Because there is still considerable morbidity and mortality associated with the process of transplantation, and because more than a thousand people die each year while on the liver transplantation list, it is evident that improved and safer liver transplantation would be valuable, as would approaches that provide for an increased number of transplantations in a timely manner. A technology that might address these issues is the development of a human liver cell line that can be employed in liver cell transplantation or in a bioartificial assist device. Developing such a cell line from human embryonic stem cells (hESC) or from other human stem cell sources would provide a valuable tool for pharmacology studies, as well as for use in cell-based therapeutics.
In the proposed studies, we will differentiate human embryonic stem cells or fetal liver cells or bone-marrow derived cells so that they act like liver cells in culture. Once it has been established that the cells are acting like liver cells by producing normal human liver proteins, and that they do not act like cancer cells, the cells will be injected into the livers of immunoincompetent mice that do not rejects human cells. Then we will evaluate whether the cells grow and thrive in the mouse livers, whether they still produce high levels of human liver-specific proteins, whether they produce tumors in the mouse livers, and whether they can replace damaged mouse liver cells with human cells. One of the ways this will be done is to label the cells with a marker gene and to image the marker gene in the livers of the mice with special x-ray machines that can distinguish a few hundred human cells in the mouse liver. Finally, we will infuse the human liver stem cells into the liver of monkeys to determine if they will grow in the monkey livers, because the monkeys are more similar to man. Such studies should be done in nonhuman primates before clinical studies are undertaken to employ these cells to replace abnormal liver cells in man. Our intent is these studies is to compare and contrast three types of stem cells to determine which will be the most effective cells to use in human studies.
If the studies are successfully undertaken, we will establish a clinically useful and safe liver cell line that could be used to repopulate an injured liver in a safer and less expensive manner than with liver transplantation; moreover, all people who had liver failure or an inherited liver disease could be treated, because there would be an unlimited supply of liver cells.
In California, as in all parts of the US, there are not enough livers available for transplantation for all the people who need them. The result is that many more people die of liver failure than is necessary. One way to improve this situation is the transplantation of liver cells rather than whole organ transplantation. We are attempting to develop liver cell lines from stem cells that will act like normal liver cells. If the cells that we develop function well and do not act like cancer cells in culture, we will then transplant them into special mouse models of liver disease and see if the human cells can rescue the mouse from its liver disease. As a final test, we will see if the cells function in primate livers. In our studies, we will compare human embryonic stem cells with adult stem cells to determine which will be the most effective cells to transplant into people. If the studies are successfully undertaken, it means that we will have a stem cell line that can then be employed in human studies to determine their safety and effectiveness.
SYNOPSIS: The overall aim is to develop a proliferative human hepatocyte–like line that can be used for transplantation. Three very different stem cell populations will be characterized/compared to determine the most effective hepatocyte-like cells in vitro and in vivo. Specific Aim 1 is to optimize their in vitro liver-specific expression. Differentiated hESC will be purified by FACS analysis and by laser microdissection and pressure catapulting (LMPC), with hepatic lineage commitment determined by transduction with a lentivirus containing a hepatocyte-specific promoter. Cells will be characterized for hepatic function and potential oncogenicity. Specific Aim 2 is to establish the in vivo potential of these three populations in NOD-SCID mice. The ability to engraft, survive, and proliferate after transplantation will be assessed by a number of approaches. Specific Aim 3 is to establish in vivo potential in nonhuman primates (rhesus macaques).
IMPACT AND SIGNIFICANCE: Liver disease affects a large segment of the population including patients with cirrhosis, hepatitis, and inborn errors of metabolism. Liver failure is a significant health problem that can be solved with liver transplants, the only potentially curative treatment for end-stage liver disease, but transplantation is associated with considerable morbidity and mortality and is limited by a shortage of cadaveric donor livers. An alternative technology might involve a cell-based therapy with hepatocytes either employing direct transplantation or implantation into bioartificial assist devices. Again, the lack of donor livers makes it difficult to obtain enough viable hepatocytes for evaluating these potentially promising therapies. New sources of human hepatocytes would increase the number of patients aided, and developing adequate functional hepatocyte cell lines would provide a valuable tool not only for cell-based therapeutics but also for pharmacology and toxicology studies. Unfortunately, while hES cells have been shown to differentiate into neural precursors, hematopoietic precursors, and muscle cells, no proliferating or replicating hepatocytes have as yet been generated from hES cells. Here the applicants propose to establish a viable human cell line with hepatocyte function that could be used for these cell-based therapies. hESC are being differentiated into hepatocytes and studied further in a head-to-head comparison with other hepatocyte-like cells derived from other human stem cell populations. The applicants propose to purify lines of differentiated hES cells by FACS and laser microdissection after transducing these cells with a lentivirus containing a hepatocyte-specific promoter. Effectively enriched cells would then be characterized and assessed for hepatic function as well as potential oncogenicity. This proposal not only optimizes a protocol for differentiating hESC to hepatocyte-like cells but also rigorously compares these derived cells with two other strong candidates for replacement sources. As such, this proposal undertakes a direct, unbiased comparison between three human liver stem cell populations including those derived from ESCs, fetal hepatocytes, and CD34+ peripheral blood cell-derived Omnicytes, seeking to identify the most effective hepatocyte cells by testing them in critical phenotypic and functional assays. The innovative aspects of the proposal include the use of pressure catapulting and triple lentiviral-based reporter constructs for isolating hepatic lineage committed cells, and cell survival tracking through the use of novel imaging approaches including MicroPET scanning. The PI has brought together a very strong team of numerous collaborators who are experienced investigators in their respective fields of expertise. In addition, the applicants have developed an array of techniques to assess the fate of these cells, including immunohistochemistry, real-time quantitative RT-PCR, in situ hybridization with a human-specific probe, transduction with a triple fusion vector, and imaging with a CCD camera and MicroPET analysis. Eventually the applicants propose to compare the in vivo potential of such cells in NOD/SCID mice to human fetal hepatocytes immortalized with hTERT and so-called Omnicytes. If successful, they plan to test these strategies to correct diseases in animal models, which, however, is not part of this application. They do intend to test the three stem cell populations in rhesus monkeys. To that end, they plan to work with the California National Primate Center using combination immunosuppression with cyclosporine and mycophenolate mofetil. Thus, not only are hESC being studied but also an appropriate translational direction is already being taken. This study has a high likelihood of reaching the patient quickly.
QUALITY OF THE RESEARCH PLAN: This proposal, whose overall goal is to critically compare three different hepatocyte stem cell populations (hESC-derived hepatocytes, hTERT-immortalized fetal hepatocytes, and peripheral blood cell CD34+ Omnicytes), is complex, but logical, well thought-out, well-written, and well-organized. It has the necessary expertise in the large group of collaborators, and has extensive and promising preliminary data. Already the applicants have developed culture conditions promoting the emergence of hepatocyte-specific function in hES cells.
Specific Aim 1 deals with optimizing conditions for in vitro liver-specific differentiation, particularly for the hESC population, focusing on achieving near normal or normal maturational phenotypes. The applicants intend to use extensive empirical cell culture condition experiments, and subsequent refinements in part based on studies of Drs. Verafaille and D’Amour, to identify the ideal culture conditions to induce further differentiation into hepatocytes. Differentiated cells will be characterized for hepatic function and potential oncogenicity. Once they have identified the appropriate mass culture conditions that produce significant numbers of hepatocyte-like cells, enrichment will be attempted to reduce oncogenic potential. They will transduce the cells with lentiviral-mediated GFP expression under the control of the human alpha-1 antitrypsin promoter/enhancer construct which will facilitate laser microdissection and pressure catapulting (LMPC) isolation and enrichment of hepatocyte cells. Differentiated hESC purified by FACS analysis and by LMPC will be tested for hepatic lineage commitment as determined by GFP expression under the hepatocyte-specific promoter. These studies will be carried out with subsequent molecular analysis at the UC Davis / Whittier molecular and diagnostic core facility. The PI and his collaborators have already obtained significant data in this regard to demonstrate the likelihood of success. Also, they are well aware of potential pitfalls of this approach and have alternative culture methods in place if the original methods don't work out, such as growing cells on murine feeder layers, etc. As for the work with FH-hTERT cells, they have established lines that have been extensively characterized. Finally, the Omnicytes will be provided to them by Drs. Gordon and Habib.
The second two aims are to some degree linearly interdependent and dependent on the optimization of the in vitro culture of the three different cell types in the first aim. Specific Aim 2 is to establish the in vivo potential of these three populations in the flanks of NOD/SCID mice using established techniques. Studies on the in vivo potential of the three stem cell populations in mice will involve successful approaches established by Dr. Gupta including nitroglycerin, fibronectin-like polymer, or cyclophosphamide. Once an effective intervention has been identified, they will use monocrotaline to induce extensive proliferation in transplanted cells. The ability of the cells to engraft, survive, and proliferate after transplantation will be assessed by a number of approaches. The feasibility of many of these approaches has already been demonstrated in the preliminary data, which is focused and highly relevant. For example, they have already done CCD camera imaging studies with which they will monitor the transplant cells.
In Specific Aim 3, having demonstrated and optimized conditions for engraftment and survival in NOD SCID mice, the applicants will move to juvenile rhesus macaque monkeys in a non-human primate model. The reagents for triple reporter lentivirus are available as is the alpha-1 antitrypsin:GFP construct for sorting of hepatocyte-like cells. The expertise for obtaining hepatocyte -like cells from each of the three stem cell populations are not only within the group of collaborators but transferred to the UC Davis group. The nonhuman primate studies will be done in collaboration with Dr. Tarantal at the California National Primate Research Center using an established immunosuppressive regimen that promises to extend survival of the transplanted hepatocyte-like cells. They will use female monkeys transplanted with male hES cells and intend to use FISH techniques and PCR techniques for the human Y chromosome sequences to assess success of engraftment. They also intend to use imaging by CCD camera. Immunosuppression will be necessary in this system and cells will be transplanted by ultrasound guided portal vein inoculation, a model which has already been established in their collaborators’ labs. These studies are logically laid out, and this mature and promising project is likely to yield results.
STRENGHTS: This is a well-written and organized proposal clearly outlining an extensive series of assays to evaluate the outcomes in all aims. The PI has an excellent track record in this field and has ample experience with hESC culture techniques. Moreover, the PI’s group has developed the protocol for obtaining hepatocytes from hESC as well as the original immortalization of the fetal human hepatocyte cells, and they have already gained the transfer of the Omnicyte culture. The PI has assembled a strong network of collaborators with expertise in the various areas of study. The overall team has experience with hES cells, immortalized human fetal hepatocytes and Omnicytes, and has produced a cell with a hepatocyte phenotype for extended periods in culture. Multiple models for liver repopulation are available among the collaborators and the transfer of these to non-human primates is on-going. The lentiviral technology is also in place. In fact, as described in more detail below, most of the techniques necessary to perform the experiments and assess outcomes are available and have been used for preliminary data. For example, the applicants have shown that use of liver-specific lentiviral vector and LMPC seems to be effective in enhancing the purity of hepatocyte-like differentiated hES cells. Further, they have done successful bioluminescence monitoring, and established an immunosuppressive regimen for juvenile nonhuman primates that can be used for transplantation of human hepatic stem cells.
The collaborators include Dr. Tarantal, a talented primate investigator who has experience with bone marrow-derived stem cells and directs a state-of-the-art ultrasound imaging and fetal therapy program. Dr. Tarantal is also developing in utero primate cell transplantation techniques, a model that could be used in subsequent studies. Immunosuppression and the primate transplantation models are already in place for the current studies. Dr. Sutcliff, Cherry and Gambhir are collaborators in biomedical imaging. Dr. Gupta has extensive experience with hepatic cell transplantation over many years, and is helping develop animal models of liver pathophysiology in primates with Dr. Tarantal. Drs. Gordon and Habib in Great Britain were the first to isolate and describe Omnicytes and have an established collaboration with the PI. This extensive network of collaborators should allow these studies to be brought to their fruitful conclusion.
With respect to the experimental plan, the head-to head comparison of the hepatocyte-like cells derived from the three different sources is key to understanding their comparative advantages. The proposed comparisons are rigorous not just for the hepatocyte phenotype but also for the stem cell and hematopoietic phenotypes. Hepatocyte-like phenotypes are to be purified using alpha-1 antitrypsin:GFP expression, but importantly the expression of hepatocyte genes will be compared to that of primary hepatocytes. There is a huge difference in cells expressing low levels of a cell-specific gene and approaching the expression level of normal primary cells. Preliminary data of purified hESC derived hepatocyte-like cells at day 30 showed 27% albumin, 72% alpa-1 antitrypsin, 86% transferrin, 199% arginine, and 13% of tyrosine aminotransferase compared to primary human hepatocytes. Another key strength of the experimental plan is that there are multiple modalities for imaging in the mice and possibly in the monkeys. While luciferase in the liver can be merely superficial enough to be detected in mice and maybe monkeys, it is reassuring that they have already found that as few as 100 cells can be detected.
WEAKNESSES: The main weakness of this proposal is that there is as yet no documented evidence that functional liver cells have been generated from hES cells other than a demonstration of liver-specific gene expression in transduced cells whose lifespan apparently is still very limited. Also, as in other studies of hES cells used in xenogeneic (and also allogeneic) hosts, the issue of graft rejection looms large in the future. There were a few weaknesses in regards to the imaging techniques as well. For example, the bioluminescence imaging of luciferase activity in the monkey liver may not be possible due to the increased tissue depth. In addition, the grant would have benefited from a more in-depth description and greater reliance on MicroPET scanning; proof of concept preliminary data to demonstrate its feasibility or utility is lacking. Finally, although alpha-1 antitrypsin, albumin and CK18 antibodies that distinguish mouse and human antigens are described in the preliminary data, it has not been demonstrated that these or other antibodies used in immunohistochemistry will be able to distinguish primate vs. human antigens.
Several relatively minor weaknesses were identified in this proposal. These include: 1) the linear interdependence of the aims and project structure, 2) the question of whether the flow sorting of large hepatocytes is possible with current flow technology, and 3) the immunohistochemistry studies on liver monkey biopsies, which may not be possible or efficient given that the small core biopsies, even if multiple sampling is done, may be subject to potential sampling error and donor cells may not be seen or may be quite rare. Lastly, it is odd that on page 21 of the proposal, the need for Stem Cell Research Oversight approval is marked “No”. The hepatocyte-like cells derived from the hESC are to be transplanted in to mice and monkeys, which should have “Introduce a covered stem cell line into an animal” checked.
DISCUSSION: This is a unique proposal, which was well though out and a pleasure to read. This world expert PI in liver cell physiology has brought together the right people to do the job in this “exciting grant” addressing liver disease, which affects a large proportion of the population. The proposal has the potential to find a strong candidate for hepatocyte replacement. The PI, who has worked with hESC to get a differentiated population, has teamed up with Dr. Gupta at Einstein, who has made immortalized fetal hepatocytes. Along with hESC-derived hepatocytes and ‘Omnicytes’ (a CD34+ cell line from peripheral blood), these lines will be the subjects of a head-to-head comparison to determine most effective hepatocyte-like cells. The head-to-head, unbiased comparison is this proposal’s biggest strength. The PI has all the technology in hand to do these studies (e.g., the CDC camera with luciferase or microPET to study in-vivo; the lentiviral vectors with RFP and TK for the microPET), and has extensive preliminary data. This proposal has an extremely careful consideration of the imaging approach, such that the applicant may experience some problems, but will have a high likelihood of success in following the cells in vivo with this imaging strategy.
Enthusiasm was lowered somewhat because there is no documented evidence that functional liver cells have been generated from hESC (only marker expression has been shown as evidence). Also, the lifespan of the cells is an unknown. The specter of graft rejection looms large and the plan to use immunosuppressant in the primate studies also limited enthusiasm. Another potential weakness is that the aims are linearly dependent, and it is unclear whether they'll have the right set of conditions to get functional hepatocytes. One discussant raised the issue regarding the poor characterization of cells from a company (Omnicytes), which aren’t associated with detailed information on their ability to repair liver. This comment was countered by the strength of the proposal in doing a head-to-head comparison, where these data will be generated.