Decades of study have demonstrated that adult human retinal pigment epithelium (RPE) cells have strong proliferative capacity in vitro, which indicates that the RPE layer has the possibility of self-repair. We have shown that adult RPE from elderly donors or donors with age-related macular degeneration (AMD) can proliferate in culture and produce a near-native, renewed RPE monolayer. Despite this, RPE cells in vivo do not regenerate the damaged cobblestone RPE layer in patients with degenerating cells, such as those with dry AMD. The environment in vivo must effectively prevent repair of the cobblestone RPE monolayer, either through lack of mitogens or inhibitory molecules or a combination of both. The overarching goal of these studies is to achieve safe, controlled proliferation of endogenous RPE cells to enable self-repair of the RPE layer in patients with AMD. The objective of this proposal is to characterize the environmental factors that positively and negatively control the proliferation of adult human RPE cells. The first specific aim is to characterize the adult human RPE cell surfaceome on dividing and non-dividing cells using an innovative mass spectrometry and bioinformatic platform. This will provide the first comprehensive analysis of the molecules on the RPE plasma membrane and reveal cell surface receptors and secreted proteins that respond to environmental factors impacting cell division. Investigating both normal and AMD RPE will provide a greater understanding of how RPE cells change with disease. The second specific aim is to examine a transcriptional network we have identified that is associated with adult human RPE cell proliferation to determine which molecules are critical. To do this efficiently, we will first employ CRISPRi, then additional functional screens. The third specific aim will examine whether exogenous factors can activate proliferation of quiescent, cobblestone human RPE, including those in situ on Bruch?s membrane explants, and those from patients with AMD. In addition to the main objective, this study will generate new knowledge about RPE molecules that can be used to target RPE in vivo. The proposed work also has the potential to improve RPE cell proliferation ex vivo for more efficient cell manufacturing. Most importantly, this study will create a foundation for safely stimulating RPE cell proliferation in vivo. Endogenous activation of RPE cell proliferation to counteract RPE cell loss in AMD has the potential to avoid surgery and immunosuppression involved in RPE cell transplantation, which would greatly benefit the elderly AMD patient population.
The retinal pigment epithelium (RPE) degenerates in diseases such as dry age-related macular degeneration (AMD), for which no therapy is currently available. Here we propose to develop a novel approach to RPE repair ?increasing the number of RPE cells by stimulating their division. We will define the environmental factors regulating adult human RPE proliferation using cutting edge methods to assess protein expression. Then, we will use targeted screens to test specific genes, growth factors and small molecules for potential to repair the RPE layer.
