The derivation and culture of human embryonic stem cells has provided new possibilities for treatment of a wide variety of human diseases because these cells have the potential to help regenerate and repair many types of damaged tissue. Diseases for which such cell-based treatments may be helpful include obstructive renal disease, a disorder for which there has been little progress made in terms of treatment. Infants with this and other inherited kidney disease may be severely compromised before birth and treatments necessary to prolong their life may be accompanied by severe side effects. This raises many difficulties not only for these young patients but also for their families. If new ways to treat these infants prior to birth can be developed, this could lead to the delivery of healthy babies at full term. The use of cells obtained from human embryonic stem cells to repair and treat damaged kidneys prior to birth offers promise to improve survival and quality of life for these babies. Since it is clear that embryonic stem cells have vast potential to form a variety of cell types, it is possible that the kinds of cells needed to provide repair could be obtained and treatments initiated prior to birth. The studies proposed will assess ways to obtain such cells and the effectiveness of such treatments. Ultimately, even small improvements in function of damaged kidneys following embryonic stem cell-based therapies may increase survival and eliminate the need for dialysis or kidney transplants. Although methods to grow embryonic stem cells and even obtain cells that could be useful for treating some human diseases have been described, the use of these cells for human therapies remains highly controversial because their safety remains untested. While these cells have great potential and promise to form cell types useful for treatment of disease, they also have the potential for uncontrolled growth and to form tissues that would be harmful. Therefore, studies must be performed and techniques must be developed to carefully examine the use of these cells in relevant models of human disease, and before they are ever considered for human treatments. The overall intent of these studies is to develop techniques that can be used to test the safety of human embryonic stem cell-based therapies, and to determine ways to evaluate the cells after they have been injected into the body. As we develop new treatments for obstructive kidney disease, we will use this model system to explore these essential safety questions related to stem cell therapies. The studies proposed will fill a critical need for new treatments for kidney disease, ways to monitor cells in patients, and develop methods to assess safety issues associated with the transfer of this research to human patients.
This proposal focuses on ways to fill the significant gap in the development of new human therapies using stem cells – transfer of ideas and techniques that are developed in laboratories to effective and safe treatments for human patients afflicted with disease. While the potential medical benefits of human embryonic stem cells may seem great, proof that these cells will not cause harm must be shown, and this must be accomplished before any patients receive treatments. Removing the barrier preventing the transfer of promising stem cell therapies to human patients will require connecting people with the expertise to develop and to evaluate such treatments. With this in mind, our studies will bring together collaborators from many areas: developmental biologists, clinicians, engineers, and those with vast experience in the study of stem cells and with preclinical models to address questions associated with a pediatric kidney disease, which is one of the leading causes of chronic renal failure in children. Kidney disease is a major cause of illness and death among infants and children with over 20,000 babies born each year with kidney problems. Approximately 5,000 have kidney failure and are on dialysis or are in need of a kidney transplant. In California alone, nearly 100 children under 10 years of age are currently awaiting available kidneys for organ transplant. The benefit to the California community is, thus, potential new therapies for the treatment of kidney disease in children, and a model system available to all researchers in which safety and efficacy of embryonic stem cell therapies can be predicted.
SYNOPSIS: The goals of this proposal are to develop a preclinical non-human primate model for testing the safety of human embryonic stem cell (hESC) transplants and to repair fetal kidney obstruction. Aim 1 will translate a murine protocol for early renal precursors to hESCs and test if these cells injected into fetal monkey kidney explants will incorporate and differentiate. Sublines of hESCs transduced with lentiviruses expressing eGFP under lineage specific promoters and a luciferase/thymidine kinase construct will be developed and tested for proper differentiation. In Aim 2, similar cells after labeling transiently (64 Cu) or lentivirally transduced(MND-luciferase-PGK- thymidine kinase) will be intrauterinely injected into fetal monkey obstructed kidneys at 90 days (term 165±10). These cells will be followed in vivo using microPET, optical imaging and ultrasound over the first 48 hrs (64 Cu) and after 10, 40 and 75 days (luciferase/TK labeled). Addition of undifferentiated hESCs will test the level of purity necessary.
IMPACT AND SIGNIFICANCE: The main goal of this project is to develop the use of non-human primates with obstructive renal disease to explore the safety of transplantation of human embryonic stem cells (hESC) to treat kidney diseases. The proposed work appears to be for the most part original and uses a multi-pronged approach (culture with renal growth factors, provision of regulatory growth proteins via lentiviral infection, radioisotopic imaging, microPET) to monitor the success of hESC differentiation into renal tissue in vitro and the fate of differentiated hESC transplantation into embryonic primate kidneys. The applicants have expertise with a highly unique model system that is a very good mimic of human disease. There are many safety issues to address in the use of hESCs to treat human disease. The exploration of this non-human primate model will provide a means of testing the safety and efficacy of differentiated hESCs to treat kidney disease. This is an innovative apsect of the proposal. The applicants will also develop methods for in vivo imaging of hESCs to determine whether they are trafficking to the site of disease. The development of the imaging techniques is highly important for the future use of hESCs. Early determination of whether the hESCs trafficked to the site of disease and whether they remained viable will be essential for safe and successful applications of hESCs. Successful completion of these studies will provide highly valuable information about translational research in the area of obstructive renal disease. Although the proposal focuses on treating kidney disease, the methods developed through this proposal will have widespread use for hESC research to treat many diseases.
QUALITY OF THE RESEARCH PLAN: The research plan is a tour de force of very high quality, very ambitious, and clearly directed towards translational research. The investigators appear to have the required technologies, knowledge, and experimental tools well in hand, and have track records that make it seem more likely than not they will be successful in carrying out the work described in the time planned. If successful, their results will be very interesting and clinically very important.
While the team of investigators is outstanding, there is considerable risk to this ambitious project. Perhaps a focus on the development of the model for testing the potential of teratomas in a non-human primate or on the non-invasive imaging of the transplanted cells would be more feasible. The question of immune response to the hESC derived cells is ignored, possibly since the immune system is still immature in the fetus and thus not likely to be a problem.
In vitro protocols will be developed to obtain cells differentiated towards early renal lineages from hESCs. Tables 1 and 2 outline conditions for growing embryoid bodies (EBs) under a variety of culture conditions (5 factors, each at 5 different concentrations) along with various types of genes that will be investigated. Once the optimal culture conditions are identified the applicants will place transduced hESCs expressing EGFP in nephrogenic culture for injection into embryonic monkey kidney explants. These experiments will determine whether the differentiated cells show integration and branching. There seem to be a very large number of variables to examine in these studies, and a plan for how these variables will be tested and how their data will be analyzed is not explicitly described.
The applicants present a short description of cell labeling with Cu-64-PTSM, but it is not entirely clear which cells from the proposed experiments will be labeled with Cu-64-PTSM. In addition, there is no description of the caveats to labeling cells with Cu-64-PTSM. For example, it is known that Cu-64-PTSM is highly toxic to tumor cells, possibly due to the delivery of Cu-64 to the cell nucleus. The applicants propose to evaluate cell viability over 48 hours of labeling. Efflux of Cu-64 will also be measured in vitro; however, the applicants do not mention that efflux of Cu-64 from the cells is likely to occur in vivo as well. If this occurs, what is the fate of the Cu-64, and how will this affect cell trafficking by this method? The time frame of Cu-64-PTSM imaging is presented in table 3 as 48 hours, and there could be extensive efflux in vivo over this relatively long time period.
STRENGTHS: There are many strengths to this proposal, including the experienced team of very productive investigators; the level of peer-reviewed funding already in hand for several of the investigators; the clear center-based, comprehensive approach to constructing the proposal; the directed goal of translational research in nonhuman primates to set the stage for eventual human application; and the focus on an important pediatric disease. In addition, the proposal presents promising preliminary data on hESC differentiation and preliminary bioluminescence imaging in infant monkeys demonstrates the ability to detect transgene expression in the abdominal area.
The PI has brought together impressive expertise in imaging, stem cells, gene therapy, kidney development and disease. The PI has an NIH Center of Excellence in Translational Human Stem Cell Research and is part of the Primate Center which provide outstanding facilities and environment for performing these experiments.
Thus, there is a unique opportunity here. The overall goal of developing a non-human primate model for testing the efficacy and safety of transplantation of hESC derived cells is vital to the field. Similarly development of non-invasive techniques to follow the cells would be a major contribution.
WEAKNESSES: Perhaps the main weakness is the inadequate development of the model for testing the efficacy and safety of in vivo introduction of hESC derived cells. Since hESCs grow far slower than murine ones, will examining the tissues only up to 75 days after introduction be adequate to rule out the potential of teratomas? A much longer observation period might be necessary to gain confidence. Additionally, it is not clear that the introduced cells will become functionally differentiated nor that it will be possible to test this.
Early renal precursors are to be sorted and then expanded in vitro but it is unclear what percentage of cells will meet this criteria and will remain at this precursor stage during expansion. Preliminary data on the expression of some of the early renal markers is promising but no comparison to actual fetal kidney nor localization of the expressing cells are given. It is unclear whether there is some low level expression in a number of cells or if a few cells spontaneously start along this path. How effectively the non-invasive imaging will be able to follow the introduced cells during the final growth and development of the kidney is unclear. Luciferase and optical imaging has a detection of only about 1 cm and is very dependent on repeated imaging of the same orientation. How many cells would need to be engrafted to be detected? Preliminary experiments on luciferase detection after injection of viral constructs or transduced MSC involve much higher labeling of cells and detection in more superficial locations than are likely with the fetal kidney model. After birth is there an issue of immune rejection of the human cells in this primate model? It is unclear why encapsulating the cells in alginate beads would be beneficial for repairing damaged kidneys.
Many of the weaknesses involve primarily certain areas of the proposal that could be clearer. This is perhaps not unexpected, given the limited number of pages allowed for the application and the ambitious, comprehensive plan of traveling from basic in vitro generation of the desired hESC renal precursor phenotype through transplantation and extensive monitoring of the fate of the transplant. Additional information that would have improved the grant include a lengthier consideration of allorejection issues of hESCs by the embryo and mother in their primate model, including preliminary data; how the number of renal precursors was chosen for injection; and whether the infusion will be into a renal artery, retrograde via a renal vein, or directly into renal tissue of the fetus, including preliminary results to assess success; and some quantitative measure of functional success of incorporating hESC-derived renal tissue into an injury-based model.
DISCUSSION: One reviewer felt ambivalent about this proposal for a number of reasons. This is the only application with a preclinical model. It has some issues but overall it’s good science. The PI is an expert on working with the monkey model and has established an NIH Center of Excellence, which is a real strength. The aims follow on from a combination of two papers by others in the field. Here, the PI will use lentiviruses to transduce hESCs and select colonies (not cells) off of FACS. The cells may not be kidney at that point, and may only be intermediate mesoderm. Overall this is a unique opportunity to develop non-human primate model, but there are many unknowns. For example, the optical imaging can detect up to 1cm in depth and there is a question of whether that is good enough to view the transformed cells. Orientation may also be a problem. In rodents, if the orientation is changed, one gets a very different readout. The main concern is whether these cells will integrate and become functional in the kidney, but to be fair, this is what the PI is trying to find out. There is also concern that the PI is not really testing safety and efficacy, since the PI is introducing undifferentiated and partly differentiated cells, then only following for 75 days; not long enough (in this model) to rule out teratoma formation. Also, there are no immune studies, and yet this is xenograft work. The PI explains that because the work was in the fetus, there shouldn't be immune issues, but there are effects in the mother.
A second reviewer felt that this proposal was a real tour de force in that the PI has brought strong collaborators to work together. Rejection issues were not well addressed, and it would be nice to have a measure of success beyond imaging. Some of the questions and concerns of the previous reviewer will be answered as they go through the experiments.
Another reviewer appreciates the non-human primate work, but was concerned by the number of variables in Aim 1, and felt this was the main weakness. What are the priorities in this proposal? The reviewer did not have the same concerns with imaging as the first reviewer, but felt that cells at a certain depth would be difficult to visualize. A key problem is that the PSTM cell trapping hasn't been repeated since it was first published. Many people have tried to repeat it and couldn't due to the high efflux of tracer from the cells. This leads to lots of signal, but not from the target cells. The reviewer felt the PI should have been aware of this issue.
Other reviewers questioned how relevant this model is from a clinical perspective. Apparently, the tubules do rescue (according to other publications), and it may be that the PI here isn't looking to repair obstructed kidney, but simply using the model to create an in-vivo differentiation induction environment.
PROGRAMMATIC REVIEW: There was a motion made during programmatic discussion to move this proposal into the "Recommended for Funding" group. The reviewers felt they might have erred on the side of being hypercritical. One reviewer expressed some enthusiasm given that this might be a good pre-clinical model. The clinical grants could deal with scale up and such later on. Another reviewer argued that the PI is dedicated to this area of studying non-human primates, and gives the application strong support because it is a unique group. There may be some issues with the proposal, but it is good science.