Retinal Pigment Epithelium Derived from Human Embryonic Stem Cells The long-term objective of this proposal is to develop applications of human embryonic stem (hES) cells in the treatment of eye diseases, including Age-Related Macular Degeneration, one of the leading causes of human blindness. This disease, as well as other retinal degenerations, is thought to involve the death of the retinal pigmented epithelium (RPE), a layer of pigmented cells in the back of the eye that nourish the photoreceptors of the retina. Preliminary work has shown that hES cells can differentiate into RPE-like cells, so a possible cellular therapy could involve transplantation of these cells in areas where native RPE has been lost. However, there is much to do before it is safe to begin human clinical trials with these cells. Currently only small numbers of cells can be produced, and it is not yet clear if they can fulfill all of the important functions carried out by the native RPE. Studies are proposed that focus on aspects of hES-RPE cell biology that will impact their potential application in ocular therapy. The first aim targets the identification of molecular mechanisms regulating the differentiation of hES-RPE cells, focusing on growth factors, extracellular matrix, matrix receptors, and transcription factors that have been implicated in RPE development in animal studies. Currently little is known about the factors that cause an undifferentiated hES cell to turn into an RPE cell. Ultimately, these studies should lead to the identification of optimal culture conditions that drive hES cells down the RPE differentiation pathway. The second aim is a comprehensive a molecular and cellular characterization of hES-RPE to assess the full extent of similarity to native RPE. Key RPE functions, including transport of nutrients, synthesis of pigment, and interaction with photoreceptors will be assessed. hES-RPE will be compared to native RPE via analysis of proteins, RNAs, and DNAs. Finally, the third aim proposes functional testing of hES-RPE in animal models to evaluate their key functions in vivo. While the proposed studies are considered high risk, they have an equally high potential gain. The experiments will provide a provisional assessment of the suitability of hES-RPE for ocular therapeutic applications.
Statement of Benefit to California:
Age-related macular degeneration (AMD) is the leading cause of blindness in elderly patients in California. An estimated 30% of people over 75 years of age are diagnosed with AMD and the disease incidence is predicted to double over the next 25 years, which represents a significant public health challenge to the State. The disease leads to the deterioration of fine acuity vision in the middle of the visual field, and can eventually lead to total blindness. The pathogenesis of this disease is poorly understood and there are no effective treatments. Loss of vision is a result of photoreceptor death, which is thought to be a consequence of the death of the neighboring retinal pigment epithelial (RPE) cells. One strategy that holds promise for the treatment of AMD is the replacement of defective RPE cells via cellular transplantation, and recently, it was demonstrated that human embryonic stem cells can be coaxed to turn into RPE-like cells (hES-RPE). This proposal will assess the suitability of these cells for possible use in human therapy for AMD. The benefit to California will be the development of a potential therapy for a blinding disease that affects millions of its citizens.
SYNOPSIS: Age-related macular degeneration occurs because of degeneration of the photoreceptors in the macular region, which is presumed to be a result of degeneration of the neighboring retinal pigment epithelium (RPE). A recent report describes restoration of some retinal function in a genetic rat model of indirect photoreceptor degeneration, the RCS rat, by hES-RPE cells. This proposal aims to develop methods that are optimal for the generation of homogeneous preparations of RPE cells from hESCs. In Aim I, the PI will identify factors (cytokine/growth factors, the extracellular matrix, and transcription factors) regulating the differentiation of hES-RPE cells. The PI proposes to develop methods to enhance the production of retinal pigmented epithelial cells from human embryonic stem cell lines. Two other groups have already demonstrated that RPE cells spontaneously arise in human ES cell cultures under differentiating conditions, and the PI proposes to test whether this process can be enhanced by culturing the cells with various BMPs, since these factors have been shown to be critical for RPE development in model systems. In Aim II, the PI will characterize these cells from a molecular and cellular perspective and compare them to native human fetal RPE. In Aim III, the PI will test the therapeutic potential of hES-RPE cells in vitro (e.g., in retinal cell or human vascular endothelial cell cultures) and in a number of animal models of retinal degeneration. SIGNIFICANCE AND INNOVATION: Macular degeneration is a formidable problem among the elderly, affecting 30% of individual over 75 years. The reports that hESCs can differentiate into RPEs and that hESC-RPE can correct retinal degeneration are exciting ones, and ones that provide a very strong rationale for this study and its potential substantial impact on health. The use of hESCs-RPE for macular degeneration is a novel direction, but the overall strategy to use developmental information to design conditions for directing human ES cells to particular fates is not, except that this has not yet been done for the RPE. The structural, molecular and functional characterization of hESCs-RPE and the planned transplantation of these cells into animals with Na iodate-induced RPE degeneration, physical RPE debridement, and corneal angiogenesis involve up-to-date techniques and creative approaches to study the usefulness of these cells. In addition, the PI's characterization of RPE function in-vitro (i.e., for their ability to form tight junctions, process retinoids, and phagocytose outer segments) is a level of analysis that is more extensive than what other groups have thus far attained. STRENGTHS: The proposal is well-written and nicely organized, proceeding in a logical and very comprehensive way to characterize hESC-RPE and compare them with fetal RPE. It is likely that new, important information will be gleaned from the studies planned in this application. The PI, Chair of the Department of Molecular, Cellular & Developmental Biology at UCSB and Director, UCSB Stem Cell Training Program of CIRM, is a productive scientist and expert in fields very relevant to the present proposal. This proposal is also highly collaborative, and the PI and collaborators have extensive experience with retinal cell biology. This is a very strong team covering experts in retinal development, RPE biology, proteomics and genomics, and hES cell propagation and biology. They have used or in some cases developed the assays for RPE and retina to be used to characterize any RPE that they derive from the human ES cells. Interestingly, the group apparently originally submitted three separate Letters of Intent, but decided to “gather forces and submit one proposal focused on hES-RPE." WEAKNESSES: This is a big, ambitious proposal which reads more like a Program Project than a SEED proposal. One worries that there are too many directions with a catalog of planned experiments that is too long involving too many people. The PI has little previous experience with human ES cells, and little experience with developmental biology upon which they base their strategy. The collaborations are impressive, but coordination of the group of scientists involved in this proposal will be challenging. There are clear risks to this proposal, e.g., that hESC-RPE may not be useful in human therapeutics. In addition, the basic strategy reflects a lack of appreciation of the various stages of development that the cells are likely to have to proceed through in order to generate RPE. Most successful attempts to direct ES cells to particular fates employ a multi-step process. Simply adding BMPs to the ES cells will likely promote their development into posterior, non-neural tissues, and the yields of RPE will likely be lower than adding nothing at all. Further, the proposal is rather descriptive in nature as details regarding many of the proposed experiments are lacking. For example, the PI has not detailed methods for the purification of RPE cells from cells that do not acquire the desired phenotype. This is critical to the interpretation of all the studies that follow from the initial derivation of these cells. Moreover, Specific Aim 2 will characterize the hESC-RPE using a protocol that Specific Aim 1 may modify. Finally, the PI notes that he replicated “the work of Klimanskaya, et al. and obtained RPE derivatives of the H9 hESC line.” Although preliminary data are not required in SEED proposals, it would have been valuable to provide a small amount of the data from these preliminary experiments. DISCUSSION: This proposal aims to create methods to generate homogeneous populations of retinal pigment epithelial (RPE) cells for ultimate therapeutic potential. This field has great potential, and the interest in this grant is based on prior publications showing that hESC can be differentiated into RPE and transferred into rat models of photoreceptor degeneration. This kind of work has been proposed and many people outside of California are working on this problem, including the Lanza group from ACT, and the field has already moved beyond the studies proposed here. This makes reviewers somewhat less enthusiastic. The approach is somewhat naive in attempting to directly differentiate cells, and reviewers feel that the applicant has not thought through the experiments very well and that the experiments likely won't work as planned. The broad scope of the proposal was a major criticism. The catalog of experiments listed amounts to a long description of relevant observational approaches, but likely too broad in scope, and it was thought to be perhaps "too collaborative" with 8 PIs and an overambitious research plan. Reviewers thought that the proposal lacked an appropriate discussion of experimental detail, and pointed out that the claims of having made RPE cells were not supported by the inclusion of relevant data. RPEs are a small percentage of the cells from hESC cultures, which are made by flushing out the pigmented cells from cultures. While the applicant proposes to expand the yield of differentiated RPE, and there may be some use in trying new development protocols, there is no indication that the applicant's lab has the expertise to generate RPE lines. If RPE cells are actually found, the applicant is very well suited to do much of the follow-up analysis, except that they do not have the relevant experience with transplantation.