Age-related macular degeneration (AMD) is the leading cause of blindness in elderly populations in the developed world. AMD is characterized by progressive loss of vision in the central visual field, which is attributed to death and dysfunction of a pigmented monolayer of epithelial cells that reside at the back of the eye, underneath the neural retina, called the retinal pigment epithelium (RPE). The RPE is a highly specialized cell type, which plays a critical role in the visual cycle and maintaining health and homeostasis of the photoreceptors. When RPE become dysfunctional, photoreceptors die and vision is impaired. There are limited treatment options for the wet, or exudative, form of AMD and currently none for the more common, dry, or atrophic, form of the disease. Therapeutic efforts have been initiated, some advancing to clinical trials, using RPE differentiated from human embryonic stem cell (hESC) or induced pluripotent stem cell (iPSC) to replace diseased RPE and rescue vision. While RPE derivation methods are improving, a bottleneck for translation to the clinic is the time it takes to generate these cells. Additionally, many aspects of RPE development and differentiation remain poorly understood. The chapters that follow in this dissertation will describe development of an accelerated 14-day method for differentiating RPE from hESC, characterization and comparison of hESC-RPE and iPSC-RPE derived using two methods, and will provide insight into signaling pathways that work to promote RPE cell fate.

The first chapter of this dissertation offers an in depth overview of RPE differentiation methods, potential obstacles to translating these methods to the clinic, and the benefits of using patient-derived iPSC combined with RPE derivation methods for modeling ocular disease. The second chapter outlines an expedited 14-day directed differentiation protocol, which was modified to include activation of the RPE-promoting Wnt/beta-catenin signaling pathway to generate high quality RPE cells from hESC. This improvement yields cultures approximately 97% positive for the RPE marker, PMEL17, at day 14. In this chapter, RPE cell identity, purity, and function are extensively characterized. The third chapter, takes a more mechanistic look at how Wnt/beta-catenin signaling works to promote an RPE fate. The fourth chapter presents a collaborative, comparative study of iPSC-RPE differentiated using two methods: the 14-day directed method and the longer spontaneous continuous adherent culture method. This chapter shows comprehensive characterization of resulting RPE and discusses differences in derivation capacity between iPSC lines and between derivation methods. Furthermore, the results presented in this chapter underscore the need for optimizing assays that accurately test the quality of pluripotent stem cell-derived RPE. The fifth and final chapter provides additional considerations and future directions to the work presented herein.

The findings presented within this dissertation hold importance for identifying and acknowledging the variability that exists between RPE derived from different iPSC and hESC lines. This is especially relevant as more iPSC- and hESC-based therapies move toward clinical trials. Additionally, the benefits of further improving and understanding RPE derivation have implications not only for generating high quality cells for therapy, but also provide a unique in vitro platform in which to study the effect signaling pathways and transcriptional networks have on RPE differentiation.