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RN1-00551-1: Engraftment and Differentiation of Adult Human Stem Cells in Embryonic Eye

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Age-related macular degeneration (AMD) is the leading cause of blindness in the United States, for which there is no cure. It has devastating effects on the quality of life as it makes it impossible for affected individuals to read and drive, and has become a major public health concern in the rapidly expanding aging population of California. AMD is particularly amenable to a potential cell-replacement therapeutic strategy because the affected areas of retinal pigment epithelium (RPE, the tissue primarily affected by the disease) can be directly visualized, and transplantation of stem cells could be performed by intraocular injections using currently available surgical techniques. A number of recent animal studies suggest a great potential for using various populations of stem cells to improve the morphological and functional capacity of the RPE. However, we are yet to develop a stem cell-based approach for the management of specific retinal diseases such as AMD, and to establish clinically acceptable treatment protocols. Patient-specific stem cell transplantation is currently the safest and, thus, preferred approach in patients with AMD at least for the near future. This implies using adult stem cells obtained from an easily accessible and abundant tissue source (such as bone marrow) from the same patient, which greatly increases their immune compatibility and overall safety. Following their isolation, these cells may be injected into the eyes of patients with age-related macular degeneration over the areas affected by the disease. However, there are two major obstacles that prevent successful RPE regeneration using adult patient-specific stem cells. When transplanted into adult tissues, these cells have a relatively low potential for developing into adult cells bearing the desired function and have relatively low efficiency to engraft within adult microenvironment. We propose to develop a robust, convenient and rapid engraftment approach using eyes of chick embryos for guiding the developmental program of human bone marrow-derived stem cells towards their development into RPE. Embryonic chick eye is more permissive for stem cell engraftment compared to the adult tissues and provides a natural habitat for the donor stem cells to follow the native RPE developmental program in the context of ocular environment. We will determine and characterize the developmental pathways for native human RPE precursor cells and transplanted adult human stem cells transplanted into chick embryos. We also propose to modulate interactions between donor stem cells and the recipient embryonic ocular microenvironment to enhance the efficiency of stem cell incorporation. The proposed embryonic chick ocular model provides the foundation for future development of a broad platform for patient-specific stem cell-based AMD treatment and, therefore, has important therapeutic implications for the leading cause of blindness in the United States.

Statement of Benefit to California (provided by applicant)

My proposed research focuses on the development of stem cell-based therapeutic approaches to age-related macular degeneration (AMD), and other diseases in which deterioration of the retinal pigment epithelium (RPE) plays a significant role. It will benefit the citizens of the state of California in several important ways. AMD is the leading cause of blindness in the United States, and in California, and it is currently incurable. It has devastating effects on quality of life and independence, and is becoming a major public health concern as California’s population ages, and the state’s older citizens live longer, and require more assistance in the activities of daily living. The socioeconomic and emotional impact of AMD is tremendous because the disease affects central vision, limiting the ability of those who suffer from it to drive or read. Identifying a successful treatment would allow tens of thousands of elderly Californians to enjoy a more functional, productive, and enjoyable years beyond retirement. Several studies have suggested the great therapeutic potential of various populations of adult stem cells derived from bone marrow for regeneration of RPE. Stem cell transplantation has already received considerable attention as a novel neurotherapeutic strategy, but so far, little work has been devoted to stem cell therapies for specific retinal disorders. We have yet to develop a stem cell approach for the management of specific retinal diseases like AMD, or to establish clinically acceptable treatment protocols. Patient-specific transplantation of adult stem cells obtained from patient’s own bone marrow or blood has many practical advantages over the use of human embryonic stem cells. Most importantly it eliminates the need for immunosuppressant drugs necessary in transplantation of cells or tissue from another individual, and carries no risk of tumor formation. The research proposed in the current application to the California Institute for Regenerative Medicine will utilize the embryonic chick model to lay the groundwork for the use of human bone marrow-derived multi-potential stem cells as a potential therapy for the blinding retinal degenerative and age-related disorders. This will benefit California’s citizens by speeding our progress towards a safe and effective treatment for AMD and other related diseases affecting vision.

Review

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 bone marrow-derived cells 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. In Aim 2, the RPE engrafting activity of BM cells will be studied further to identify which population(s) of BM contains the engraftment function. Finally, in Aim 3, the applicant will attempt to enhance the engraftment efficiency of transplanted human bone marrow cells by approaches to modulate the interactions between the donor stem cells and the recipient chick embryonic microenvironment.

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 (2) the 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/her 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 where there is a good chance of diabetes, an oxidizing microenvironment, inadequate nutritional sources for the transplants, and largely uncharacterized problems that change signals to stem cells with aging. Although the application 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 characterized. Significantly, prior studies cited by the applicant suggest that these cells are poor candidates for cell replacement, and so would not be the population of choice for therapy. Human embryonic stem (hES) 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 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. 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. 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. Significantly, 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. 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 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 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. 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 using methods that 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 is unclear. It is unclear that these injuries model AMD. The numbers of groups/conditions for many of the experiments are unclear and the presentation suggests they are over-ambitious. In the preliminary experiments, the data are hard to interpret. The description of the proposed experiments in this aim was insufficient given that one of the end-points is a very difficult analysis from explanted, transplanted chick embryos.

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. Important basic feasibility issues in the proposal that suggest the timeframe is unrealistic.

QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant has great potential to develop a productive physician-scientist career, and should be encouraged to incorporate stem cell biology into his/her work. S/he has a terrific training background, and clearly understands the basic science of the retina. The PI’s strength is the strong clinical training and post-doctoral research experience, which speak through the grant as it is written with a very sophisticated understanding of the retina.

The applicant received an MD from a foreign medical and dental school, and a PhD from an institution in California. S/he has trained as a physician and scientist at several top tier institutions in California, and became an Assistant Professor in the departments of ophthalmology and biological chemistry at the current institution in 2006. His/her funding support appears to come from setup funds supplied by the institution.

Although the PI is an ophthalmologist, there will be a learning curve necessary for execution of these technical experiments. 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 lists approximately 10 publications in the years 1997-2006. S/he appears to have no previous experience with the chick xenograft model described in this proposal, but s/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, s/he will rely heavily on a collaborator who will visit the applicant’s laboratory for several weeks from a remote location in order to train staff and troubleshoot the system

The applicant outlines a career development plan focused around developing cellular therapies for retinal disease. This goal has led the applicant to stem cell biology. The candidate’s plan is to develop and test pre-clinical models, and then to use his/her clinical expertise to translate these for human therapy. S/he will utilize the resources of the stem cell research center at the institution to develop knowledge of stem cell biology and receive constructive criticism about ongoing experiments.

INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The institution 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 the applicant has to spend vs. laboratory time. In addition, the candidate’s career development is supported by mentorship interactions with the faculty oversight committee of the stem cell research center, who will meet with the applicant 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 translational science expertise that will assist the PI in obtaining and using human cells and in biostatistical analysis of the data.

The applicant’s institution has a track record of excellence in developmental biology and regenerative medicine, and is recognized for excellence in developmental biology. The university also has made an investment in stem cell science with the recent establishment of a new stem cell research center. Specific expertise in stem cell biology is indicated by the receipt of previous CIRM awards by this institution, and by their intention 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 (AMD) is an important clinical problem. The PI proposes to use human bone marrow stem cells to generate RPE cells, an AMD disease target, using chick embryo eye as model. However, reviewers were not enthusiastic about this proposal since it is already known that mouse bone marrow stem cells do not engraft. At least one reviewer questioned the usefulness and purpose of using the 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, why use bone marrow stem cells to repair RPE?

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:

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