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RN1-00559-1: Isolation of Fetal Hepatic Stem Cells for In Utero Transplantation
Recommendation: Not recommended for funding
Public Abstract (provided by applicant)
This research project is aimed at developing a new form of liver transplantation to treat birth defects that prevent normal liver function using liver cells isolated from discarded fetal tissues. It is now possible to detect many inherited diseases early in pregnancy. It may also be possible to treat diseases that prevent normal stem cell function by transplantation of healthy stem cells before birth. There are potential advantages to such a therapy such as early treatment of disease and a lower risk of rejection because the fetal immune system is not developed. This proposal studies a number of methods aimed at testing the effectiveness of transplanting liver stem cells into a fetus to treat liver disease. Often transplanting healthy cells offers treatment or a cure for many genetic diseases as well as other illnesses caused by viral infection or cancer. However, a lack of available or suitable donor tissue prevents such therapy in many cases. Fetal tissues are a source of stem cells available from elective abortions that are generally discarded. Harvesting liver cells from these tissues to treat patients with liver diseases offers the possibility of increasing the pool of donor cells that are available transplantation. This proposal studies the feasibility of banking fetal liver for transplantation.
Statement of Benefit to California (provided by applicant)
Liver diseases caused by viral infection, drugs or inherited disease affect many thousands of Californians. Often, transplanting healthy cells offers treatment or a cure for many of these diseases, but a lack of available or suitable donor tissue prevents such therapy in many cases. Use of fetal stem cells offers the hope of generating a greater supply of tissues for cellular therapy. The development of prenatal liver transplantation would also offer additional means to treat birth defects that affect normal liver function. The successful outcome of this work will offer new hope to many Californians suffering from liver diseases. This will improve lives and save money on long-term health care costs associated with these diseases. Development of the technologies and expertise to bring these novel forms of therapy from the laboratory bench to hospital bedside will also keep California in the forefront of the biotechnology industry, will attract talented scientists and clinicians to California and will create high-paying jobs.
Review
SYNOPSIS: The project focuses on developing human fetal hepatic stem cells and intrauterine transplantation (IUT) as a prenatal cellular therapy to treat a wide variety of liver disorders. The project is organized into three specific aims. In the first aim, the Principal Investigator (PI) will use cell surface markers and cell sorting to identify a population of hepatic stem cells in the human fetal liver. In the second aim the PI intends to test two strategies of in utero therapy including direct injection of fetal hepatic stem cells into the liver, and a second strategy to inoculate hematopoietic cells in utero to induce immunological tolerance followed by postnatal liver cell transplantation of liver cells from the same donor. The third aim is to develop clinically applicable methods for procuring, processing and banking human fetal hepatic precursors.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This proposal will use the novel approach of in utero transplantation to assay the effectiveness of pre-natal stem cell therapy for the liver and blood systems. Inherent in this study is the identification of novel populations of hepatic precursor cells and new methods for cell isolation from the liver. If successful, these studies could encourage future clinical trials of in utero transplantation for human liver disease. The application to human disease is primarily focused on neonatal liver diseases including inborn errors of metabolism or neonatal liver failure. In general, these are relatively uncommon conditions. If human fetal hepatic stem cells would be used as a donor source for adult liver disease a significant degree of expansion would be necessary, an aspect that is not directly addressed by this proposal.
This application does not articulate well the challenges that need to be overcome to develop in utero patient therapy for hepatic diseases for fetuses or infants. For example, one delivery strategy presumes cells will not migrate from the injected organ, while the other delivery method presumes that cells will migrate to an appropriate environment and stay there for differentiation, but the analysis does not account for either of these scenarios.
The major innovation in the work is the use of fetal injection to test stem cell potential in the context of all the normal developmental cues provided by the uterine environment. The first aim to study subpopulations of hepatic stem cells by flow cytometry appears realistic and doable. The investigators have some preliminary data to support these investigations. Multiple markers will be used which is important as one marker probably would not be sufficient. Although there is a good deal of enthusiasm for the experiments to determine the relative frequency of progenitors, it is not clear from the proposal that this will be adequately accomplished because of a lack of methodological detail.
With respect to the overall research plan, there were general concerns cited by reviewers. First, a significant weakness throughout is that “efficiency of transplantation” will be examined by liver function tests, but these tests are never outlined. Second, in the abstract and aims, a number of markers are discussed that will be used for sorting and analyzing different potential hepatic stem cell populations, then in the research design section, more markers are brought in, but the plan is unclear. Will the PI use various combinations of high, low, and no expression to define the optimal stem cell populations (with all the markers proposed)? A calculation of the numbers of different cell types to be analyzed would help with the feasibility of these studies. A table with the planned populations would make it easier to discuss feasibility issues in this work. Finally, a lot of literature suggests that bone marrow derived stem cells can cure animal models of inherited metabolic disease affecting the liver, but at the cost of cell fusion to hepatocytes, not really true regeneration of hepatocytes from marrow. There should be some acknowledgement of the issues inherent in these reports.
Specific concerns within the first aim were further described. The PI will test the capacity of the different cell populations, including putative hepatic stem cells, to grow and develop into hepatocytes and cholangiocytes using a variety of not completely specified culture methods to assess the growth and differentiation potential. It is quite clear that the basic culture requirements have not yet been worked out. Many options are hinted at – feeder layers, growth factors, seeding density – yet these experiments take a long time to optimize in one stem cell population let alone in the number of stem cell populations discussed in the application. In addition, the PI proposes to determine the frequency of stem cell progenitors within sorted populations, and to definitively establish the multilineage potential of putative hepatic stem cells. Functional assays, which are key to the success of such prospective isolation strategies, are also proposed, based on in vitro culture. Sufficient alternative approaches are discussed to back up these experiments; however, a better functional analysis of the differentiated cells produced would have strengthened the proposal.
Stability of the phenotype in culture may also be problematic, but the PI does not propose a solution, and the ability to expand cells under certain culture conditions has not been shown to date. The PI acknowledges that the cells may be hard to grow; however s/he does not outline a systematic plan of attack for making them grow. The animal work calls for enough cells to make the expansion of the stem cells without a change in their phenotype a limiting factor for the rest of the proposed work. An in vivo clonogenic assay is what is really needed.
Finally, although the PI is correct in saying that little is known about the difference between fetal and adult hepatic stem cells, there is quite a bit of information about hepatic stem cells in the literature, and the PI is certainly aware that at least one hepatic stem cell population may have been defined because the relevant papers are cited in the proposal. Does the PI feel that these stem cells are not adequate for this purpose? If so, there should be some discussion convincing the reviewers that the effort needed to precisely identify a new stem cell population (and the effort in optimization is a big one) is justified.
The second aim, to develop strategies of in utero therapy, is primarily a technical aim that is not hypothesis-driven. The investigator appears to be largely familiar with these models from previous work. In one strategy, an allogeneic murine model system will be used to compare the efficacy of transplanting fetal vs. adult liver cells. The comparison of fetal vs. adult hepatic populations is of interest; however, it is not clear how donor cells will be distinguished from host cells in mouse-to-mouse transplants. These experiments will use bulk fetal liver populations, so the PI cannot distinguish between changes in precursor frequency and changes in precursor activity/survival after transplant. The PI proposes to analyze engraftment only in the short term. This is a limitation of this strategy, since long-term engraftment potential will not be assessed.
Fetal hepatic stem cells transplants (via IUT) in immunodeficient mice will be assessed. Methods to detect human cells in a mouse system are readily available and the xenotransplant studies will help to define the functional properties of the sorted human cell populations defined in the first aim, although if in vivo function does not correlate with the marker expression used for cell identification, then these studies may require a separate prospective isolation strategy.
One reviewer felt that the allogeneic model makes more sense to pursue for its clinical importance (translational relevance) than the xenogeneic model. The allogeneic model could capitalize on prior transplant research in rodent models, which is not taken into account in the proposal. The idea of isolating human fetal hepatic stem cells is appealing, but the case for proceeding with xenogeneic transplants is not supported by the proposal.
The second strategy to be tested is IUT of hematopoietic cells to induce immunological tolerance, followed by a postnatal liver cell transplantation. Advice from a collaborator will be sought in these fetal transplantation experiments. The same allogeneic murine IUT model will be used; however, it is not clear if the animals will receive preconditioning. After IUT hematopoietic engraftment (donor cell types were not specified) animals will be tested for chimerism at one month of age. It is not clear from these experiments how the proposed methods will advance the field or improve upon what has already been achieved and previously published.
There were further concerns with the endpoints and analysis of the data to be obtained in the second aim. The main endpoints of analysis are engraftment and hepatic function. Will non-hepatic organs be examined for these cells? It is certainly reasonable to assume that the cells will be present in many end organs with one delivery strategy. Also, when statistical tests are cited, they are cited in isolation of the tests. Will all the end-points of analysis be suited to Student’s t-testing? This seems unlikely since there are non-parametric data sets being generated. Is Mann-Whitney testing appropriate for analysis of the proposed assays listed in this part of the application?
In the third aim, the PI will collaborate with two individuals to develop clinically applicable methods for the procurement, processing and banking of human fetal hepatic precursors. They have made an observation that a proposed method could reduce the incidence of microbial contamination which has clinical relevance. It is not clear from the proposal that methods of generating cell suspensions and enzyme preparations for isolation have been determined. In general, this aim is a technical exercise that includes testing various methods of cryopreservation and thawing to determine the best way to store fetal liver specimens in order to establish a tissue bank, yet many techniques exist that could be readily applied to culturing and cryopreserving hepatic stem cells. One reviewer notes that each stem cell population requires special handling including freezing and thawing procedures that are often ignored in favor of generic protocols for handling cells in the lab. However, the variables that are important in this process of optimization of long-term storage are not addressed by the PI. Furthermore, the utility of this bank has not yet been determined and depends in part on success in the first 2 aims, which would demonstrate a clinical need for fetal liver cell banking. In the absence of such data, this aim seems premature.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant received a PhD in Cell Biology and Genetics in 1992, and worked as a postdoc in human immunology from 1992-1995. The applicant worked as a researcher in a laboratory relevant to the project from 1995-2000, and then as an Assistant Adjunct Professor from 2001-2006. Subsequently, the PI was recruited to the current institution. It has been 15 years since completing a PhD.
The PI has spent most of his/her career as a research scientist in the area of hematopoietic biology. It is clear in reading through the publications listed that the PI became interested in liver stem cells a few years ago, and started to publish in the area of liver development 3-4 years ago. The applicant has been reasonably productive as a research scientist, and appears qualified to extend his/her knowledge of hematopoietic development into a new stem cell system. The applicant lists several first and last author papers in recent years that are relevant to the in utero transplantation system proposed for study. The applicant currently has one short non peer-reviewed publication related to hepatocyte transplantation and only one published paper since 2004. The applicant has extramural funding from a foundation and the NIH to study in utero transplantation and an unrelated topic. The applicant has no extramural funding for hepatocyte transplantation.
The applicant has trained in several laboratories focused on bone marrow and fetal liver hematopoiesis. The PI’s expertise and experience with working with human fetal liver cells is an advantage, and the PI has shown extensive preliminary data cataloging gene expression in sorted hepatic subsets.
The applicant’s effort will be 100% devoted to research and s/he has received a strong financial commitment from the institution with an initial three-year startup package. The applicant’s career development receives a high degree of oversight, and the career development plan is primarily based on the institution’s metrics by which an advisory panel will monitor progress and development as an independent researcher. The applicant’s performance is also evaluated once every 2 years by an external advisory committee through written documentation and site visit. The applicant has set several goals for him/herself, including publication of at least 1 high impact paper per year. The applicant describes funding milestones as well, related to recruitment of extramural funds. Despite the emphasis on evaluation, this descriptive plan does not speak to obtaining additional experience in fields related to his/her active research. The applicant will be an adjunct professor at a neighboring institution and will have contacts with several laboratories as well as the institution and stem cell research center.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant has received ample laboratory space and start up funds to begin this research. The institute has taken a mentorship role in helping to define goals and achieve milestones. In addition, they have invested in significant new equipment to support this research. The applicant also maintains contacts with investigators at a second institution that strengthen the research program. A clear commitment from the second institution is not indicated but there appears to be some commitment to this association from the applicant institution. An adequate track record for establishing and advancing the careers of independent investigators at the applicant institution is not evident.
The host institution is affiliated with a second institution, with access to their core facilities, library and administrative oversight staff. The host institution has an overall interest in cell therapy, and interacts with the second institution’s GMP facilities. The host institution (by the c.v.’s enclosed) has a record of hiring scientists who develop their own programs of research, but the institution became independent only recently, so there is not much of a long-term history to evaluate. The institution does not yet have an animal facility.
The PI proposes to devote 50% time to the work, which likely means that the PI also plans to contribute in a hands-on way. Employers acknowledge this percentage commitment and appear supportive. The PI has created affiliations with a lot of senior level consultants who can give intellectual advice but will certainly not be conducting experiments.
DISCUSSION: Discussion centered on some major difficulties with the research plan. Although developing a novel progenitor line would be beneficial, it is not clear what markers would be used and there is no plan for distinguishing host and donor in the transplant model(s). Since there is no defect in the donor liver, it will be difficult to assess liver function. There is no clinical context to the proposal and no plan to look at what diseases damage the liver. Banking studies are premature, since it is unclear that the cells are useful. In consideration of these major concerns, the reviewers could not enthusiastically support the proposal.
The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:
- None