Funding opportunities

Isolation of Fetal Hepatic Stem Cells for In Utero Transplantation

Funding Type: 
New Faculty I
Grant Number: 
Funds requested: 
$1 762 430
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
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: 
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 Summary: 
SYNOPSIS: The project focuses on developing human fetal hepatic stem cells and intrauterine transplantation 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 PI will use cell surface markers and cell sorting to identify a population of hepatic stem cells in the human fetal liver.The stated hypothesis is to “test the hypothesis that human fetal hepatoblasts and hepatic stem cells can be identified by their levels of CD34, CD66a, CD203c and CD326.” Using these markers to sort the cells from fetal liver, the PI proposes to test their potential for generating hepatocytes and cholangiocytes. In Aim 2 the potential of the isolated cells will be tested in an animal model with injection of the cells into fetal liver or placental vessels. 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 hosts will be allogeneic or xenogeneic (human cell transplants), and the endpoint of analysis is “efficiency of transplantation”. Aim 3, which is aligned with the PI’s independent blood systems research institute, 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. 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 injection of cells into the liver presumes they will not migrate from there, and the injection of cells in the placenta vessels is presumably made under the assumption that the cells will migrate to an appropriate environment and stay there for differentiation. But the analysis does not account for either of these scenarios. Only liver will be examined. The idea of correcting liver diseases in utero is an interesting one, but the PI has not chosen interesting animal models, suggesting that there is not a good understanding of the clinical needs in this area. For example, hemophilia does not cause damage to the hepatocytes despite several statements in the grant to the contrary. It is true that patients with hemophilia who receive a liver transplant also have their hemophilia cured (because the donor liver hepatocytes are capable of synthesizing the missing clotting factor), but liver transplants are usually done because the patients have another disease (such as viral hepatitis) that damages their liver. In fact, it is completely conceivable that gene therapy approaches with clotting factors produced by any cell could work for this disease—no liver stem cells or hepatocytes are required. The reviewers cited concerns regarding feasibility of the project, based on concerns of limited preliminary data. 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. Aim 1 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 would probably not be sufficient. Although there is a good deal of enthusiasm for the experiments to perform clonal assays to determine the relative frequency of progenitors, it is not clear from the proposal that this will be adequately accomplished because of lack of methodological detail. For example, the preliminary data show isolation of some cell populations from human fetal flow cytometry experiments, but there are no data to show if they grow at all in culture. The timing of the flow analysis is also unclear from the presentation. The wealth of data on surface marker expression has not yet been linked to culture survival or differentiation. These would seem to be the basic data needed for this proposal to warrant support. There is significantly less enthusiasm for Aims 2 and 3 which are purely technical aims. Because of scant preliminary data and a naïve approach to the clinical issues, the work is not likely to move the field forward. Finally, the advances in tissue engineering really haven’t translated into a huge repository of growing fetal liver cells, so there is a feasibility issue running throughout the proposal that requires some preliminary data. With respect to the overall research plan, there were general concerns cited by reviewers. First, one significant weakness throughout is that “efficiency of transplantation” is going to 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 Aim1 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 based on their markers, 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. 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 grant. In addition, the PI proposes to perform in vitro clonal assays to determine the frequency of stem cell progenitors within the FACS sorted populations, and to definitively establish 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 differeniated cells produced (more refined than just AFP and CK19 staining) 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 also not been shown to date. The PI acknowledges that the cells may be hard to grow. However he does not outline a systematic plan of attack for making them grow. Since the animal work calls for 106 cells per pup (10 pups), the expansion of the stem cells without a change in their phenotype is 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 some information about hepatic stem cells in the literature, and the PI is certainly aware of some of the groups who feel that they have defined at least one hepatic stem cell population --because their papers are cited in the proposal. Does that PI feel that these stem cells (from Fausto group and others) are not adequate for his purposes? 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. Specific concerns with Aim 2 are discussed below. Aim 2, 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 his previous work in the Fetal Treatment Center research laboratory under the direction Dr. Harrison at UCSF. In this first strategy, an allogeneic murine model system will be used to compare the efficacy of transplanting fetal vs. adult liver cells. To assess engraftment and chimerism, flow cytometric analyses using HLA markers will determine the percentage of donor cells. These experiments will use bulk fetal liver populations, rather than the sorted populations, and will analyze engraftment only in the short term. This is a limitation of this strategy, since long-term engraftment potential will not be assessed. The comparison of fetal vs. adult hepatic populations is of interest; however it is not clear how donor cells will be distiguished from host cells in mouse-to-mouse transplants. Also, since sorted populations will not be used, the PI cannot distinguish between changes in precursor frequency and changes in precursor activity/survival after transplant. Fetal hepatic stem cells transplants (via IUT) in immunodeficient SCID mice wil be assessed. Methods to detect human cells in a mouse system are readily available and again will be based on HLA-specific antibodies and flow cytometry. The xenotransplant studies will help to define the functional properties of the sorted human cell populations defined in Aim 1, 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. Durability of engraftment will be assessed out to 6 months. 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 Dr. Farmer of Fetal Treatment Center at UCSF 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 not specified, but may be unfractionated bone marrow) animals will be tested for chimerism at one month of age by flow cytometry. They will test transplantation of liver cells from donor strain and third party mice. 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 Aim 2. 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 they will be all over the place with the intravessel injections. And since the host animals have normal liver function to start with, how will any improvement in liver function be assayed in the normal hosts? 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 (page 5)? This seems unlikely since there are non-parametric data sets being generated. Lower down on page 5, are all the assays that precede the listing of the Mann-Whitney testing appropriate for this kind of analysis? Specific concerns within Aim 3 are discussed below. In Aim 3, the PI intends to develop clinically applicable methods for the procurement and banking of fetal liver cells. The PI will collaborate with Drs. Read and Reed at BSRI to develop clinically applicable methods for the procurement, processing and banking of human fetal hepatic precursors. They have made an observation that a single purification step is required to greatly reduce the incidence of microbial contamination which has clinical relevance. They plan to test a number of methods of generating cell suspensions and a variety of enzyme preparations. Although they currently use Liberase, they do not seem to be aware of the recent BSE issue with Liberase enzyme. It is clear from the discussion on page 8 that the most appropriate enzyme to use for isolation is not yet clear. In general, this aim is also a technical exercise and includes testing for various methods for 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: Dr. Muench received his PhD in Cell Biology and Genetics from Cornell in 1992, and worked as a postdoc in human immunology at DNAX from 1992-1995. He worked as a researcher at the Fetal Treatment Center Research Lab at UCSF from 1995-2000, and then as an Assistant Adjunct Professor from 2001-2006. Subsequently, the PI was recruited to Blood Systems Research Institute in San Francisco in 2006. It has been 15 years since completing his PhD. The PI has spent most of his career as a research scientist in the area of hematopoietic biology, and it is clear in just reading through the publications listed that he became interested also in liver stem cells a few years ago, and he started to publish in the area of liver development 3-4 years ago. He has been reasonably productive as a research scientist, and appears qualified to extend his knowledge of hematopoietic development into a new stem cell system. He lists several first and last author papers in recent years that are relevant to the in utero transplantation system he proposes to study. He began the position at BSRI in 2006, and currently has one short non peer-reviewed publication related to hepatocyte transplantation and only one published paper since 2004. Dr. Muench has extramural funding from the National Blood Foundation and the NIH to study in utero transplantation and ontogenic in erythroid gene expression. He has no extramural funding for hepatocyte transplantation. He 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 he shown extensive preliminary data cataloging gene expression in sorted hepatic subsets. Dr. Muench’s effort will be 100% devoted to research and he has received a strong financial commitment from the BSRI with an initial three year startup package. Dr. Muench's career development receives a high degree of oversight, and his career development plan is primarily based on the BSRI metrics by which his advisory panel will monitor his progress and development as an independent, researcher. His performance is also evaluated once every 2 years by an external advisory committee, through written documentation and site visit. He has set several goals for himself, including publication of at least 1 high impact paper per year. He 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 related fields of his active research. Dr. Muench will be an adjunct professor at UCSF and will have contacts with several laboratories as well as the UCSF Liver Center and Human Embryonic Stem Cell Research Center. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: Dr. Muench has received ample laboratory space and start up funds to begin his research. The institute has taken a mentorship role in helping him to define goals and achieve milestones. In addition, they have invested in significant new equipment to support his research. He also maintains contacts with investigators at UCSF that strengthen his research program. A clear commitment from UCSF is not indicated but there appears to be some commitment from the BSRI. An adequate track record for establishing and advancing the careers of independent investigators at the BSRI is not evident. The Blood Systems Research Institute is a division of Blood Systems, Inc., and is affiliated with UCSF, with access to their core facilities, library and adminitrative oversight staff. The Institute has an overall interest in cell therapy, and interacts with UCSF GMP facilities. The Blood Systems Research Institute (by the c.v.’s enclosed) has a record of hiring scientists who develop their own programs of research within the Institute, but the company became independent only in 2004, so there is not much of a long-term history to evaluate. Blood Systems Research Institute does not yet have an animal facility and there is no approved protocol for the survival surgical procedures, which could delay the research. This work would also require IRB approval for collection of stem cells from aborted human fetuses, and no approval is enclosed with the grant. The PI proposes to devote 50% of his time to the work, which likely means he also plans to contribute in a hands on way. His employers acknowledge this percentage commitment and appear supportive. The experiments will mostly be executed by a technician and post-doc (do they have other duties at Blood Systems)? The PI has surrounded himself 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. Banking studies are premature, since it is unclear that the cells are useful. 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. In consideration of these major concerns, the reviewers could not entusiastically support the proposal.

© 2013 California Institute for Regenerative Medicine