Human Adult Retinal Pigment Epithelial Stem Cell–Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue

The RPE is a monolayer of cells between the neural retina and choroidal blood supply, and is critically important for photoreceptor function.1–3 For example, in vivo light/dark transitions trigger RPE physiological changes that homeostatically maintain the chemical composition and volume of the extracellular spaces separating the apical membrane/photoreceptor and basolateral/Bruch membrane interfaces.4–6 Damage to the RPE with aging and disease can lead to vision loss, as seen in age-related macular degeneration (AMD), Best's disease, retinitis pigmentosa, and other retinal degenerative diseases.7–11 In vitro, primary cultures of human RPE represent an attractive model for the study of RPE-related diseases, because they allow the independent pharmacologic manipulation of the retinal and choroidal sides of the monolayer while monitoring the polarized physiological responses to specific disease-producing agents.12–16 Retinal pigment epithelium cell lines, such as ARPE-19, a spontaneously immortalized line,17 have proven useful for in vitro studies, but are limited in their ability to mimic molecular and physiological functions of primary RPE.18–24 Primary cultures of fetal human RPE (fhRPE) are a highly valuable model, since they exhibit many of the known functions of mammalian and native human RPE (nhRPE). Several protocols have been developed to improve culture reproducibility and allow assessment of a wide variety of RPE functions.15,24–36 However, cultures from adult human RPE (ahRPE) may be physiologically more representative of native mature tissue. Prior pioneering work has demonstrated that ahRPE can proliferate successfully in culture. However, these cultures lose their native physiology, tending to undergo an epithelial-mesenchymal transition and form fibroblast-like progeny more readily than fhRPE.37–41 Recently, we have shown that the adult RPE layer includes a subset of cells that can be activated in vitro to self-renew and produce large numbers of new RPE cells.42 We have developed a method to culture ahRPE, which, based on the preliminary examinations so far, suggest an improvement of the preservation of morphology.43 These RPE stem cell (RPESC)–derived RPE cultures can be isolated from donor eyes, cultured, and expanded to produce confluent monolayers of cobblestone RPE. The goal of this study was to determine whether this culture method is a significant advance in preserving native physiology over other attempts to culture adult human RPE. We examined in depth the physiology of ahRPE cultures and found they share many features with fhRPE and nhRPE. These studies suggested the use of ahRPE as an in vitro model of RPE physiology and disease. Since the ahRPE cultures can be generated successfully from aged human donors, even nonagenarians, they offer the opportunity to compare RPE characteristics during aging and between nonaffected and diseased donors. These “disease in a dish” models can be useful for therapeutic compound screening. In addition, our studies indicated ahRPESC-derived RPE represent a viable cell source for replacement of atrophic cells in patients suffering from RPE degenerative disease.