Basic Biology III
Vision is arguably our most important sense. While loss of vision is rarely life threatening, there is a tremendous loss of quality of life and there are substantial associated socioeconomic costs. A large fraction of the disorders that cause blindness result from the dysfunction and/or degeneration of retinal pigmented epithelium (RPE). The RPE is a simple monolayer of cells adjacent to the sensory retina that both supports and participates in normal retina function. Recent efforts to restore vision by translocation or transplantation of RPE from areas outside of the central visual field to diseased areas have resulted in marked improvement in sight. Unfortunately, for practical, economical, and technical reasons it is unlikely that this procedure will gain widespread usage. However, with the recent discovery that human embryonic stems cells can spontaneously differentiate into cells that bear marked semblance to RPE cells it should be possible to overcome these shortcomings. Lending further support to this approach is the demonstration that stem cell derived RPE-like cells can restore sight when transplanted into rats with vision loss resulting from RPE dysfunction. As a consequence, significant resources are now being directed at bringing a stem cell based therapy for the treatment of RPE degenerations to the clinic. Despite the promise of a stem cell based therapy for the treatment ocular degenerations, there are hurdles that need to be overcome. Most notably it will be essential to routinely and reproducibly produce large amounts of well characterized RPE cells for the treatment of potentially millions of patients. While initial characterizations of the stem cell derived RPE cells show that they express genes characteristic of RPE cells, our more recent analysis using improved methods indicate that there is more variation between stem cell-derived RPE cells than commonly appreciated. Whether the differences are an inherent property of the non-directed, spontaneous differentiation process used to derive these cells or as yet undetermined variables has not been resolved. This project seeks to determine the fundamental basis underlying the observed variability in RPE-like cells derived from embryonic stem cells and the molecular mechanisms that account for the these differences. Finally, using existing knowledge and information gained from these studies, the influence of potential regulators of RPE differentiation will be investigated to ascertain if they can act to direct RPE differentiation and lead to faithful, reproducible generation of stem cell derived RPE cells. In addition to having direct implications regarding the development of a stem cell based therapy for the treatment of RPE and retinal degenerations, these studies should provide significant insights into the molecular mechanisms controlling normal development and maintenance of the RPE and could lead to the development of novel non-stem cell based treatment strategies.
Statement of Benefit to California:
Numerous disorders resulting in blindness involve the dysfunction and degeneration of the retinal pigmented epithelium (RPE), a monolayer of cells that lies beneath the retina that is essential for retinal function and maintenance. The most prevalent of these diseases, age-related macular degeneration (AMD), is the leading cause of blindness for those over the age of 60 years. By age 85, upwards of 30% of the white population will exhibit some degree of AMD and it has been estimated that by 2020 as many as 50% of all individuals will be affected by some form of AMD in their lifetime. Not only are there devastating sociological impacts of the disease on a population that is ill-equipped to adapt to a vision loss, but there are enormous economical impacts as well. It can be estimated that the total costs associated with AMD in California are in excess of 5 billion dollars per year. While there is a treatment for a subpopulation of patients with complicating rampant blood vessel growth and anti-oxidant cocktails have shown promise in slowing the progression of the disease, there is no effective curative treatment for the majority of those afflicted with AMD. Recent efforts to replace the diseased RPE in AMD by relocation or transplantation have shown potential for the treatment of all patients with AMD, however the availability of tissue or cells for transplantation is limiting. One promising solution is the use of stem cell-derived RPE cells and it has been shown that transplantation of human stem cell derived RPE can restore vision in the retinal dystrophic rat. It is anticipated that initial clinical trials will begin in the near future. However, before a stem cell based therapy can be of widespread utility technical hurdles related to the large scale production of stem cell-derived RPE will have to be overcome. This proposal seeks to characterize the molecular processes controlling the in vitro differentiation of RPE from pluripotent embryonic stem cells and to identify specific methods that promote the faithful development of the RPE phenotype. In doing so, we hope to provide the framework and basis to enable the application of this promising therapeutic approach on a large scale. In addition to being directly applicable to the derivation of RPE from stem cells, the molecular mechanisms discovered through these studies will likely be relevant to the normal development and maintenance of RPE cells in vivo and could provide the basis for the development of non-stem cell based therapies. It is also likely that the general principles that regulate the derivation of RPE from stem cells will be shared in the development of other cell types from stem cells and will have general applicability, beyond ocular biology.
Project Synopsis: This proposal seeks to determine the fundamental basis underlying the observed variability in retinal pigment epithelial (RPE)-like cells derived from embryonic stem cells, and the molecular mechanisms that account for these differences. The applicant proposes to identify the molecular basis of faithful and unfaithful differentiation of stem cell (SC)-derived RPE from human embryonic stem cells (hESC) using an integrative systems biology approach. Three aims are proposed. Aim 1 will examine whether variable gene expression among SC-derived RPE lines is an inherent property of spontaneous differentiation; Aim 2 will assess the role of external variables on the development of authentic RPE; finally, Aim 3 will characterize the epigenetic changes associated with SC-RPE differentiation. An overall goal is to aid in the design of procedures for the efficient and reliable production of SC-RPE for therapeutic application. Significance and Innovation: - The applicant makes a case that SC-derived RPE lines will be therapeutically useful for the treatment of AMD, a significant health burden in an aging population, and presents substantial preliminary data demonstrating that variation exists with the current paradigm of spontaneous RPE generation from SC. - Reviewers questioned whether this variation is functionally significant or if it represents an acceptable degree of noise. If there is no observed functional correlate with the variation in expression, it calls into question the significance. Without a better understanding of this fundamental question, reviewers questioned the benefit to be gained from the huge data sets to be generated by the proposed experiments. This was a major criticism of the proposal. - The proposed study has some innovation, as it will use novel bioinformatics to undertake a more detailed assessment of genetic variability in SC-RPE isolates. Feasibility and Experimental Design: - The overall approach is very unfocused and there is no indication of how the investigators will use the information gathered to better understand the generation or fate determination of SC-RPE cell lines. - Reviewers believed the overall amount of work proposed was not achievable in the proposed timeframe given the size and bandwidth of the team. - The experimental design had several weaknesses, in particular, relating to cell purity and the culture conditions. Reviewers pointed out the need for a robust and reproducible, preferably GMP, protocol for generating high quality RPE cell lines. Without a high level of quality control, the study is unlikely to provide useful data. - There was a concern regarding the validity of the preliminary data, as the applicant commented that differences may be due to contamination by non-RPE cell types or to the fact that fetal RPE cells do not represent the adult RPE phenotype. - Specific Aim 3 and its focus on correlating DNA methylation patterns and variability in gene expression is the most interesting part of the application, to identify candidate mechanisms/genes for regulating expression. Those data, if the correlation has sufficient power, would be of value to the field by highlighting potential molecular targets for manipulating gene expression patterns. - The design of the experiments is appropriate to determine whether differences in gene expression represent stochastic consequences of spontaneous generation, source material, or other technical aspects of SC use. However, the reviewers argued that the applicants will not establish the biological significance of the differences. Principal Investigator (PI) and Research Team: - There were concerns about the strength of the team. The PI is primarily a biochemist, albeit with a long record of publication in the area of AMD. The systems biology approach seems to be a recent shift in emphasis and is an area in which the PI has not yet published. - Although a number of talented collaborators have been recruited to the team, there was concern that overall, the team was lacking in necessary expertise. - The facilities available to the PI, and the institute within which the PI is embedded, were thought to be considerable strengths of the application. - The budget appears appropriate. Responsiveness to the RFA: - The proposal attempts to get at the basic mechanisms underlying differentiation of ESCs, and is therefore within the scope of the RFA.