terms of the number of patients undergoing transplantation at that center. However, other resources are in place such as a relevant animal model research center, and stem cell and translational studies programs. The proposed planning approach was regarded to be sound. The PI proposes weekly meetings to flesh out the research plan with timelines and deliverables. They plan for recruiting a Project Manager and an External Advisory Committee as well as designating an Internal Advisory Board. They already have a structure and infrastructure that should facilitate their putting together a cohesive Disease Team for Liver Therapies. Reviewer Synopsis This proposal is submitted by Mark Zern, Professor of Medicine at UC Davis. The aim is to establish new therapies for Liver Cell replacement through the use of differentiated hES cells or human fetal hepatocytes immortalized with telomerase. The planning will develop an organizational structure that will enhance collaboration within a group including basic and clinical investigators. The first deliverable will be consistent methods to differentiate and isolate pure hepatocyte-like cells from hES cells. A second deliverable will be preclinical studies to investigate efficacy and safety. The final component will be development of clinical scale-up and GMP-level testing of cells. The goal is to develop a Phase I trial. Ultimately the hope is to fill the unmet need for successful treatment of end-stage liver disease. Reviewer One Comments Concept: This proposal seeks to establish a liver cell therapies team to develop a viable human hepatocyte-like cell line that will be used for cell-based therapies. The group will attempt to develop such cells employing two different strategies; first, using a cell line from human embryonic stem cells and secondly, from immortalized human fetal hepatocytes immortalized with telomerase reconstitution. These cells will then be used for cell transplantation. While I think there is little doubt that this group will be able to assemble a liver cell therapies team, I have serious concerns regarding their overall strategy to turn this into an effective treatment for patients with chronic liver disease. The major reason why people need liver transplantation in this country relates to the development of cirrhosis with subsequent complications of portal hypertension and in some cases the development of hepatocellular carcinoma. It is my belief that this therapy will not be effective for these patients. Yet, it could potentially be effective for patients with acute liver failure where there is rapid destruction of liver cell mass or for patients with inborn errors of metabolism expressed in the liver that produce extra hepatic disease. However, this represents a relatively small subset of patients. Finally, while there is little doubt that chronic liver disease is an important issue, I do want to correct several misstatements in their introduction. First, there has been no increase in the number of patients on the active waiting list in the United States since 2002. Moreover, the number of patients listed for end stage liver disease for hepatitis C is actually decreasing and the only increase has been in patients who have developed the complication of hepatocellular carcinoma. I also have concerns as to whether the cells that will be developed by this strategy will truly be able to exhibit all differentiated cell functions usually expressed by hepatocytes. While this is acknowledged by the principal investigator, it does not seem to be considered as a likely serious problem as I think it will be. As I stated, the goal is to assemble the team. Some of the strengths of this proposal are that there is clearly great expertise in stem cell biology and hepatocyte transplantation in the proposed team. In addition, multiple members of the team have already established a track record of being able to work well together. Principal Investigator: The principal investigator is clearly a talented scientist with a long track record of funding in the area of hepatocyte biology. He has previously demonstrated the ability to generate immortalized hepatocytes from stem cells in fetal hepatocytes. He has assembled an impressive number of collaborators with extensive expertise in the field of stem cell biology, clinical trial design and execution. Two members of the team already have experience in hepatocyte transplantation although that experience is extremely modest. He also has individuals identified that will assist with the development of an appropriate bioreactor to scale up cell line production to the level where it could be used as a clinical tool. Finally, the PI is well funded. Two of the concerns that I have is that UC Davis is not recognized to be a particularly active liver disease center, particularly in terms of the number of patients undergoing transplantation at that center. Finally, it is not clear as to what degree the individuals stated have experience with the clinical trials as the principal investigator rather than just simply being supporting characters. Planning Approach: The planning approach appears to make sense given the very serious concerns listed above. Step one will be the development of appropriate cell lines. Step two will be to demonstrate efficacy of cell lines to treat animal models of liver disease. It is worth noting that there is no discussion in this proposal as to which animal models will be used. Step three is the development of clinical scale of GMP testing with the best cell lines available for human trials. The fourth step is to initiate clinical trials. As I stated above, I do have concerns as to in which clinical indication these cell lines will be clinically efficacious. Reviewer Two Comments Concept: Chronic liver disease is currently best treated by transplantation but there is a dearth of available organs and the procedure for transplant is daunting. Thus a cell-based therapy would be advantageous if fully differentiated hepatocytes were available. Dr Zern was awarded a Comprehensive Grant last summer for comparing hESC-derived cells, telomerase-immortalized fetal hepatocytes, and oval cells. Here they will continue optimizing the differentiation of the hES cells, compare to the fetal cells in vitro and in vivo and then take the best cells to clinical trials. This group has the expertise necessary for bringing the basic science to the non-human primate trials. This Disease Team will also consider how to eliminate teratoma potential by sorting or engineering a suicide gene, scale-up for adequate tissue quantities, and genetically engineer to facilitate longitudinal tracking by imaging. Yet the acceptability of such maneuvers by the FDA must be considered early on. The question of maturity of the science must be asked with this much emphasis on optimizing hepatocyte differentiation and even choosing what cells to use. There is no mention about how the cell delivery would be done, yet this would seem a critical part of clinical trials. Will single cells, aggregates, encapsulated cells, cells on biocompatible scaffolds be used? Additionally it would seem that perhaps some interaction with Geron, who reported in 2003 that they were able to differentiate hepatocytes from hES cells, should be included. Principal Investigator: Dr. Zern is a well-respected expert on liver and the use of stem cells for replacement of liver. He is Professor of Medicine and Director of Transplant Research Program at UC Davis. He has served as chair for various review groups including NIH study sections and liver foundations. He has been Chief of the Gastroenterology Division at Roger Williams Hospital (Brown) and Jefferson Medical College. As director of the Transplant Research Program at UC Davis he has worked collaboratively with many scientists on campus and in the Medical technology program at Livermore National Lab. He has had continuous NIH funding since 1979 and has a CIRM Comprehensive Grant for preclinical studies comparing three different human hepatic stem cells including hESc. He has helped design a clinical trial in Cairo for autologous CD34+ cell transplantation in patients with end stage liver disease. The Co-PIs, Drs Nolta and Tarantal, who have collaborated with the PI, add expertise and experience in organizing multi-investigator projects. Planning Approach: Drs Zern, Nolta and Tarantal form the executive portion of the team and provide extensive leadership experience. In addition they have the resources of the California National Primate Research Center, the UC Davis Stem Cell Program and the Clinical Translational Studies Center. They have enlisted experts in molecular imaging for follow-up, development of scale-up and GMP testing, and several experts on the clinical aspects for regulatory compliance and trial design. There are weekly meeting to flesh out the research plan with timelines and deliverables. They plan for recruiting a Project Manager and an External Advisory Committee as well as designating an Internal Advisory Board. They already have a structure and infrastructure that should facilitate their putting together a cohesive Disease Team for Liver Therapies. They should focus on what gaps there still are, e.g., delivery of cells, acceptance of genetically altered cells by the FDA at this planning stage in order to make the best possible final application. Reviewer Three Comments Concept: End-stage liver disease is devastating and calls out for new cellular therapies. The field of liver stem cells and hepatocyte differentiation from hES cells is young but maturing. The PI and his collaborators constitute a suitable group to pursue the translational problem. It is conceivable that a Phase I study might be developed by 5 years of basic and preclinical work, especially if integration into a group drives progress at a faster rate. Principal Investigator: The PI has considerable experience and accomplishments in the field of gastroenterology. He has been continuously funded by NIH since 1979, including an NIH R01 on hES cell differentiation into hepatocytes, as well as a CIRM comprehensive research grant on liver stem cells. Planning Approach: A major portion of the planning grant budget relates to having 5 consultants attend 2 meetings to help advise the UC Davis group on developing their program. Dr. Zern has evidence that tert-immortalized fetal hepatocytes may be suitable cells for liver reconstitution. In addition, the group has access to collaborators with expertise in stem cell biology in the mouse. Moreover, they have in-house expertise on GMP facilities. The planning is sound and led by an experienced investigator in the liver field.