SYNOPSIS: The overall goal of this application is to develop methods for treating age-related macular degeneration by stem cell transplantation to repair the damaged retinal pigment epithelium (RPE). To this end, the applicant proposes to develop a xenotransplantation system in which human cells (either CD34-enriched bone marrow or cultured bone marrow MSCs (obtained from Darwin Prockop) will be transferred into the eyes of chick embryos. In the first Aim, the applicant will assay the ability of human bone marrow (BM) cells to differentiate into RPE after injection into the developing chick eye (injections will be done at multiple stages of development). The efficiency of engraftment and possibility of cell-cell fusion will be assessed by immunostaining. In Aim 2, the RPE engrafting activity of BM cells will be studied further by cell fractionation to identify which population(s) of BM contains the engraftment function. This will be done using proteomic and gene expression analysis of human cells re-isolated from engrafted chick eyes. Finally, in Aim 3, the applicant will attempt to enhance the engraftment efficiency of transplanted human bone marrow cells by either pre-treatment of the cells to be transplanted with laser irradiation, or by irradiation or mechanical damage of the recipient chick eye. These studies will again be read out by immunostaining approaches, and also by gene profiling. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: Age-related macular degeneration (AMD) is a leading cause of blindness, and strategies to prevent or reverse vision loss in patients suffering from AMD are acutely needed. The major issues in translating stem cell treatment for therapy of macular degeneration are (1) suitable cell source/type and the (2) aged microenvironment into which they will be placed. In this respect, the use of stem cell therapy for this degenerative disease is very timely. This proposal aims to develop a novel xenotransplant model for analyzing the ability of human bone marrow cells to generate retinal pigment epithelium (RPE). The experiments are high risk, but the applicant has formed collaborations to complement his expertise. An overarching concern with the strategy proposed is that the xenotransplant system will not properly recapitulate the human eye or human disease, particularly as birds do not develop AMD. The use of potentially autologous cell sources is a great idea, however, a big hurdle in this regard is the average age of the (real, clinical) autologous donors. Even if fetal or young adult marrow cells can undergo ‘robust’ differentiation into RPE (yet to be shown) and engraft in the chick model, there is no guarantee that autologous cells from an old human would do the same thing. Similarly, no matter what the cell behavior is in the embryonic chick eye environment, there is certain to be different behavior in the old human eye (with a good chance of diabetes) with a oxidized microenvironment, inadequate nutritional sources for the transplants, and largely uncharacterized problems that change signals to stem cells with aging. Although the grant is likely to tell us something (and an important something) about the molecular mechanisms of differentiation to the RPE lineage, the choice of cells and host do not represent the most clinically relevant studies to push toward translation. This caveat calls into question the ability to translate discoveries made in this system, particularly as they relate to interactions of the transplanted cells with the microenvironment, which will not accurately model the human AMD situation. In addition, the rationale described by the investigator for using human bone marrow as a source of replacement cells is not compelling - the primary concern regarding the choice of replacement cells should be their robust capacity to engraft and regenerate the tissue of interest, not the ease with which they can be isolated or stained by antibodies. Significantly, prior studies cited by the applicant in which the ability of bone marrow cells to generate RPE in adult recipients was analyzed suggest that these cells are poor candidates for cell replacement, and so would not be the population of choice for therapy. Human embryonic stem cells, on the other hand, undergo extremely robust differentiation to RPE, and large numbers of RPE cells are easily obtained from hES cells. The molecular underpinnings of differentiation (this time species-specific for human) could easily be studied in vitro using hES cells. And the cells have been transplanted into mouse models of blindness with excellent results. Human embryonic stem cells are not autologous, granted, but they could be HLA-typed and appropriate immunosuppression could be used for clinical transplantation. Significantly, prior studies cited by the applicant in which the ability of bone marrow cells to generate RPE in adult recipients was analyzed suggest that these cells are poor candidates for cell replacement, and so would not be the population of choice for therapy. In addition, it is unclear how well the transplanted bone marrow cells will compete with endogenous RPE precursors, which already support robust regenerative activity in the primitive chick eye. At the very least the PI should show some preliminary data using the chick model for transplantation. There are important basic feasibility issues in the proposal that suggest the timeframe is unrealistic. Importantly, the PI needs to develop expertise with the model. Furthermore, it is not at all clear that marrow-derived stem cells will be efficient generators of RPE, even in the conducive environment of the chick eye. Also, the fact that cells can be identified in tissue culture dishes does not make a clear case that the cells will be detectable easily in the animal studies. Another concern is that both Aim 2 and Aim 3 depend on the successful demonstration in Aim 1 that human bone marrow cells engraft to generate RPE in the chick eye. If the xenotransplant model does not show robust engraftment, Aims 2 and 3 are not relevant. This is particularly a concern in light of published experiments suggesting a poor potential of bone marrow cells to function in analogous adult animal models The success of the genomic and proteomic approaches proposed under Aim 2 depends on the ability to re-isolate sufficient numbers of human cells after engraftment, which is not demonstrated. Also, data needs to be presented on the amount of protein material that will be available for proteomic analysis. More importantly, the ability to distinguish relative representation of chick vs. human proteins needs to be clarified in the proteomic studies, which will yield fragments with considerable overlap. How rich are the chick proteomic databases for this purpose? The PI states that the protein pellet will be divided into membrane and non-membrane fractions, implying that they will be separately analyzed in the proteomics analysis, which makes the abundance issue even more concerning. The PI acknowledges that the amount of tissue for RNA is going to be limited, yet does not discuss the necessary amounts of protein (from a single eye?) that would facilitate proteomics analysis. Similarly, there are certainly new proteomics techniques available at UCI so that some consideration of precise methodologies, statistics, and the software for pathway analyses deserves discussion. As the plan stands, the proteomics work does not appear to be feasible. Finally, the applicant proposes to confirm the identified targets in this study by immunostaining and FACS, but this will depend on the availability of antibody reagents, which may not exist for the targets of interest. The rationale for the pre- and post-transplantation injury experiments proposed in Aim 3 is unclear. Do these injuries model AMD? It is unlikely that the microenvironment of the developing chick eye resembles closely the microenvironment in the eye of an aging human, and so the usefulness of information obtained from these studies is questionable.The numbers of groups/conditions for many of the experiments are unclear and the presentation suggests they are over-ambitious. How many cells will be transplanted per eye? (Presumably the non-transplanted eye is a control?). How much tissue is extracted per eye? In the preliminary data presentation, we are given raw data only as single numbers (98% vs. 100%) with no indication of standard deviation vs. error, number of experiements, so that the data are hard to interpret. The numbers of transplants necessary for each experiment is not clear. How many laser times and frequencies will be tested, given that one of the end-points is a very difficult analysis from explanted, transplanted chick embryos? The PI states that if the laser experiments don’t work to precondition the cells for better engraftment, then they will try the hypoxic preconditioning. What is the end-point of the laser experiments (given all the conditions to be tested) that will make them move on to hypoxic preconditioning? There is no acknowledgement that hypoxic preconditioning details are different for every cell and organ type, and also differ considerably by gender. The timing (length of time from hypoxia to implantation) is critically important, as well as the precise level of hypoxia and the precise length of time the hypoxia is held constant. The design of the experiments is not such that these issues will be sorted out. And why go on to hypoxic preconditioning when the few preliminary data presented suggest that laser and hypoxia are working in different directions in the cells? Overall, this is an interesting proposal from a qualified physician-scientist. However, the clinical context could be better served by rethinking this proposal to be more translationally oriented. There are important basic feasibility issues in the proposal that suggest the timeframe is unrealistic. First, the PI needs to develop expertise with the model. Second, it is not at all clear that marrow-derived stem cells will be efficient generators of RPE, even in the conducive environment of the chick eye. Third, there is not clear delineation of the numbers of experiments under each aim, but reading between the lines, there are just too many conditions in each experimental protocol. Fourth, the technical issues are not addressed re: proteomics approaches, hypoxic preconditioning, and others. As an aside, it is disappointing that the RPE work from human embryonic stem cells is never acknowledged in this proposal, especially given that this is a CIRM application. If this were an RO1, the study section would likely ask the grant to be modular, but the budget is for twice the usual modular budget amount per year. Personnel devoted: PI (40%), co-PI (0% for all years except year 4, 5%), proteomics collaborator asked for 3% on year 3 only, technician (50%) responsible for cell culture and grafts, technician (100%) with no listing of animal experience in the qualifications, post-doc (100%) for Aim 3, and a part-time undergraduate. If the experiments were more focused, the budget could be more realistic. QUALIFICATIONS AND POTENTIAL OF THE PRINICIPAL INVESTIGATOR: The applicant has great potential to develop a productive physician-scientist career, and should be encouraged to incorporate stem cell biology into his work. He has a terrific training background, and clearly understands the basic science of the retina. The PI’s strength is his clinical training and post-doctoral research experience, which speak through the grant as it is written with a very sophisticated understanding of the retina. Although the PI is an ophthalmologist, there will be a learning curve necessary for execution of these experiments. (neurosurgeons do not necessarily translate into great mouse neurosurgeons). Presumably a technician or post-doc will be carrying out some of the work, so there may be a greater learning curve there. These are not insurmountable given the PI’s background, but should have been an open part of the plan. The applicant received his MD in 1989 from Moscow Medical and Dental School, and his PhD in 2002 from the Molecular Biology Institute at UCLA. He has trained as a physician and scientist at several institutions in California, including UCSF and UCLA. He became an assistant professor in the Department of Ophthamology and Biological Chemistry at UC-Irvine in 2006. Dr. Lerner lists 9 publications in the years 1997-2006. His funding support appears to come from setup funds supplied by UC-Irvine. He appears to have no previous experience with the chick xenograft model he proposes to develop, but he has published first-author papers on retinal cell biology and has a clinical knowledge of AMD and other retinal diseases. For establishment of the chick xenograft model, he will rely heavily on collaboration with Dr. Joel Glover, who will visit Dr. Lerner's laboratory for several weeks from his home in Oslo in order to train staff and trouble-shoot the system The applicant outlines a career development plan focused around developing cellular therapies for retinal disease. This goal has led him to stem cell biology. His plan is to develop and test pre-clinical models, and then to use his clinical expertise to translate these for human therapy. He will utilize the resources of the Gross Stem Cell Research Center at UC-Irvine to develop his knowledge of stem cell biology and receive constructive criticism about his ongoing experiments. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: UC-Irvine has provided ample laboratory space, equipment, core facilities, administrative assistance, and a relatively small start-up package to support the applicant's research. It is unclear how much clinical time he has to spend vs. laboratory time. In addition, Dr. Lerner's career development is supported by mentorship interactions with the Faculty Oversight committee of the Gross Stem cell Research Center, with whom he will meet bi-annually. The scientific interactions that the PI will obtain through this center will be very helpful in supporting the proposed studies. The institution also provides important clinical collaborations and resources, including the UCI Institute for Clinical Translational Science, that will assist the PI in obtaining and using human cells and in biostastical analysis of the data. UC-Irvine has a track record of excellence in developmental biology and regenerative medicine, and has housed a national Center of Excellence for Developmental Biology for the past 25 years. The university also has made and investment in stem cell science with the recent establishment of the Gross Stem cell Research Center. Specific expertise in stem cell biology is indicated by the receipt of this institution of previous CIRM awards, including a CIRM Training Grant, and by the intention of the university to hire several additional faculty in the stem cell field over the next several years. Several relevant core facilities are available. DISCUSSION: Age-related macular degeneration is an important clinical problem. The PI proposes to use human bone marrow stem cells (BMSC) to generate RPE cells (AMD disease target), using chick embryo eye as model. However, it is already known that mouse bone marrow stem cells do not engraft. At least one reviewer questions the usefulness and purpose of using another model - selecting a system to fit the cells is not a very convincing approach. Also, given that retinal pigment epithelium (RPE) can be generated from hESC (which the applicant fails to mention), why use bone marrow stem cells to repair RPE?