UCLA

The RPE is a polarized epithelium, whose apical surface faces the outer segments of photoreceptor cells in the neurosensory retina, and performs numerous functions that are essential for healthy vision. In this dissertation, I investigated aspects of RPE biology that are critical for cellular homeostasis and, when perturbed, may contribute to RPE dysfunction and retinal degeneration. In particular, I focused on the RPE’s phagocytic function in which the cells internalize shed disks of photoreceptor outer segments and subsequently degrade the resultant phagosomes, thereby maintaining photoreceptor health. Using in vitro models of RPE, phagocytosis assays were performed to test the kinetics of outer segment degradation using state-of-the-art live-cell imaging. The kinetics of this process were further tested in a mouse model of dominant Stargardt 3 macular degeneration, which showed that the digestion of mutant outer segments is delayed and thus contributes to photoreceptor degeneration. This data implicated the impaired motility of phagosomes in the RPE as a major contributor to their delayed degradation, thereby identifying a sensitive measurement of a parameter that is critical to overall RPE health. Given that the RPE is a primary site of insult in retinal degeneration, cell transplantation may be a viable treatment to replace damaged RPE cells in human patients. To that end, we generated human RPE cells from induced pluripotent stem cells and extensively characterized the critical aspects of their cell biology. Importantly, we demonstrated that when these cells manifest phagosome degradation kinetics, comparable to that in vivo, and, when transplanted into a mouse model of retinal degeneration, they are capable of integrating into the host monolayer and partially rescue the photoreceptor from degenerating. Lastly, we developed a rapid method to differentiate a human immortalized RPE cell line such that the cultures exhibit many of the characteristics of their in vivo counterparts, and we propose that when these cells are properly differentiated, they can be used to draw meaningful inferences about RPE physiology and pathology.