Ocular diseases affect millions world-wide and dramatically influence the quality of life. Although much is known about ocular biology and disease pathologies, effective treatments are still lacking. The eye is well suited for application of emerging cell-based therapies. This dissertation will explore the development of stem cell-based treatments for age-related macular degeneration (AMD), a prevalent ocular disease in the elderly. Retinal pigmented epithelium (RPE), a cell type implicated in AMD, has been derived from both human induced pluripotent stem cells (iPSC) and human embryonic stem cells (hESC). Rapidly advancing research has generated various methods of RPE differentiation and several transplantation strategies. The following chapters will provide an in-depth overview and characterization of hESC-RPE and iPSC-RPE across two methods of derivation.
Pluripotent stem cell derived RPE have unique cellular characteristics. This dissertation will also examine various RPE behaviors upon manipulation with a synthetic kinase inhibitor and describe the creation and production of exogenous reporters that can be used for real-time monitoring of cellular transplantations.
Human embryonic stem cell derived retinal pigmented epithelium (hESC-RPE) is in clinical trials for the treatment of macular diseases. Currently, these cells take over three months to derive and subsequent months to mature and characterize. After only four to five passages the cells begin to undergo an epithelial-to-mesenchymal transition and are unsuitable for cellular therapies. We show in this dissertation that inhibition of Rho-associated coiled-coil kinase (ROCK) using specific inhibitor, Y-27632, can increase the lifespan and proliferation rate of hESC-RPE in culture. hESC-RPE maintain typical cuboidal morphology, gene expression, protein localization, factor secretion, and phagocytic abilities up to passage 13 following ROCK inhibition.
In addition, this dissertation will elucidate other effects of ROCK inhibition on hESC-RPE. ROCK inhibition is known to affect cytoskeletal rearrangements and we will show that ROCK inhibition can promote wound closure and attachment through increased cell cycle activation and cell spreading respectively. Elucidating these effects and beginning to understand the mechanisms of wound healing and attachment is critical when considering ROCK inhibition as a novel treatment for geographic atrophy, a late stage phenotype of AMD, and as a potential combinational therapy with cellular replacement to enhance engraftment.
In the final chapter, the creation of a molecular tool basket will be described. As hESC-RPE progress through the phases of clinical trials, it will be important to gain a better understanding of how the cells behave in vivo to address any potential caveats within the clinical trials. Fluorescent reporters that monitor transplanted cell migration, function and mitotic stage have been created to examine, in real time, how the stem cell derived transplant is affected by the endogenous retinal milieu.
The findings within this dissertation have tremendous implications in understanding the variability between hESC-RPE and iPSC-RPE and the various methods of derivation as these cellular therapies move into the clinics. In addition, the benefits of ROCK inhibition on hESC-RPE and iPSC-RPE could lead to combinational therapies and help produce large intermediate cell banks if clinical trials prove efficient and a large scale cell production is required.